1. health and fitness
2. disease
3. cancer

The organizers gratefully acknowledge and appreciate support
from the following sponsors of NESDB 2014:
The organizers would also like to thanks Drs. Michel Barresi, Elizabeth Deschene and
Ryan Cowan for their help with illustrations, Web design and program booklet
assembling.
NESDB 2014 PROGRAM
FRIDAY APRIL 11TH SESSION I: 1:00-6:15
1:00-1:15
WELCOME AND INTRODUCTORY REMARKS BY VALENTINA GRECO AND
CLARISSA HENRY
WELCOME ADDRESS BY VIVIAN IRISH (SDB PAST-PRESIDENT)
CHAIR:
MARY DONOHOE
1:15-1:35
SCOTT WEATHERBEE (YALE) : USING
FORWARD
GENETICS
TO
IDENTIFY
DEVELOPMENTAL GENES
1:35-1:50
JESSICA GRAY (HARVARD) : EVOLUTION
1:50-2:05
LAURA LOWERY (BOSTON COLLEGE) : GROWTH CONE-SPECIFIC FUNCTIONS OF MT
OF MIRNA SIGNALING IN DEVELOPMENT:
INSIGHTS FROM THE HEMICHORDATE SACCOGLOSSUS KOWALEVSKII
POLYMERASE
OUTGROWTH
2:05-2:20
XMAP215 IN RESTRICTING MT DYNAMICS AND PROMOTING AXON
VAIBHAV PAI (TUFTS) : ENDOGENOUS
INSTRUCTIVELY PATTERN NEURAL TISSUE VIA
PROLIFERATION
GRADIENTS
OF
RESTING
POTENTIAL
NOTCH SIGNALING AND REGULATION OF
2:20-2:50
COFFEE BREAK
CHAIR:
ALICIA EBERT
2:50-2:55
ABOUT SDB BY JOEL SMITH (MBL, SDB JR FACULTY REP)
2:55-3:15
LIONEL CHRISTIAEN (NYU): TRANSCRIPTIONAL
3:15-3:30
VALERIA YARTSEVA (YALE) : CHARACTERIZATION
CONTROL
SPECIFICATION IN THE ASCIDIAN CARDIOPHARYNGEAL MESODERM
OF
CELL
FATE
OF MRNA CLEARANCE DURING
THE MATERNAL-TO-ZYGOTIC TRANSITION
3:30-4:05
FAST TRACK: CHARLES NELSON (UMASS), DENISE ZANNINO (AMHERST),
SPENCER MASS (SUNY); JAMES GAGNON (HARVARD), KIMBERLEY JOHNSON
(SMITH), HELEN COUSIN (UMASSAMHERST) DANIEL MCINTYRE (NYU)
*NOTE: POSTERS 1-7 AUTHORS*
4:05-4:35
COFFEE BREAK
CHAIR:
KAI MESA
4:35-4:55
MARY BAYLIES (SLOAN-KETTERING) : MOTORS, ADAPTORS
AND
SIGNALING
DRIVE MYONUCLEAR POSITIONING AND MUSCLE FUNCTION
4:55-5:10
AKASH SRIVASTAVA (MBL) : DIFFERENTIAL
ROLES OF BETA-CATENIN INHIBITOR
CHIBBY1 IN PANCREAS DEVELOPMENT AND TRANSDIFFERENTIATION OF LIVER TO
PANCREAS
KEYNOTE ADDRESS: 5:15-6:15 - ANDREAS JENNY INTRODUCTION
SPYROS ARTAVANIS-TSAKONAS (HARVARD): THE NOTCH SIGNALING SYSTEM: PARADIGM
FOR COMPLEXITY
DINNER: 6:30-7:45
POSTER SESSION AND MIXER: 8:00-10:00 (AUTHORS 1-43 NUMBERED POSTERS PRESENT)
SATURDAY APRIL 12TH
BREAKFAST: 7:00-8:30
SESSION II: 9:00-12:00
CHAIR:
CRAIG MAGIE
9:00-9:20
HERMANN STELLER (ROCKEFELLER) : REGULATION
OF STEM CELL NUMBER BY
APOPTOSIS
9:20-9:35
KAREN ALIM (HARVARD) : ADAPTION OF FLUID FLOW IN THE SLIME MOLD PHYSARUM
POLYCEPHALUM
9:35-9:50
ELIZABETH DESCHENE JACOX (YALE) :
9:50-10:05
YUSUFF ABDU (NYU) : CELLULAR MORPHOGENESIS
ΒETA-CATENIN ACTIVATION REGULATES
TISSUE GROWTH VIA A NON-CELL AUTONOMOUS MECHANISM WITHIN THE HAIR STEM
CELL NICHE
OF
C. ELEGANS GERM CELLS
THROUGH ENDODERM-ASSISTED CANNIBALISM
10:05-10:30
COFFEE BREAK – SPONSORED BY RAINBOWGENE
CHAIR:
DUSTIN UPDIKE
10:30-10:50
SUSAN MANGO (HARVARD) : DYNAMIC CHROMATIN
DURING PLURIPOTENCY AND
DIFFERENTIATION
10:50-11:05
CELINA JULIANO (YALE) : PIWI-PIRNA PATHWAY FUNCTION IN THE STEM CELLS OF
IMMORTAL
11:05-11:20
HYDRA
MATTHEW DUNN (SUNY STONY BROOK) : IDENTIFYING STEM CELLS IN THE SEA
ANEMONE NEMATOSTELLA VECTENSIS
11:20-11:35
ANTOINE BORENSZTEJN (BROWN) : CONTROL
11:35-11:55
CASSANDRA EXTAVOUR (HARVARD) : FROM
OF BOTH CELL MAINTENANCE AND
CELL MIGRATION DURING DROSOPHILA OOGENESIS BY JAK/STAT AND BMP SIGNALING
EGG TO IMMORTALITY:
THE
EVOLUTION OF THE GERM LINE
LUNCH: 12:00-1:00
OUTREACH TABLES HAVE BEEN ORGANIZED FOR
WITH UNDERGRADUATE CLASS AND HIGH SCHOOL STUDENTS.
SPEAKERS TO INTERACT
SESSION III: 1:00-5:00
CHAIR:
CAROLINE BURNS
1:00-1:20
ANNA-KAT HADJANTONAKIS (SLOAN-KETTERING) : CELL
FATE CHOICE IN THE
MOUSE BLASTOCYST: THE EMERGENCE OF PLURIPOTENCY.
1:20-1:35
TONY JOUDI (BOSTON UNIVERSITY) : CHARACTERIZING ENDOCYTOSIS
IN
DYING
EGG CHAMBERS OF DROSOPHILA MELANOGASTER
1:35-1:50
LEILA JAHANGIRI (HARVARD): THE AP-1
TRANSCRIPTION FACTOR COMPONENT
FOSL2 POTENTIATES MYOCARDIAL DIFFERENTION FROM THE ZEBRAFISH SECOND
HEART FIELD
1:50-2:10
WHITNEY HABLE (UMASS DARTMOUTH) : POLYCHLORINATED
BIPHENYLS
DISRUPT EMBRYOGENESIS IN INTERTIDAL MARINE ALGAE
2:10-2:30
COFFEE BREAK – SPONSORED BY NIGHTSEE
CHAIR:
CHRISTOPHER WYLIE
2:30-2:50
MIKE BARRESI (SMITH) : DIVIDE AND CONQUER NEUROGENESIS: A KINESIN MOTOR
POWERS NEURAL STEM CELL DIVISION IN THE ZEBRAFISH NEURAL TUBE.
2:50-3:05
CHITRA DAHIA (HOSPITAL FOR SPECIAL SURGERY) : WNT AND SHH SIGNALING
ARE KEY PATHWAYS REGULATING POSTNATAL MOUSE DISC DEVELOPMENT AND AGING
3: 05-3:20
KATHERINE ROGERS (HARVARD) : A
LATE REQUIREMENT FOR
LEFTY-MEDIATED
NODAL INHIBITION
3:20-3:40
KATE LEWIS (SYRACUSE) : INTERNEURON SPECIFICATION
IN THE
ZEBRAFISH
SPINAL CORD
3:40-4:10
COFFEE BREAK
EDUCATION SESSION: 4:10-5:10 - MICHELLE SMITH
KEYNOTE ADDRESS: 5:15-6:15 - KIM TREMBLAY INTRODUCTION
SUE ACKERMAN (JACKSON LABS) : MULTICOPY RNA GENES AND THEIR ROLE IN THE CNS
DINNER: 6:30-7:45
POSTER SESSION AND MIXER: 8:00-10:00 (AUTHORS OF 44-86 NUMBERED POSTERS PRESENT)
SUNDAY APRIL 13TH
BREAKFAST: 7:00-8:30
SESSION IV: 9:00-12:00
CHAIR:
BRYAN CRAWFORD
9:00-9:20
HANNES BUELOW (EINSTEIN) : SHAPING SENSORY
DENDRITES
– SKIN-DERIVED
CUES DO THE JOB.
9:20-9:35
ETHAN DAVID COHEN (ROCHESTER) : COOPERATIVE WNT5A/WNT11
9:35-9:50
BENJAMIN MARTIN (SUNY STONY BROOK) : POST-GASTRULATION MESODERMAL
SIGNALING
INHIBITS CANONICAL WNT SIGNALING AND PROMOTES CARDIAC PROGENITOR FATE VIA
THE CASPASE-DEPENDENT DEGRADATION OF AKT
PATTERNING
9:50-10:10
MAGDALENA BENZANILLA (UMASS AMHERST) : STEERING PLANT CELL DIVISION
10:10-10:40
COFFEE BREAK
CHAIR:
ETHAN DAVID COHEN
10:40-10:55
ADAM MARTIN (MIT) : COLLECTIVE
CELL BEHAVIOR PROMOTES EPITHELIAL
INVAGINATION
10:55-11:10
RUTH JOHNSON (WESLEYAN) : THE
SIGNALING IN DEVELOPING EPITHELIA
11:10-11:30
BUSINESS MEETING
POSTER AWARDS: 11:30-11:45
BOXED LUNCH: 12:00
ADAPTOR PROTEIN
CINDR FINE-TUNES JNK
Oral
Presentations
Using Forward Genetics to Identify Developmental Genes
Weatherbee, Scott
Yale University, New Haven, CT, USA
Developmental biology and genetics are intimately linked. Classic developmental
studies relied heavily upon forward genetics to identify many of the key genes that drive
development. However, this approach has been relatively underutilized in the mouse, in
part due to the length of time that it takes to map and identify the affected locus. Recent
advances in high-throughput sequencing have greatly improved the ease of cloning
mutations and have made mouse forward genetics much more tractable. We use
forward genetics in mice as an unbiased approach to identify genes required for organ
development and have identified several novel genes that are essential for limb,
nervous system and skin development.
I will focus on a subset of these genes that are critical for the formation and function of a
small cellular organelle called the cilium. The cilium is involved in a variety of
developmental processes, including left-right specification, organogenesis, and tissue
patterning. In the past decade, work from multiple labs has identified and elucidated the
function of a handful of ciliary proteins, however many remain unknown or
uncharacterized. We have identified Mks1 and Tmem107 as essential for cilia formation
and for normal cilia protein content. We further identified Ttc26 as a key factor that
regulates cilia microtubule architecture. In terms of development, we have further
shown that Tmem107, Mks1 and Ttc26 are required for Shh patterning in the limb and
neural tube. Together, these studies expand the known repertoire of cilia genes, help
define how cilia form and provide a better understanding of human diseases that result
from defective cilia.
Oral presentation 1
Evolution of miRNA signaling in development: Insights from the hemichordate
Saccoglossus kowalevskii
Gray, Jessica (USA); Freeman Jr, Robert M (Boston, MA, USA); Gerhart, John
(Berkeley, CA, USA); Kirschner, Marc (Boston, MA, USA)
miRNAs act as post-transcriptional regulators of gene expression networks in a number
of developmental processes, and multiple evolutionary expansions of miRNAs are
associated with increasing complexity. However, despite growing evidence for miRNAs
in the development of model organisms and genome-wide small RNA studies in
numerous species, data is lacking for a functional role of miRNAs in the development of
non-model organisms. The question remains whether the evolution of miRNA targets
and functions have driven the evolution of developmental pathways or if they are
instead uniquely regulated in different lineages. We are investigating the developmental
expression and function of miRNAs in the direct-developing hemichordate
Saccoglossus kowalevskii. Hemichordates and vertebrates share a common ancestor
and many developmental signaling pathways, making it an ideal model for uncovering
how ancestral miRNAs may have contributed to evolution of development in
deuterostome lineage. Small RNA sequencing shows that Saccoglossus miRNAs are
dynamically expressed throughout development, suggesting potential roles in a number
of developmental processes. Target predictions are being combined with functional
perturbations to identify developmental roles for individual miRNAs. An initial functional
screen has revealed roles for neural miRNAs and confirmed a conserved role for miR-1
in muscle development. The targets and functions of conserved and non-conserved
Saccoglossus miRNAs will be compared with their homologs and functional
counterparts in vertebrates. Our data provide a first exploration of miRNA function in
hemichordate development and will contribute to understanding how the role of miRNA
regulation in development has changed through evolution.
Oral presentation 2
Growth cone-specific functions of MT polymerase XMAP215 in restricting MT
dynamics and promoting axon outgrowth
Lowery, Laura A.; Stout, Alina; Faris, Anna; Danuser, Gaudenz; Van Vactor, David
Boston College, Boston, MA, USA
Proper neural connections, essential to nervous system function, depend upon precise
navigation by the growth cone during development. A fundamental problem in growth
cone biology is how guidance pathways are integrated to coordinate cytoskeletal
dynamics and drive accurate axonal navigation. To address this, we focus on the plusends of microtubules (MTs), which explore the growth cone and play a key role in
steering. Plus-end dynamics are regulated by a conserved family of proteins called
‘plus-end-tracking proteins’ (+TIPs). Yet, it is unclear how +TIPs interact with each other
and with MTs to control MT behavior, especially in the developing nervous system.
Here, we address this question by investigating the function of the traditional MT
polymerase, XMAP215, during neurite outgrowth. When XMAP215 function is reduced,
neurite outgrowth is severely compromised both in vivo and ex vivo. Rather than being
due to reduced axon outgrowth velocity, which might be expected upon knocking down
a MT polymerase, this phenotype instead occurs due to an increased rate of
spontaneous axon retractions. By using automated, quantitative imaging analysis
following acquisition of high-resolution live-imaging data of tagged +TIPs within cultured
Xenopus laevis growth cones, we were surprised to discover that partial knock-down of
XMAP215 leads to a 20% increase in MT plus-end velocity, specifically within growth
cones. This is unexpected given that XMAP215 is known to have MT polymerase
activity. Interestingly, XMAP215 function depends on cell type, as knocking it down in
epidermal epithelial cells leads to reduced MT polymerization, consistent with its known
role in other systems. There are at least two possible mechanisms by which the
increase in MT plus-end velocity may occur in growth cones, either by affecting the
polymerization/ depolymerization rate directly, or by changing MT translocation rates
through the regulation of MT interaction with other cellular structures. We have tested
between these two models using quantitative fluorescence speckle microscopy, and we
have determined that XMAP215 perturbation leads to changes in MT translocation rates
in embryonic growth cones. Collectively, our findings reveal that XMAP215 functions as
more than a simple MT polymerase in motile growth cones and sets the stage for future
investigation of XMAP215 function during MT and F-actin organization during neural
development.
Oral presentation 3
Endogenous gradients of resting potential instructively pattern neural tissue�
via Notch signaling and regulation of proliferation
Pai, Vaibhav (Tufts University, Medford, USA); Lemire, Joan M. (Tufts University,
Medford, MA, USA); Pare, Jean-Francois (Medford, MA, USA); Lin, Gufa (University of
Minnesota, Minnesota, USA); Chen, Ying (University of Minnesota, Minnesota, USA);
Levin, Michael (Medford, USA)
Biophysical forces play important roles in pattern formation of the embryonic brain and
other organs. Previously we showed that endogenous bioelectric signals are crucial eye
patterning signals, being necessary and sufficient for eye induction. Here, we identify a
new role for endogenous bioelectricity as an instructive factor during brain patterning in
Xenopus laevis. Early frog embryos exhibit a characteristic polarization of a key group
of cells in the neural tube; disruption of this bioelectric gradient induces specific brain
defects with changes in the expression of early brain markers, causing anatomical
mispatterning of the brain. This effect is mediated by voltage-gated calcium signaling
and gap-junctional communication. Neural cell proliferation within the developing brain
is regulated by both, the local Vmem states within the neural tube, and the Vmem states of
distant regions. Misexpression of the constitutively-active form of Notch, a suppressor of
neural induction, impairs the normal voltage polarization pattern and neural patterning;
moreover, reinforcing proper polarization rescues brain defects induced by activated
Notch signaling. Interestingly, ectopic polarization implemented by misexpression of one
ion channel’s mRNA induces ectopic neural tissue well outside the neural field. We also
found that polarization states synergize with the actions of reprogramming factors that
are known to promote an undifferentiated cell state to direct ectopic tissues toward
neural fate in vivo. These data identify a new functional role for bioelectric signaling in
brain patterning, characterize the interaction between Vmem and key biochemical
pathways (Notch and Ca2+ signaling) during organogenesis of the vertebrate brain, and
suggest voltage modulation as a tractable strategy for intervention in certain classes of
neural birth defects.
Oral presentation 4
Transcriptional control of cell fate specification in the ascidian cardiopharyngeal
mesoderm
Florian Razy-Krajka, Wei Wang, Nicole Kaplan, Karen Lam and Lionel Christiaen
New York University, New York, NY, USA
In vertebrates, the pharyngeal subset of head muscles shares a common clonal origin
with cardiomyocytes in early embryos. Retrospective clonal analyses demonstrated that
the derivatives of the first heart field initially diverge from common progenitors of the
pharyngeal muscles and second heart field derivatives. However, the complexity of
early vertebrate embryos hinders high-resolution analyses of initial fate choices within
the cardiopharygeal mesoderm. The ascidian Ciona intestinalis offers a unique
opportunity to study cardiopharyngeal fate specification with cellular resolution in a
simple chordate model. We found that, on each side of the tail bud embryo, two bipotent
cardiopharyngeal progenitors undergo stereotyped asymmetric cell divisions to
sequentially produce first and second heart precursors and pharyngeal muscle
precursors following a conserved clonal pattern. We used targeted molecular
manipulations, Fluorescence Activated Cell Sorting (FACS), whole genome transcription
profiling and fluorescent in situ hybridization to study the transcriptional dynamics
underlying heart vs. pharyngeal muscle fate choices in ascidians. We found that
multipotent cardiopharyngeal progenitors are transcriptionally primed by expressing a
mixture of early cardiac and pharyngeal muscle regulators. This multilineage priming is
resolved following asymmetric cell divisions through regulatory cross-antagonisms,
whereby Tbx1/10 inhibits GATA4/5/6 and heart specification in the pharyngeal muscle
precursors, thus promoting COE/EBF expression and muscle fate specification; while, in
the heart precursors, NK4/NKX2-5 inhibits Tbx1 and COE/EBF thus permitting
GATA4/5/6 expression. In the pharyngeal muscles, COE/EBF then promotes activation
of a MyoD/MRF-associated muscle differentiation program and the maintenance of
muscle precursors' stemness through Notch-mediated lateral inhibition of differentiation.
Finally, we identified the early signaling events that differentially regulate the initial heart
vs. muscle fate choice and polarize the regulatory cross-antagonism between the heart
and pharyngeal muscle precursors. In summary, we present a detailed characterization
of cellular and molecular underpinnings of conserved heart vs. pharyngeal muscle fate
choices with a spatio-temporal resolution unprecedented in chordate model systems.
Oral presentation 5
Characterization of mRNA clearance during the maternal-to-zygotic transition
Yartseva, Valeria; Takacs, Carter; Lee, Miler; Vejnar, Charles; Giraldez, Antonio
Yale University, New Haven, CT, USA
Early embryonic development is directed by mRNAs present in the oocyte at the time of
fertilization. During the maternal-to-zygotic transition (MZT), a large number of these
maternal mRNAs must be removed to ensure the transfer of control to zygoticallyencoded developmental programs. Previous work in our laboratory has led to the
identification of a zygotic factor, the microRNA miR-430, as a major regulator of
maternal mRNA decay (Giraldez et al., 2006). However, despite the influence of miR430,
many
maternal
mRNAs
are
cleared
through
as-yet
unknown
mechanisms. Through transcriptional profiling under a variety of chemical and genetic
perturbations, we have identified classes of maternal mRNAs under distinct modes of
zygotic regulation. Taking a transcriptome-wide reporter approach, we have identified
cis-regulatory elements that direct the clearance of these maternal transcripts. Finally,
genetic and biochemical approaches are being undertaken to identify the causative
factors mediating mRNA clearance. By studying how early embryos clear previous
transcriptional states, we hope to gain insights into the cellular reprogramming that
drives early embryogenesis.
Oral presentation 6
Motors, Adaptors and Signaling drive Myonuclear Positioning and Muscle
Function
Victoria K. Schulman1,2, Eric S. Folker2, Mary K. Baylies1,2
1. Weill Cornell Graduate School of Medical Sciences, New York, NY 10065
2. Sloan-Kettering Institute, Developmental Biology Program, New York, NY 10065
Muscles are composed of multinucleated cells in which nuclei are spaced to maximize
internuclear distance. Clustered and mislocalized myonuclei are highly correlated with
muscle disorders; yet, it is unknown whether mispositioned myonuclei are a cause or
consequence of disease. We have found that myonuclear positioning is a microtubuledependent process, requiring Kinesin and Dynein. In developing Drosophila muscles,
Dynein and Kinesin work together to move nuclei in a single direction by two separate
mechanisms that are spatially segregated. First, the two motors work together in a
sequential pathway that acts from the cell cortex at the muscle poles. This mechanism
requires Kinesin-dependent localization of Dynein to cell cortex near the muscle pole.
From this location Dynein can pulls microtubule minus-ends and the attached myonuclei
toward the muscle pole. Second, the motors exert forces directly on individual nuclei
independently of the cortical pathway. These motor activities are polarized relative to
the direction of myonuclear translocation: Kinesin acts at the leading edge of the
nucleus, whereas Dynein acts at the lagging edge of the nucleus. To identify how these
pools of motors are controlled spatially and temporally, we screened known motor
regulators. We found that Sunday Driver (Syd) is expressed in muscle tissue, and
disruptions in syd lead to mispositioned myonuclei. Syd interacts with both motors, and
when Kinesin or Syd is mutated, Dynein fails to localize properly at the muscle poles.
These data indicate that Syd positions myonuclei by regulating Kinesin-dependent
localization of Dynein. These defects are rescued by expressing JIP3, the mammalian
ortholog of Drosophila Syd. JIP3 is a JNK-Interacting Protein, and indeed, Syd interacts
with Bsk, the Drosophila homolog of mammalian JNK1/2. Further, impaired JNK
signaling mimics syd mutants. Loss of JNK signaling restricts Syd and Dynein to the
perinuclear space, whereas overactive JNK signaling permits proper transport of these
factors to the muscle poles but prevents their downstream functions. Thus, precise
coordination of de/phosphorylation via the JNK signaling pathway is necessary to
position myonuclei. Finally, muscle-specific depletion of Syd reduces muscle output, but
locomotion is rescued by JIP3 expression. Thus, we implicate Syd as a novel regulator
of myogenesis and demonstrate that Syd-mediated JNK signaling is required for proper
intracellular organization and ultimately tissue function.
Oral presentation 7
Differential roles of beta-catenin inhibitor Chibby1 in pancreas development and
transdifferentiation of liver to pancreas
Srivastava, Akash; Horb, Marko
Marine Biological Laboratory, Woods Hole, MA, USA
Transdifferentiation of liver to pancreas is a promising strategy for generating ectopic
pancreatic tissue from other tissues present in the body. Using transgenic Xenopus
tadpoles we previously showed that transcription factor Pdx1-VP16 converts liver cells
into all pancreatic cell types (both endocrine and exocrine), while Ptf1a-VP16 converts
liver cells to only exocrine cells. However which genes are activated by Pdx1-VP16 or
Ptf1a-VP16 in this process was unknown. We identified the gene expression changes
that occurred immediately after these genes were expressed in the liver. This was
achieved by driving the expression of Pdx1-VP16 and Ptf1a-VP16 in Xenopus tadpoles
using the liver-specific TTR (transthyretin) promoter, which is activated in transgenic
tadpoles at NF 44. We performed microarray to compare the genes expression profile of
NF 44/45 transgenic livers with that of control liver samples. Chibby1, a Wnt-beta
catenin inhibitor gene, was found to be upregulated in Ptf1a-VP16 expressing
transgenic livers. Functional analysis of Chibby1 shows that it has two differential roles
in normal pancreas development and transdifferentiation of liver to pancreas.
Oral presentation 8
The Notch Signaling System: Paradigm for Complexity
Artavanis-Tsakonas, Spyros
Harvard Medical School, Cambridge, MA, USA
Notch signaling is highly pleiotropic, and can play a critical role in differentiation,
apoptotic, and proliferative events depending on the developmental context. I will
attempt to review and illustrate the biological complexity and how we try to deconvolute
this complexity in genetic and molecular terms.
Oral presentation 9
Regulation of Stem Cell Number by Apoptosis
Steller, Hermann
Howard Hughes Medical Institute, Rockefeller University, NY, USA
Adult stem cells (SCs) are essential for tissue homeostasis and wound-repair. However,
their proliferative capacity must be tightly regulated to prevent the emergence of
unwanted and potentially dangerous cells, such as cancer cells. We found that mice
lacking the pro-apoptotic ARTS protein have elevated numbers of hematopoietic and
stem cells hair follicle SCs (HFSCs). The increased number of apoptosis-impaired stem
cells has profound consequences for both the risk of tumor development and wound
repair.
On the one hand, impairment of SC apoptosis predisposes mice to
lymphomagenesis, suggesting that cell death plays an important role as a frontline
defense against cancer by restricting the number of normal stem cells. On the other
hand, inhibition of apoptosis in HFSCs causes dramatic improvements in wound healing
and regeneration of hair follicles. Furthermore, inactivation of XIAP, a direct target of
ARTS, abrogates these phenotypes. Finally, apoptotic cells are also the source of
critical local signals that can stimulate both proliferative and death signals. This work
reveals both autonomous and non-autonomous roles of apoptosis in the regulation of
stem cell number.
Oral presentation 10
Adaption of fluid flow in the slime mold Physarum polycephalum
Alim, Karen; Amselem, Gabriel; Peaudecerf, François; Brenner, Michael P.; Pringle,
Anne
Harvard University, Cambridge, MA, USA
The network-forming slime mold Physarum polycephalum lacks any central coordination
center, yet it shows often-termed intelligent dynamics in the way it grows and adapts its
network morphology. Our work investigates the role of fluid mechanics for transport and
signal transfer during the morphological dynamics of this network-like slime mold. We
combine experimental observations of the fluid flow and its driving force with the
development of the theoretical concept of transport by peristaltic flow in a network. This
synergy allows us to show that the slime mold actively controls its internal fluid flow by
establishing a peristaltic wave. This peristaltic wave always spans the total extent of an
individual independent of its size. Thus, we find that the slime mold actively adapts its
flows as to maximize transport. The quantitative description of flows in P. polycephalum
enables a new view on the slime molds growth dynamics during the encounter of food
or toxins and how their location can be “remembered”, an important step to perform an
informed decision during an individuals network growth and adaptation.
Oral presentation 11
β-catenin activation regulates tissue growth via a non-cell autonomous
mechanism within the hair stem cell niche
Deschene Jacox, Elizabeth1; Myung, Peggy2; Rompolas, Panteleimon1; Zito, Giovanni1;
Sun, Thomas Yang1; Taketo, Makoto3; Satomoe, Ichiko1; Greco, Valentina1
1
Yale University, Department of Genetics, New Haven, CT, USA
Yale University, Department of Dermatology, New Haven, CT, USA
3
Graduate School of Medicine, Kyoto University, Kyoto, JPN
2
Wnt/β-catenin signaling is critical for tissue regeneration and cancer. However, it is
unclear how β-catenin controls stem cell behaviors to coordinate growth. Using time
lapse imaging of live mice, we show that genetic activation of β-catenin specifically
within hair follicle SCs generates new organized axes of hair growth through oriented
cell divisions and coordinated cellular displacement. Additionally, β-catenin activation is
sufficient to induce growth within the stem cell pool independently of mesenchymal
niche signals previously shown to be required for hair follicle regeneration. Strikingly, we
demonstrate that wild-type cells contribute to induced hair-like growths and that βcatenin mutant cells act non-cell autonomously to activate Wnt signaling within wild-type
cells via Wnt ligand up-regulation. Collectively, this study demonstrates a novel
mechanism by which Wnt/β-catenin signaling controls stem cell-dependent tissue
growth non–cell autonomously and carries broader implications toward understanding
mechanisms of tumor growth and regeneration.
Oral presentation 12
Cellular Morphogenesis of C. elegans Germ Cells Through Endoderm-Assisted
Cannibalism
Abdu, Yusuff
NYU School of Medicine, New York, NY, USA
Cells remodel to carry out specialized functions and to dispose of unneeded
components. We have discovered a novel mechanism of morphogenesis where large
portions of a cell are removed by neighboring cells. During C. elegans embryogenesis,
the primordial germ cells (PGCs) extend large protrusions (lobes) that are shortly after
embedded in adjacent endodermal cells; the PGC lobes disappear before the embryo
hatches. Using live fluorescent imaging, we observed that lobes are cannibalized and
subsequently digested by the endoderm, and that in mutants lacking endoderm, PGC
lobes persist. Lobe cannibalism required the cell corpse engulfment gene ced-10/rac,
which we showed functioned in the endoderm. Null mutations in other cell corpse
engulfment genes, such as like ced-1/CD91 and ced-5/DOCK180, did not affect lobe
cannibalism, suggesting that ced-10/rac acts in a novel pathway to promote lobe
cannibalism. In order to find other regulators of lobe cannibalism, we carried out a largescale chemical mutagenesis screen and isolated 13 mutants that have defects in the
removal or digestion of PGC lobes, but unlike ced-10/rac, do not cause defects in cell
corpse engulfment. We are using whole genome sequencing methods to clone the
mutants and characterize their role in lobe cannibalism.
Oral presentation 13
Dynamic Chromatin during pluripotency and differentiation
Mango, Susan
Harvard University, Cambridge, MA, USA
The transition from naive precursor towards the differentiated state is characterized by
sequential waves of gene expression that are governed by regulatory transcription
factors. The goal of our research is to understand how transcriptional circuitry dictates
the progression from pluripotency, to cell-fate specification and ultimately differentiation.
We have defined a window in early embryogenesis when cells are developmentally
plastic, while after this stage cells are restricted in their cell-fate potential. We have used
cell biological and genomic approaches to determine the configuration of chromatin
surrounding genes during development. This analysis revealed large-scale alterations in
chromatin morphology in pluripotent vs. differentiating cells. I will discuss our latest
research on chromatin morphology and transcription factor function.
Oral presentation 14
PIWI-piRNA pathway function in the stem cells of immortal Hydra
Juliano, Celina (USA); Reich, Adrian (Department of Molecular Biology, Cell Biology,
and Biochemistry, Brown University, Providence, RI, USA); Liu, Na (Yale Stem Cell
Center and Department of Cell Biology, Yale University School of Medicine, New
Haven, CT, USA); Götzfried, Jessica (Yale Stem Cell Center and Department of Cell
Biology, Yale University School of Medicine, New Haven, CT, USA); Zhong, Mei (Yale
Stem Cell Center and Department of Cell Biology, Yale University School of Medicine,
New Haven, CT, USA); Uman, Selen (Yale Stem Cell Center and Department of Cell
Biology, Yale University School of Medicine, New Haven, CT, USA); Reenan, Robert
(Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University,
Providence, RI, USA); Wessel, Gary (Department of Molecular Biology, Cell Biology,
and Biochemistry, Brown University, Providence, RI, USA); Robert, Steele (Department
of Biological Chemistry and the Developmental Biology Center, University of California,
Irvine, Irvine, CA, USA); Lin, Haifan (Yale Stem Cell Center and Department of Cell
Biology, Yale University School of Medicine, New Haven, CT, USA)
Although individual animals cannot escape mortality, their genetic material is passed to
offspring through the germ line. PIWI proteins and their bound small RNAs (piRNAs)
maintain genomic stability in the germline, most notably by repressing transposon
expression. Interestingly, Hydra vulgaris appears to challenge this traditional concept of
mortality. The stem and progenitor cells of an adult Hydra are in a constant proliferative
state and excess cells are shed in part through asexual budding to create genetically
identical clones. No signs of aging or decreased stem cell function have been observed.
Hydra consists of three distinct lineages and conversions between these lineages do
not occur. The ectodermal and endodermal epithelial lineages are strictly somatic and
are supported by a unipotent stem cell. The interstitial lineage is supported by
multipotent stem cells that give rise to several somatic cell types and the germ line. We
find that Hydra has two PIWI proteins, Hywi and Hyli, that are expressed in the
cytoplasm of all three stem cell types. Furthermore, hywi is an essential gene in the two
strictly somatic lineages. Hydra piRNAs have the same characteristics as bilatarian
piRNAs, with primary and secondary piRNAs bound specifically to Hywi and Hyli
respectively. To identify putative targets of the pathway, we mapped the piRNAs to a
Hydra transcriptome. A large percentage of the piRNAs map to transposons and these
data suggest that post-transcriptional repression of transposons by the PIWI-piRNA
pathway is deeply conserved in metazoans. In addition, we isolated piRNAs specific to
each lineage to identify lineage-specific targets of the pathway. We find that in addition
to transposon regulation, the pathway likely regulates a unique set of non-transposon
genes in each lineage. Cnidarian gene sets, such as that of Hydra, exhibit the same
complexity found in vertebrate genomes, thus the identification of piRNA pathway
targets in our work will likely provide important insights into the function of this pathway
in more complex animals.
Oral presentation 15
Identifying Stem Cells In The Sea Anemone Nematostella vectensis
Dunn, Matthew; Thomsen, Gerald
Stony Brook University, Stony Brook, NY, USA
A better understanding of the properties and behavior of stem cells across Metazoa will
shed light on key shared stem cell characteristics and therefore improve our abilities to
utilize stem cells in regenerative medicine. We are using the sea anemone
Nematostella vectensis to understand regenerative mechanisms in Cnidaria, an ancient
group of animals with extraordinary regenerative abilities. Nematostella can regenerate
any amputated body structure, but whether stem cells and pluripotency genes regulate
regenerative processes in this animal is unknown. By identifying the Nematostella
orthologs of many known mammalian stem cell marker genes and examining their
expression during regeneration, we hope to identify potential stem cells, determine how
they contribute to regeneration, and assess the regenerative functions of pluripotency
genes. While some pluripotency markers, such as c-Myc and Lin28a, have direct
orthology across the animal phylogeny, other genes (Nanos, Oct4, Piwi-like, etc.) have
duplicated in various lineages, making orthology assessment difficult. We present our
findings in identifying sea anemone pluripotency marker genes, and their expression
during regeneration.
Oral presentation 16
Control of both cell maintenance and cell migration during Drosophila oogenesis
by JAK/STAT and BMP signaling
Borensztejn, Antoine; Akiyama, Takuya; Wharton, Kristi
Brown University, Providence, RI, USA
The formation of viable germ cells is critical for the perpetuation of a species. As in most
organisms, in Drosophila, specific interactions between germline and somatic cells are
critical for the development of gametes. During oogenesis, specific sets of somatic
follicle cells in conjunction with germline cells comprise an egg chamber, from which a
single mature egg is produced. Each set of follicle cell serves a particular function in the
production of a viable egg. The stalk cells ensure that each egg chamber is separated in
a long chain of maturing egg chambers, while the polar and border cells produce
specialized structures in the egg shell that are essential for fertilization and egg viability.
In both cases, the formation of a specific number of cells, as well as their maintenance
during oogenesis, is known to be critical for egg production. For example, it has been
shown the precise number of two polar cells, crucial for polar and border cell migration,
is achieved through JAK/STAT-regulated apoptosis (Borensztejn et al. 2013). We find
that the precise number of stalk cell is also achieved through apoptosis but in this case
apoptosis is not promoted by JAK/STAT signaling but rather inhibited by it. Interestingly,
we find that BMP signaling is also required for the maintenance of stalk cells through an
anti-apoptotic mechanism to ensure that egg chambers remain separate and do not
merge. We considered the possibility that the different response of polar cells versus
stalk cells to JAK/STAT signaling may depend on active BMP signaling. We will discuss
our findings on JAK/STAT and BMP signaling cross talk in regulating stalk cell number
and in polar cell/border cell migration.
Oral presentation 17
From egg to immortality: The evolution of the Germ Line
Extavour, Cassandra G.
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge MA
02138
Germ cells in many animals, including Drosophila, are specified by maternal deposition
of localized “germ plasm” that contains molecular determinants of germ cell fate.
However, basally branching animals, including basal arthropods, are classically
described as lacking germ plasm. Using molecular markers and functional genetics, we
tested the hypothesis that a zygotic, inductive signaling mechanism may have been
used ancestrally for germ line specification within insects and arthropods. In two
hemimetabolous insects and a spider, we provide evidence that, in contrast to the
Drosophila paradigm, gene products of multiple “germ plasm genes” are not
asymmetrically localized during oogenesis or early cleavage stages, and maternal
contributions of vasa and piwi are not required for germ cell formation. We show that the
oskar gene, which is both necessary and sufficient for specifying functional germ cells in
Drosophila, evolved before the divergence of higher insects. We propose that oskar
may have played an ancestral role in nervous system development, and that its role in
germ cell specification is a recent evolutionary innovation likely due to co-option of
oskar from a somatic function into a germ line specification pathway. Finally, we provide
evidence that in a basally branching insect that lacks germ plasm, germ cells appear to
arise from a subpopulation of abdominal mesoderm, and that this process requires BMP
signaling. Given the commonality of BMP signaling in mouse and cricket germ cell
induction, we suggest that BMP-based germ cell formation may be a shared ancestral
mechanism in animals.
Oral presentation 18
Cell fate choice in the mouse blastocyst: the emergence of pluripotency.
Hadjantonakis, Anna-Kat
Developmental Biology Program, Sloan-Kettering Institute, New York, USA
The blastocyst is a universal stage of mammalian embryonic development. It is the
embryonic stage at which a bona fide pluripotent cell population – the epiblast (EPI) – is
established. Prior to implantation into the maternal uterus the blastocyst comprises
three cell lineages: pluripotent EPI, primitive endoderm (PrE) and trophectoderm.
During blastocyst development, cells of the inner cell mass (ICM) undergo a binary cell
fate decision, either acquiring a pluripotent identity and committing to the EPI lineage, or
becoming PrE. By combining live imaging and quantitative single-cell analyses with
mouse genetics, and utilizing embryo-derived stem cells, we are investigating
mechanisms regulating ICM cell fate choice. Progress with these studies will be
presented.
Oral presentation 19
Characterizing Endocytosis in Dying Egg Chambers of Drosophila melanogaster
Joudi, Tony (Boston, USA); Meehan, Tracy (Boston, USA); Timmons, Allison (Boston,
USA); McCall, Kim (Boston, USA)
Phagocytosis is the crucial process that allows for the efficient removal of dying or dead
cellular material.
Dysregulation of phagocytosis has been known to lead to
autoimmune diseases such as lupus. Phagocytosis is one type of endocytosis, which is
characterized by the turnover of small GTPases, Rab proteins, which associate with
endocytic vesicles. It has been established in the model C. elegans that endocytic
vesicle maturation occurs through the initial association of Rab5, followed by
replacement by Rab7, and finally the acidification of the phagosome into an
autophagosome associated with LAMP proteins. The Drosophila melanogaster ovary
serves as a physiologically relevant model of engulfment by non-professional
phagocytes induced by naturally occurring stresses, such as starvation. Upon
starvation, germline-derived nurse cells undergo apoptosis and the surrounding layer of
somatically-derived follicle cells synchronously enlarge and engulf the dying nurse cells.
D. melanogaster utilizes both professional and non-professional phagocytes,
highlighting the relevance of our system compared to that seen in
mammals. Surprisingly, we found that an antibody raised against active caspase Dcp-1
marked vesicles present in the follicle cells during the very initial phases of
engulfment. To initiate our studies of endocytosis in engulfment, we investigated colocalization of Dcp-1 with Rab5 and Rab7. I hypothesize that that Rab5 associates with
early vesicles, but does not associate with the acidophilic marker LysoTracker. Rab7
was demonstrated to occasionally co-localize with Lysotracker, supporting the
hypothesis that Rab5 may serve as an early endocytic marker, and Rab7 marks late
endosomes, preceding lysosomal fusion. We have established the D. melanogaster
ovary as a model for studying endocytosis during engulfment. Future work will utilize
the genetic benefits of D. melanogaster to perform a dsRNA screen to identify which
components of endocytosis are required for engulfment.
Oral presentation 20
The AP-1 transcription factor component FOSL2 potentiates myocardial
differention from the zebrafish second heart field
Jahangiri, Leila (USA); Guner-Ataman, Burcu (Harvard Medical School, Charlestown,
MA, USA); Adams, Meghan (Harvard Medical School, Charlestown, MA, USA); Burns,
Caroline (MGH/HMS, Charlsetown, MA, USA); Burns, Geoffrey (Harvard Medical
School, Charlestown, MA, USA)
During embryogenesis, cardiac progenitor cells constituting the anterior second heart
field (SHF) contribute myocardial cells to the primitive ventricle during a finite
developmental window. However, the cellular and molecular mechanisms that regulate
the rate and duration of myocardial cell accretion remain unknown. Here, we
demonstrate that a component of the AP-1 transcription factor complex, Fos-related
antigen 2 (Fosl2), potentiates myocardial differentiation from the recently-discovered
zebrafish SHF. Fosl2 transcripts overlap the site of myocardial cell accretion at the
arterial pole of the linear heart tube. Using TALEN-mediated genome editing, we
isolated two null alleles of fosl2 that abrogate protein expression and cause
indistinguishable cardiac phenotypes. Mutant animals appear grossly normal until they
develop moderate pericardial edema. Quantification of cardiomyocyte numbers at three
developmental stages uncovered a ventricular cardiomyocyte deficit that emerges
during SHF-mediated ventricular growth. Using a photoconversion assay, we learned
that mutant embryos initiate myocyte accretion appropriately but thereafter, exhibit
sluggish cardiomyocyte differentiation that results in the observed ventricular deficit.
Null embryos eventually compensate by prolonging SHF-mediated myocardial accretion
over a longer developmental window. As a consequence, the subsequent formation of
SHF-derived smooth muscle also lags. The ventricular cardiomyocyte deficiency is
accompanied by accumulation of undifferentiated SHF progenitors demonstrating that
Fosl2 is required for promoting the progenitor-to-myocyte transition. Conversely,
overexpression of Fosl2 results in decreased SHF progenitors and their myocardial
progeny, a phenotype consistent with precocious myocardial differentiation at the
expense of self renewal. Lastly, Fosl2 directly represses expression of the SHF marker
ltbp3 suggesting that it facilitates myocardial differentiation, at least in part, by
downregulating determinants of the progenitor state. Taken together, our data uncover
an essential role for Fosl2 in augmenting the rate of myocardial differentiation
specifically from SHF progenitors. Based on these observations, human Fosl2
mutations might cause or modify congenital heart defects resulting from pertubations in
SHF biology.
Oral presentation 21
Polychlorinated biphenyls disrupt embryogenesis in intertidal marine algae
Hable, Whitney
University of Massachusetts Dartmouth, Dartmouth, MA, USA
Chronic pollutant exposure has the potential to critically disrupt development and
population success for sessile organisms in marine communities. Fucoid algae are
marine rockweeds that populate the intertidal zone, providing nursery and feeding
habitats to commercially important fishes. They are well adapted to harsh environmental
extremes, like salinity and temperature fluctuations, and as residents of coastline
habitats, they are further challenged by surface water runoff and waste from commercial
industries and residential communities. Despite a 1977 moratorium, polychlorinated
biphenyls (PCBs) remain prevalent in intertidal sediments; they are extremely stable,
and have a low solubility in seawater. While studies have documented negative health
effects on fish, shellfish, birds and mammals, little is known about how PCBs affect the
development of immobile species in the intertidal environment. We compared the
effects of relevant concentrations of a PCB mixture that is common at contamination
sites, in two species of fucoid algae, Silvetia compressa and Fucus vesiculosus.
Although initial developmental events were unaffected, cell division was delayed and
growth rate was reduced, with more severe effects in F. vesiculosus. Microtubule arrays
were abnormal in F. vesiculosus and although normal in S. compressa, they revealed a
delay in the progression of the spindle through mitosis, indicating PCBs may seriously
disrupt algal recruitment. Studies are in progress to examine development and success
of algal populations residing in the New Bedford Harbor, which is heavily contaminated
with PCBs. Preliminary data indicate that population densities and biomass are greatly
diminished compared to more pristine locations.
Oral presentation 22
Divide and conquer neurogenesis: A kinesin motor powers neural stem cell
division in the zebrafish neural tube.
Barresi, Michael
Smith College, Northampton, MA, USA
As the embryonic neural stem cell, radial glial division must be tightly regulated to
generate the correct number of neuronal and glial progeny. Kif11(Eg5) is a kinesin
motor protein suggested to stabilize and separate the bipolar mitotic spindle. We show
that radial glial cells express kif11 in the ventricular zone, and loss of Kif11 causes
radial glial mitotic arrest as a result of monoastral spindle formation. M-phase radial glia
accumulate over time at the ventricular zone in kif11 mutants. Mathematical modeling of
the radial glial accumulation in kif11 mutants not only confirmed an ~226x delay in
mitotic exit, but predicted two modes of increased cell death. We experimentally
confirmed these predictions by documenting an increase in the apoptosis marker, antiactivated Caspase-3, which was inversely proportional to a decrease in cell
proliferation. In addition, treatment with the Kif11 blocker, S-trityl-L-cysteine (STLC), at
different stages of neural development uncovered two critical periods that required Kif11
function for stem cell progression through mitosis. We explored how neurogenesis may
be affected by radial glial mitotic arrest due to kif11 loss, and found specific reductions
in oligodendroglia and secondary interneurons and motorneurons. Despite the many
cell number changes in kif11 mutants, we document unchanged cell densities within the
neural tube over time, suggesting that a mechanism of compensatory regulation exists
to maintain overall proportions. We propose Kif11 functions during mitotic spindle
formation to facilitate the progression of radial glia through mitosis, which leads to the
maturation of progeny into specific secondary neuronal and glial lineages in the
developing neural tube.
Oral presentation 23
Wnt and Shh signaling are key pathways regulating postnatal mouse disc
development and aging
Sarah Loh1, Eric Mahoney2, Christopher Wylie2, and Chitra Dahia1
1
Tissue Engineering, Regeneration and Repair Program, Hospital for Special Surgery,
New York, NY; 2Division of Developmental Biology, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH
Intervertebral disc (IVD) degeneration is a major musculoskeletal disorder affecting
almost 80% of people at some point of their life. IVDs are strong fibrocartilagenous
joints that connect adjacent vertebrae of the spine. Each disc has a central gelatinous
core, the nucleus pulposus (NP), surrounded by a multilayered annulus fibrosus (AF)
between the vertebral bodies, and a cartilagenous endplate (EP) over their surfaces.
With aging, IVDs degenerate, often resulting in severe neurological consequences.
Current treatments for disc degeneration focus on surgical repair, which is mostly
palliative. An ideal treatment would be to reverse the degenerative process using the
mechanisms of their normal formation and differentiation. However, these processes
are not well understood. We have used the mouse lumbar disc as a model to study the
cell signaling pathways that control postnatal disc development, differentiation, and
aging. Previously, we showed, using both in vivo gene targeting and small molecule
agonists and antagonists in vitro, that Shh expressed by NP cells (which are derived
from the embryonic notochord) is a key regulator of postnatal disc growth,
differentiation, and maintenance of extracellular matrix synthesis. Here we show that
Shh signaling is in turn regulated by canonical Wnt signaling, and that Shh feeds back
on the level of Wnt signaling in the IVD. Both Shh and Wnt signaling levels are downregulated at the end of the growth period. This coincides with reduction of expression of
differentiation markers, and extracellular matrix components in the aging discs.
However, we show that this down-regulation can be reversed. Re-activation of the Wnt
or Shh pathways in older discs in vitro re-activates the synthesis of NP markers, and
extracellular matrix markers that are reduced with age. These data suggest that
biological treatments of degenerative disc disease may be feasible, using the same
signals that are used during normal disc growth and differentiation.
Oral presentation 24
A late requirement for Lefty-mediated Nodal inhibition
Rogers, Katherine; Dubrulle, Julien; Gagnon, James; Schier, Alexander
Harvard University, Cambridge, MA, USA
Secreted activator/inhibitor pairs drive much of the pattern formation that occurs during
development. The Nodal/Lefty activator/inhibitor pair patterns mesendoderm during
vertebrate embryogenesis. lefty is a target of Nodal signaling, and expression of both
nodal and lefty normally commences during the late blastula stage. Previous studies
with morpholinos suggested that Lefty loss results in dramatic upregulation of Nodal
target genes as early as the late blastula stage. However, we show that most Nodal
targets are not dramatically upregulated until early gastrulation in embryos mutant for
both zebrafish leftys. The exception was lefty itself: both leftys were strongly
upregulated in lefty double mutants at the late blastula stage, concurrent with the onset
of Nodal expression. Modeling and NanoString data suggest that leftys respond to
Nodal signaling with unusually rapid transcription kinetics, exhibiting transcription rates
>50-fold higher than ~90% of Nodal target genes. Our results suggest that Leftymediated Nodal inhibition is required later in development than previously thought,
possibly due to the relatively slow response kinetics of Nodal target genes.
Oral presentation 25
Interneuron Specification in the Zebrafish Spinal Cord
Lewis, Kate
Syracuse University, Syracuse, NY, USA
Interneurons constitute most of the neurons in the CNS and they function in almost all
neuronal circuits. However, we still know very little about how interneurons with specific
functional characteristics develop and form appropriate neuronal circuitry. All of the
evidence so far suggests that the properties of distinct interneurons are determined by
the transcription factors that the cells express as they stop dividing, become post-mitotic
and start to differentiate. However, in many cases, it is still unclear which transcription
factors specify particular properties, at least partly because we do not yet know the full
complement of transcription factors expressed by any population of vertebrate spinal
interneurons.
My lab is interested in 3 distinct yet related questions:
1. How do different classes of spinal interneurons form in their correct numbers and
positions?
2. What is the complete complement of transcription factors expressed by specific
spinal interneurons and how do these transcription factors regulate the distinct
functional characteristics of these cells?
3. How much evolutionary plasticity is there in the genetic networks that specify spinal
interneurons in different vertebrates and how have any differences evolved?
To address these critical gaps in knowledge we are using and developing a variety of
techniques. For example, we are constructing transgenic zebrafish lines that label
specific neurons. This enables us to FAC-sort and expression-profile these cells. We
are also identifying transcription factors that are strong candidates for specifying
particular interneuron properties by comparing and contrasting expression profiles of
zebrafish interneurons with similar and different properties. To test the functions of
specific transcription factors, we are knocking them down using mutant lines,
morpholinos and dominant-negative constructs. To investigate spinal cord evolution we
are examining transcription factor expression in zebrafish, frogs, amniotes and sharks
and investigating differences between spinal cord signaling pathways and transcription
factor function in zebrafish and amniotes.
Here I will present some of our more recent results.
Oral presentation 26
Multicopy RNA genes and their role in the CNS
Ackerman, Susan L.
Howard Hughes Medical Institute and The Jackson Laboratory, Bar Harbor, ME, USA
Neurodevelopmental and neurodegenerative disorders affect many people, yet the
cause of these disorders is largely unknown. Using a forward genetic approach in mice,
our lab has identified several novel molecular mechanisms that underlie CNS
development and neuron death. Importantly, this phenotype-driven approach allows the
identification, without a priori assumptions, of molecules critical to these processes.
Recently, we have identified mutations in functional RNAs that specifically led to
degeneration of cerebellar granule cells in the adult brain. Surprisingly, these mutations
reside in functional RNAs that are found in multiple copies in the mammalian genome,
suggesting that these genes are not genetically redundant. Indeed a mutation in one of
a cluster of genes encoding identical U2 snRNAs resulted in pre-mRNA splicing defects
and cerebellar degeneration. Surprisingly, expression of the mutated snRNA was
regulated in a spatial and temporal fashion with highest expression in the mature
cerebellum. These, and other results suggest that mutations in multigenic functional
RNAs have important ramifications for neurological disorders, particularly for
neurodegenerative disease.
Oral presentation 27
Shaping Sensory dendrites – Skin-derived cues do the job.
Buelow, Hannes
Albert Einstein College of Medicine, Bronx, NY, USA
Neuronal development requires the elaboration of axons and dendrites both of which
are likely patterned by both autonomous and non-autonomous cues. For example, it has
been known for some time that sensory dendrites depend on cues from their
environment to control their growth and patterning. However, little is known about
dendrite-substrate interactions during dendrite morphogenesis. We discovered MNR1/menorin, which is part of the conserved Fam151 family of proteins and is expressed in
the skin to control the elaboration of ‘‘menorah’’-like dendrites of mechanosensory
neurons in Caenorhabditis elegans. Our biochemical and genetic evidence show that
MNR-1 acts as a contact-dependent or short- range cue in concert with the neural cell
adhesion molecule SAX-7/L1CAM in the skin and through the neuronal leucine-rich
repeat transmembrane receptor DMA-1 in sensory dendrites. This work describes a
previously unknown pathway that provides spatial information from the skin to pattern
sensory dendrite development non-autonomously.
Oral presentation 28
Cooperative Wnt5a/Wnt11 signaling inhibits canonical Wnt signaling and
promotes cardiac progenitor fate via the Caspase-dependent degradation of AKT
Cohen, Ethan David
Rochester, USA
Wnt proteins, ligands that regulate cell behavior via a canonical pathway that induces ßcatenin dependent transcription, promote the expansion of the second heart field (SHF)
progenitors that contribute most cardiomyoyctes. However, activating ß-catenin also
causes loss of the SHF, revealing the need to precisely control ß-catenin. We recently
reported that two divergent Wnt ligands, Wnt5a and Wnt11, cooperatively restrain ßcatenin from disrupting SHF development. Yet while these data indicate that
Wnt5a/Wnt11 are needed to control ß-catenin in the SHF, the mechanism these ligands
use to inhibit ß-catenin are unclear. A previous study showed that Wnt11 inhibited ßcatenin and promoted cardiomyocyte maturation via Caspases. Consistent with these
data, recombinant Wnt5a and Wnt11 proteins increase Caspase activity in ES cells and
staining for active Casp3 is reduced in Wnt5a/Wnt11 double mutants. Furthermore,
treating ES cells with Caspase inhibitor blocks the ability of Wnt5a/Wnt11 to promote
SHF gene expression in vitro while injecting pregnant dams with Caspase inhibitor
reduces mRNA for cardiac progenitor markers in gestating embryos. Caspase inhibitors
also block the loss of ß-catenin protein and reporter activity caused by Wnt5a/Wnt11
treatment. Interestingly, we have found that Wnt5a/Wnt11 induce the Caspasedependent degradation of AKT and treating ES cells with AKT inhibitors increases the
levels of SHF markers to levels similar to those observed after Wnt5a/Wnt11
treatment. Together, these data suggest that Wnt5a/Wnt11 inhibit the canonical Wnt
pathway and promote SHF development via the Caspase-dependent degradation of
AKT.
Oral presentation 29
Post-gastrulation mesodermal patterning
Kinney, Brian Row; Richard; Martin, Benjamin
Stony Brook University, Stony Brook, NY, USA
Vertebrate embryos form their body through a process called posterior growth, where
the head forms first and the rest of the body develops progressively towards the
posterior end. After gastrulation, a population of axial stem cells in a structure at the
posterior-most end of the embryo called the tailbud, fuels the process of posterior
growth by contributing cells to newly forming tissues. Axial stem cells make a basic
germ layer decision between neural ectoderm and mesoderm, but it is unclear how and
to what extent the newly formed mesoderm is patterned. We used heat-shock inducible
transgenes to temporally inhibit or activate the canonical Wnt, FGF, and BMP pathways
during tailbud stages and examined mesodermal cell fate. We found that Wnt and FGF
act at different steps to promote dorsal (paraxial) mesoderm formation and inhibit
ventral (vascular endothelium), while BMP has a reciprocal effect, promoting ventral and
inhibiting dorsal mesoderm formation. Our results indicate that axial stem cells generate
basal mesoderm, which is then patterned through a complex interaction between Wnt,
FGF, and BMP signaling.
Oral presentation 30
Steering Plant Cell Division
Bezanilla, Magdalena
University of Massachusetts, Amherst, MA, USA
Plant cells divide using the phragmoplast, a microtubule-based structure that directs
vesicles secretion to the nascent cell plate. The phragmoplast forms at the cell center
and expands to reach a specified site at the cell periphery, tens or hundreds of microns
distant. The mechanism responsible for guiding the phragmoplast remains largely
unknown. Using both moss and tobacco, we show that myosin VIII associates with the
plus ends of phragmoplast microtubules and together with actin guides phragmoplast
expansion to the cortical division site. Our data lead to a model whereby myosin VIII
links phragmoplast microtubules to the cortical division site via actin filaments. Myosin
VIII's motor activity along actin provides a molecular mechanism for steering
phragmoplast expansion.
Oral presentation 31
Collective cell behavior promotes epithelial invagination
Martin, Adam; Vasquez, Claudia; Mason, Frank (Cambridge, USA); Tworoger, Michael
MIT, Cambridge, MA, USA
Tissue shape requires that dynamic cell behaviors collectively sculpt tissues. A cell
shape change involved in epithelial remodeling is apical constriction, which promotes
epithelial sheet folding and cell invagination, such as during gastrulation. Apical
constriction is driven by force generated by the motor myosin II (Myo-II), whose activity
is regulated by Rho-associated coiled-coil kinase (ROCK). During Drosophila
gastrulation, apical constriction occurs in a series of contractile pulses that are
asynchronous between cells in the tissue. Contractile pulses are associated with cycles
of apical Myo-II foci assembly and disassembly that constrict cells in an incremental
manner. How upstream signaling organizes contractile pulses and the importance of
pulsatile behaviors to tissue morphogenesis is not understood. Here, we show that
upstream signals that regulate contractility exhibit spatial organization within the apical
domain, with ROCK being polarized to foci near the center of the cell apex. ROCK
exhibits pulsatile behavior that is associated with apical Myo-II and mutants that
uncouple Myo-II activation from ROCK or depletion of the phosphatase that reverses
Myo-II phosphorylation disrupts contractile pulses. Thus, direct coupling between
dynamic signals and Myo-II activation organizes contractile Myo-II pulses in both space
and time. Mutants that disrupt contractile pulses still undergo apical constriction.
However, constriction is continuous and is associated with separation and tearing
between adjacent cytoskeletal networks during tissue invagination. We propose that
contractile pulses are required for collective cell behavior by maintaining tissue integrity
during morphogenesis.
Oral presentation 32
The adaptor protein Cindr fine-tunes JNK signaling in developing epithelia
van Rensburg, Sam; Yasin, Hannah; Feiler, Christina; Johnson, Ruth
Wesleyan University, Middletown, CT, USA
Correct function of the conserved adaptor protein Cindr is crucial for the development of
epithelial tissues. For example, reducing Cindr in the pupal eye disrupts the correct
positioning of accessory cells as the eye is patterned. In the larval and pupal wing, cells
with reduced Cindr activity are similarly unable to maintain correct positions but instead
delaminate and migrate away from their original positions. We propose that Cindr
assembles protein complexes necessary to establish and maintain stable cell positions
(niches) in epithelia. Amongst these complexes is one that functions to limit JNK
activity. Cindr binds directly to the JNK signaling kinase Basket and represses JNK
signaling that would otherwise induce epithelial cell migration and cell death. The
interaction between Cindr and JNK is conserved. In genetic interactions we observed
that the effects of reducing Cindr in epithelia are modified by multiple components of the
JNK signaling cascade. Together, our genetic and biochemical data suggest that Cindr
is required to fine-tune JNK signaling and we are testing several models to determine
the mechanism Cindr uses to limit JNK.
Oral presentation 33
Posters
mir-14 regulates autophagy during developmental cell death via IP3 signaling
Nelson, Charles; Ambros, Victor; Baehrecke, Eric
UMass Medical School, Worcester, MA, USA
Macroautophagy (autophagy) is a lysosome-dependent cellular degradation process
that has been implicated in multiple age-associated diseases, including cancer and
neurodegeneration. Autophagy is involved in both cell survival and cell death, but little is
known about the mechanisms that control this catabolic process during these distinct
cell fates. During Drosophila development, the precisely timed death of the larval
salivary glands is dependent upon proper induction of autophagy. By contrast, the fat
body augments autophagy as a cell survival response to nutrient deprivation. Here, we
identify the first microRNA, miR-14, found to be both necessary and sufficient for
autophagy. Loss of miR-14 prevented induction of autophagy during salivary gland
death, but had no effect on starvation-induced autophagy in the fat body. Moreover,
mis-expression of miR-14 was sufficient to prematurely induce autophagy in the salivary
gland, but not in the fat body. Importantly, an RNAi based screen discovered that miR14 targets the gene IP3 kinase 2 thereby regulating this developmental specific
autophagy through the IP3 signaling pathway. Our results provide the first
demonstration of in vivo regulation of autophagy by a microRNA, and identify IP3
signaling as a new factor controlling autophagy in dying cells during development.
Poster 1
prdm12b specifies the p1 progenitor domain and reveals a role for V1
interneurons in swim movements
Zannino, Denise (USA); Downes, Gerald (University of Massachusetts- Amherst,
Amherst, MA, USA); Sagerstrom, Charles (University of Massachusetts- Worcester,
Worcester, MA, USA)
The dorsoventral (DV) axis of the neural tube is divided into 11 domains, each
expressing a unique transcription factor code and giving rise to a unique cell type. For
example, V1 interneurons (IN) arise from the p1 domain. Perturbation in DV patterning
leads to miss-specification, or loss, of particular cell types. We discovered a relatively
uncharacterized gene, Prdm12b, with expression limited to the p1 domain. Prdm12b is
a putative transcription factor belonging to a class of genes characterized by a Pr
containing domain and multiple zinc fingers. To determine the role of Prdm12b in DV
patterning, we used antisense morpholino oligonucleotides (MO) designed to block the
translation of Prdm12b. We find that loss of prdm12b function leads to loss of V1 INs,
identified by enb1 expression. Loss of V1 INs also leads to deficits in touch evoked
escape responses, as these INs play inhibitory roles in locomotion circuits. Specifically,
control embryos touched on the head arch their bodies into a single c-bend, bringing the
head adjacent to the tail, thus orienting the head away from the stimulus, and then swim
away in a stereotypical manner. MO-injected embryos exhibit more c-bends, longer
durations of response, and swim shorter distances than control MO-injected embryos.
Additionally, the left-right alternation of body bends appears delayed. Loss of prdm12b
function also leads to misexpression of the DV markers nkx6.1 and pax3, expressed
respectively ventrally and dorsally to prdm12b, as well as to a reduction of olig2
expression in the pMN domain of the hindbrain. Taken together, prdm12b regulates DV
patterning, is required for V1 IN specification, and plays an important role in escape
responses.
Poster 2
Low dose exposure to BPA increases blastema growth in regenerating planaria
Miller, Hannah (SUNY New Paltz, Beacon, NY, USA); Mlynarska, Izabela (SUNY New
Paltz, New Paltz, NY, USA); Young, Micaela (Smith College, Northampton, MA, USA);
Salvino, Elizabeth (SUNY New Paltz, New Paltz, NY, USA); Chilingaryan, Nicole (SUNY
New Paltz, New Paltz, NY, USA); Mass, Spencer (SUNY New Paltz, New Paltz, NY,
USA)
The environmental endocrine disruptor Bisphenol-A (BPA), used in the manufacture of
polycarbonate plastics, thermal printing systems, epoxy resins and other industrial
processes has been shown to depress and delay regeneration in a variety of flatworms
at high doses. In this work we examine the effects of much lower concentrations which
are more environmentally relevant and find a concentration dependent increase in
blastema growth with decreasing dosage. In vertebrate systems, weak estrogen
receptor (ER) agonists like BPA are known to repress ER responses at high doses and
increase ER responses at low doses in a manner very similar to the decrease and
increase in growth we observe in regenerating planaria. Prior work in our lab has
suggested that bisphenol compounds are interacting with an ER-like pathway in
planaria, and our current finding is consistent with this mechanism.
Poster 3
Cas9 protein-mediated oligonucleotide insertion overcomes allele biases in
zebrafish
Gagnon, James (Harvard University, Somerville, MA, USA); Schier, Alex (Harvard
University, Cambridge, MA, USA)
The CRISPR/Cas9 system has been implemented in a variety of model organisms to
mediate site-directed mutagenesis. A wide range of mutation rates has been reported,
at a limited number of genomic target sites. To uncover the rules that govern effective
Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for
mutagenesis using a streamlined and cloning-independent method. We successfully
generated mutations in 85% of our targets with mutation rates that vary across several
orders of magnitude. We discovered that many poor or unsuccessful single-guide RNAs
(sgRNAs) initiate with a 5’ adenine. These failures are likely a consequence of transcript
heterogeneity at the 5’ end, and their activity can be improved by several distinct
approaches that rescue 5’ end homogeneity, such as base switching and promoter
swapping. In addition to capturing inherent Cas9 sequence preferences, we discovered
that independent cleavage events had a strong tendency to induce the same DNA
repair result. While our large deep sequencing dataset unexpectedly illuminated this
feature of DNA repair mechanisms, non-random allele generation is also a weakness in
that non frame-shifting mutations can dominate. To resolve this issue, we increased
Cas9 cleavage levels by direct injection of purified protein and exploited this increased
efficiency to induce consistently high insertion rates of DNA sequences including
multiple stop codons. Our improved methods and detailed protocols make Cas9mediated mutagenesis an attractive approach for labs of all sizes.
Poster 4
Understanding the molecular mechanisms governing neural stem cell division
required for proper neurogenesis in the embryonic zebrafish spinal cord
Johnson, Kimberly (Molecular and Cellular Biology, UMass Amherst, Amherst, MA,
USA); Moriarty, Chelsea (Biological Sciences, Smith College, Northampton, MA, USA);
Bashiruddin, Sarah (Biological Sciences, Smith College, Northampton, MA, USA);
Barragan, Jessica (Biological Sciences, Smith College, Northampton, MA, USA); von
Saucken, Victoria (Biological Sciences, Smith College, Northampton, MA, USA); Burton,
Sean (Department of Biology, UMass Amherst, Amherst, MA, USA); Stein, Rachael
(Biological Sciences, Smith College, Northampton, MA, USA); Parsons, Michael
(Department of Surgery, Johns Hopkins University, Baltimore, MD, USA); Mumm, Jeff
(Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA); Karlstrom, Rolf
(Department of Biology, UMass Amherst, Amherst, MA, USA); Devoto, Stephen
(Biology Department, Wesleyan University, Middletown, CT, USA); Barresi, Michael
(Biological Sciences, Smith College, Northampton, MA, USA)
Development of the nervous system rests on the precise regulation of neural stem cell
proliferation and progenitor cell patterning. Radial glia serve as the embryonic neural
stem cell, giving rise to both neuronal and glial progeny. In a previous study, we
identified a class of zebrafish mutants that have increased radial glial cell bodies in
proliferative zones of the neural tube. Using these mutants, we hope to shed light on the
regulation of radial glial cell proliferation and differentiation. Mutation of kif11, a kinesin
motor, resulted in mitotically arrested radial glial cells, reduced cell survival, and
reductions in oligodendroglia, secondary interneurons, and motorneurons. Using a new
transgenic radial glial labeling line, gfap:nls-mCherry, we demonstrate that subsets of
later born neurons are radial glial derived. In addition, we directly test the requirement of
radial glia for these specific cell fates by employing a Nitroreductase ablation system to
temporally eliminate radial glia. We generated a gfap:nfsb-mCherry transgenic line to
specifically ablate Gfap+ radial glia through the use of the prodrug Metronidazole, and
are currently characterizing the effects of radial glial cell ablation on cell type
differentiation. Lastly, we also identified wnt5b as an important player in radial glial
development, as mutations in wnt5b caused an increase in mitotically active radial glia.
We are currently testing how the non-canonical Wnt5b ligand is functioning to regulate
radial glial development. These diverse approaches will provide a comprehensive
analysis of both the intracellular and extracellular mechanisms regulating the division
and subsequent contributions of radial glia throughout neurogenesis.
Poster 5
Role of lbh during Xenopus Craniofacial Development
McLinden, Gretchen; Powder, Kara; Albertson, Craig; Cousin, Helene
University of Massachusetts, Amherst, MA, USA
The lbh gene encodes a transcription factor expressed in the limb bud and heart of the
developing mouse embryo. It is also expressed in the first branchial arch, embryonic
precursor of facial structures. While lbh function during limb and heart development has
been investigated, nothing is known about lbh role during craniofacial development. We
have shown that lbh knock down in zebrafish lead to severe craniofacial defects without
affecting chondrogenesis. To understand the origins of these defects, we are
investigating the role of lbh using a multiple species approach. Here we describe the
three main findings we uncovered in the amphibian Xenopus laevis.
First, lbh is expressed in the cranial neural crest cell before and during their migration
and is down regulated after their migration. Second, lbh knock down using translational
morpholino inhibits CNC migration but not induction. Third, the misexpression of
Xenopus lbh induces a very modest inhibition of CNC migration while the misexpression
of a long-jawed (Metriaclima zebra) and short-jawed (Labeotropheus fuelleborni) lake
Malawi cichlid lbh produces a significant change in the number and the quality of the
migration of the cranial neural crest. These observations could help us understand the
mechanisms behind the craniofacial developmental plasticity and the microevolution of
lake Malawi cichlids.
Poster 6
Niche Function and stem Cell Activation in the C elegans Gonadal Primordium
McIntyre, Daniel (USA); Nance, Jeremy (NYU Medical Center, New York, NY, USA)
One of the most conserved aspects of stem cell biology is the close association of stem
cells with niche cells – supporting cells that regulate stem cell activity and protect these
cells from external influences. To understand how a niche functions, we have studied
the germline stem cell niche in the nematode worm, C. elegans. In this simple genetic
system, two germline stem cells and two somatic gonadal precursor cells (niche cells)
interact predictably during embryogenesis to form the gonadal primordium. Niche
formation is critical for the survival of the germline stem cells, and ultimately controls
these cells’ switch between quiescent and activated states. We have taken two
approaches to investigate how this process occurs. First, using laser ablations we are
examining the fate of germ cells that do not make contact with somatic gonadal
precursor cells. Germ cell survival depends on early contact with the niche cells and we
are working to uncover the molecular mechanisms underlying this phenotype. Second,
previous research from our lab has implicated adhesion molecules including E-cadherin
in gonadal primordium formation. We are taking advantage of the recent advances in
genome editing technology to conditionally disrupt E-cadherin function in the niche and
assay the role it plays in stem cell activation. The results will show if these molecules
have signaling as well as adhesive roles in the germline stem cell niche. Niche cell
function bears directly on many aspects of human health, including regenerative
medicine and cancer biology, and the high degree of conservation previously observed
between worms and mammals indicates our results will be applicable to problems in
human stem cell biology.
Poster 7
Distinct profiles of myelin distribution along single axons of pyramidal neurons in
the neocortex
Giulio Srubek Tomassy1, Daniel R. Berger2, Hsu-Hsin Chen1, Jeff W. Lichtman2 and
Paola Arlotta1*
1
Department of Stem Cell and Regenerative Biology, Harvard University, 2 Department
of Molecular and Cellular Biology, Harvard University
Myelin is a defining feature of the vertebrate nervous system. Variability in the thickness
of the myelin envelope is a structural feature affecting the conduction of neuronal
signals. Conversely, the distribution of myelinated tracts along the length of axons has
been assumed to be uniform. Here, we traced high-throughput electron microscopy
(EM) reconstructions of single axons of pyramidal neurons in the mouse neocortex and
built high-resolution maps of myelination. We find that individual neurons have distinct
longitudinal distribution of myelin. Neurons in the superficial layers displayed the most
diversified profiles, including a new pattern where myelinated segments are
interspersed with long, unmyelinated tracts. Our data indicate that the profile of
longitudinal distribution of myelin is an integral feature of neuronal identity and may
have evolved as a strategy to modulate long-distance communication in the neocortex.
Poster 8
Regulated cell cycle dependent transcriptional silencing promotes germline stem
cell differentiation.
Flora, Pooja
University at Albany, SUNY, Albany, NY, USA
Loss of stem cell differentiation has implications in aging and in diseases such as
cancer. Thus, it is critical to identify factors that mediate stem cell self-renewal and
differentiation. The germ line is the ultimate stem cell as it is both totipotent and
immortal. Hence, paradigms established in the germ line can be extrapolated to other
stem cell systems. Drosophila germline stem cells (GSCs) are a great model system to
understand differentiation as its germline is well characterized. During Drosophila
melanogaster embryogenesis, polar granule component (pgc), a global transcriptional
silencer, represses somatic transcription in primordial germ cells to promote germ line
fate. Surprisingly, we have discovered that Pgc is also expressed during oogenesis. We
found that Pgc is expressed in the stem cell daughter, cystoblast (CB), for a part of its
life cycle suggesting that it may be expressed in a cell cycle dependent manner. To
determine in which stage of the cell cycle Pgc is being expressed, we used different cell
cycle markers simultaneously with a reporter for Pgc and found that majority of the CBs
expressed Pgc in the early G2 phase. Additionally, we have shown that in loss of pgc,
cell cycle is disrupted and majority of the CBs accumulate in the G1/S-phase indicating
the role of Pgc in regulating cell cycle during differentiation. In addition, qPCR analysis
showed Cyclin B, a regulator of G2 phase, was down-regulated in pgc mutants, and
reduction of Cyclin B was sufficient to cause a delay in CB differentiation. Therefore, our
results favor the idea that transcriptional silencing is needed to “clear” residual stem cell
program, such as altering cell cycle in the GSC daughter, prior to differentiation.
Poster 9
Identification of tissues involved in dorsal pancreas induction
Tremblay, Kimberly; Angleo, Jesse
University of Massachusetts, Amherst, MA, USA
The definitive endoderm arises during mammalian gastrulation as a simple epithelial
sheet that will give rise to the entire gut tube and associated organs. We previously
used a fate mapping approach to locate the dorsal pancreas progenitors in the naïve
endodermal sheet of the mouse embryo. The pancreas is specified from the naïve
endoderm by inductive signals secreted from adjacent mesoderm. To identify the
tissues responsible for dorsal pancreas specification, we assessed the spatiotemporal
association of the pancreas progenitors and neighboring tissues. We find that all
mesoderm adjacent to the naïve pancreas progenitors at E8.25 are located anterior to
the pancreas buds at E9.5 and that the extent of migration is unique to distinct
mesoderm populations. The notochord is the mesoderm-derived tissue most tightly
coupled to the pre- and post-pancreatic endoderm. Because notochord-derived signals
have been implicated in dorsal pancreas development, we next tested the hypothesis
that the notochord is required for appropriate dorsal pancreas induction by ablating the
region of the notochord associated with the dorsal pancreas progenitors and culturing
these embryos through the pancreatic bud stage (E9.5). We find that notochord ablated
embryos form a dorsal and ventral pancreas bud, but that both buds fail to express
Pdx1. Notochord ablation after the onset of Pdx1 expression has no discernable affect
on pancreas development. Taken together, we have shown that the notochord
associated with the dorsal pancreas precursors is required for normal pancreas
development including expression of Pdx1 and believe that these novel insights can be
used in the generation of insulin-secreting pancreatic beta cells.
Poster 10
Collective migration along a self generated chemokine gradient
Venkiteswaran, Gayatri (New York University School of Medicine, New York, NY, USA);
Lewellis, Stephen (New York University School of Medicine, New York, NY, USA);
Wang, John (New York University School of Medicine, New York, NY, USA); Reynolds,
Eric (Berlin, DEU); Nicholson, Charles (New York, USA)
Migrating cells reach their destinations by following concentration gradients of an
attractant. However, mechanisms by which attractant gradients are generated remains
uncharacterized. We addressed this problem by using a chemokine-GFP fusion and a
signaling sensor to analyze the distribution of Sdf1 during the migration of the zebrafish
lateral line primordium. The primordium in zebrafish embryos is a cohort of about 200
cells that expresses the chemokine receptor Cxcr4 and migrates over a stripe of uniform
expression of Sdf1. We find that Sdf1 signaling by a small fraction of the total Sdf1 pool
generates a linear Sdf1-signaling gradient across the primordium. We show that the
signaling gradient is generated at the rear of the primordium by continuous
sequestration of Sdf1 protein from the stripe by the alternate Sdf1-receptor Cxcr7.
Finally we show that an external source of Cxcr7 around the rear of the primordium is
sufficient to maintain the Sdf1 signaling gradient across it. Our study describes an
elegant mechanism of collective cell guidance by a self generated gradient of a
guidance molecule.
Poster 11
Nanog, Pou5f1 (Oct4) and SoxB1 are required for widespread gene activation
during the maternal-to-zygotic transition
Bonneau, Ashley; Miler Lee, Carter Takacs, Ariel Bazzini, Kate DiVito, Elizabeth
Fleming, Antonio Giraldez
Yale University, New Haven, CT, USA
Following fertilization, maternal factors deposited during oogenesis direct the initial
stages of development in a transcriptionally quiescent embryo. At the maternal-tozygotic transition (MZT), developmental control shifts and the zygotic genome becomes
transcriptionally active and the maternal products are degraded. In zebrafish, zygotic
genome activation (ZGA) is required for gastrulation and the formation of the three germ
layers as well as the clearance of maternal mRNAs, with clearance mediated by zygotic
expression of the conserved microRNA miR-430. Although the MZT is a conserved
aspect of animal development, the specific factors that regulate ZGA in vertebrates
remain largely unknown. In order to address this question, we adopted a loss-offunction approach to elucidate the roles of the core transcription factors Nanog, Pou5f1
(Oct4) and SoxB1 in activating early zygotic transcription. These factors have been
previously shown to regulate cell specification, patterning, and morphogenesis during
zebrafish development and are highly translated in the early embryo. Using morpholinos
to repress translation of nanog and soxB1 and a maternal-zygotic pou5f1 mutant, we
report developmental arrest prior to gastrulation and a failure to activate more than 80%
of zygotic genes, including miR-430. These results indicate that Nanog, Pou5f1 and
SoxB1 are essential for initiating the zygotic program during the MZT and for the
clearance of the previous maternal program by activating miR-430 expression.
Poster 12
Caspase requirements for cell competition
Abhijit Kale, Wei Li and Nicholas E. Baker
Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461
Cell competition, discovered in Drosophila, is a phenomenon whereby the survival of
cells in developing organs is found to depend on the genotype of their near neighbors.
In the classic example, cells heterozygous for any of many ribosomal protein genes are
lost from mosaics that contain wild type cells, even though these Rp/+ cells are in
principle viable, in fact entirely Rp/+ animals survive. In other examples of cell
competition, even wild type cells can be out-competed by certain genotypes. Cell
competition involves caspase-dependent apoptosis of cells under competition. Here,
we report the genetic requirements of competitive cell death in Drosophila. Similar to
most developmental apoptosis, we find that competitive cell death depends on
proapoptotic genes reaper, grim and head involution defective, and on effector
caspases. Distinct from most developmental apoptosis, however, competitive apoptosis
can begin using the initiator caspase Strica/Dream, and involves significantly lower
activity of the initiator caspase Dronc. These findings suggest a distinct mechanism for
initiating cell death during cell competition, perhaps related to the cell communication
that is involved.
Poster 13
What are the molecular mechanisms that govern mammalian touch receptor
remodeling?
Kara L. Marshall1, Blair A. Jenkins2,3, Ellen A. Lumpkin1,2,4
1
Departments of Dermatology, 2Neurobiology & Behavior and 4Physiology & Cellular
Biophysics, Columbia University, New York, NY
3
Columbia University Medical Scientist Training Program
An array of touch receptors in the skin inform us about the features of objects in our
environment and are essential for complex behaviors such as feeding and mating.
Slowly adapting type I (SAI) afferents innervate specialized epidermal cells to form
Merkel cell-neurite complexes, which are needed for detecting texture and edges with
high acuity. These touch receptors have unique electrophysiological properties and
well-characterized morphology. Mammalian hairy skin continuously cycles between
states of hair growth, regression and rest, and the stages of this cycle occur in
synchronized waves in postnatal mice. This provides an excellent model system in
which to study changes in neuronal architecture during normal fluctuations in skin
structure.
Though our data suggest that tactile afferents remodel in order to maintain sensitivity
during skin changes, the molecular mechanisms that mediate this remodeling remain
unknown. The number of Merkel cells and SAI afferent branches changes dramatically
during hair growth and rest phases. The Bone Morphogenetic Protein (BMP) pathway
plays an essential role in the transition between resting and growth phases.
Additionally, overexpression of BMPs causes a decrease in cutaneous innervation
density. Thus, we hypothesize that BMP signaling plays a role in afferent remodeling
during skin turnover. To test this hypothesis, we have begun to outline the timing of hair
cycle-induced sensory afferent remodeling and assess the expression of BMP receptors
in the skin and in afferents. This research will not only elucidate touch receptor
remodeling mechanisms in adult skin, but could also shed light on the developmental
processes that mediate afferent outgrowth, targeting and refinement.
Poster 14
The C. elegans SNAP complex component SNPC-4 coats piRNA domains and
globally affects piRNA abundance
Kasper, Dionna; Wang, Guilin; Gardner, Kathryn; Johnstone, Timothy; Reinke, Valerie
Yale University School of Medicine, New Haven, CT, USA
Reproductive fitness depends upon the essential responsibility of germ cells to transmit
unblemished genetic information to progeny for many generations. Germ cells employ
Piwi-interacting RNAs (piRNAs), a highly conserved class of small non-coding RNAs
primarily expressed in the germ line, to defend against mutagenic activity of foreign
sequences such as transposable elements. Although tens of thousands of distinct
piRNA species exist, they arise from a remarkably limited number of discrete genomic
regions across organisms. However, the extent to which clustering of these small RNA
genes contributes to their highly coordinated expression remains unclear. We show that
C. elegans SNPC-4, the DNA-binding subunit of the small nuclear RNA activating
protein complex (SNAPc), binds piRNA clusters in a germline-specific manner and is
required for global piRNA expression. SNPC-4 not only binds at discrete sites through
its established sequence motif, but also exhibits widely distributed binding across piRNA
domains. Discrete peaks are not found at most piRNA genes, but occur frequently at
RNA polymerase III-occupied tRNA genes, which have been implicated in chromatin
organization in yeast and human. Given its unique binding pattern, SNPC-4 likely
promotes robust piRNA expression by establishing a permissive chromatin environment
at clusters specifically in the germ line. Importantly, our work provides novel
mechanistic insight into how to produce a substantial repertoire of non-coding RNA
effectors required for an effective silencing response against non-self elements so that
germline immortality is preserved.
Poster 15
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
Tessa G. Montague, José M. Cruz, James A. Gagnon, George M. Church, Eivind
Valen, Alex Schier
Harvard University
Major advances in genome editing have recently been made possible with the
development of the TALEN and CRISPR/Cas9 methods. The speed and ease of
implementing these technologies has led to an explosion of mutant and transgenic
organisms. A rate-limiting step in efficiently applying TALEN and CRISPR/Cas9
methods is the selection and design of targeting constructs. We have developed an
online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to expedite the design
process. CHOPCHOP accepts a wide range of inputs (gene identifiers, genomic regions
or pasted sequences) and provides an array of advanced options for target selection. It
uses efficient sequence alignment algorithms to minimize search times, and rigorously
determines off-target binding of single-guide RNAs (sgRNAs) and TALENs. Each query
produces an interactive visualization of the gene with candidate target sites displayed at
their genomic positions and color-coded according to quality scores. In addition, for
each possible target site, restriction sites and primer candidates are visualized,
facilitating a streamlined pipeline of mutant generation and validation. The ease-of-use
and speed of CHOPCHOP make it a valuable tool for genome engineering.
Poster 16
Investigating the Role of Maskin in Early Neuronal Development
Belinda Nwagbara, Anna Faris, Matthew Evans, Patrick Ebbert, Charles Baker, Tiffany
Enzenbacher, Laura Anne Lowery
Nervous system function is heavily dependent on making proper axonal connections
during early neuronal development. A fundamental question that has emerged in axon
guidance is how cytoskeletal dynamics are coordinated by guidance pathways to
promote precise navigation. Microtubule (MT) plus-ends, in particular, play a very
important role in the axonal growth cone (the dynamic structure at the tip of the growing
axon). Studies have shown that a conserved family of proteins known as plus- endtracking proteins (+TIPs) are essential for regulating MT plus-end dynamics. However,
little is still known on how exactly +TIPs interact with MTs and each other to control MT
behavior during embryonic development.
Maskin, a member of the TACC family (transforming acidic coiled coil domain) is a +TIP
that has been implicated in directing MT dynamics in a number of systems. However,
not much is known regarding the effect maskin may have in the growth cone. Using high
spatial and temporal resolution imaging, here we investigate the role of Maskin during
neurite outgrowth by reporting its localization dynamics for the first time in growth
cones. We also have examined the effects of Maskin localization using domain deletion
analysis, to determine which domains are essential for MT plus-end localization.
Together, these studies begin to address the function and interaction of Maskin in the
growth cone.
Poster 17
Tactile Sensing in the Bat’s Winged Hand
Marshall KL1*, Chadha M2*,
Lumpkin EA1, 3, 5
DeSouza LA3, Sterbing-D’Angelo SJ4, Moss CF2,
4
,
1.
Departments of Dermatology and 5. Physiology & Cellular Biophysics, Columbia
University, New York, NY, 2. Program in Neuroscience and Cognitive Science (NACS)
and Department of Psychology, University of Maryland, College Park, MD, 3. Program in
Neurobiology & Behavior, Columbia University, New York, NY 4. Institute for Systems
Research (ISR), University of Maryland, College Park, MD
Bats accomplish agile flight using modified hand-wings, with elongated digits connected
by thin, flexible skin. Wing skin is endowed with microscopic hairs that modulate flight,
which is proposed to occur through tactile receptors that provide feedback about airflow.
Sensorimotor circuits develop through coordinated extension of motor and sensory
innervation into surrounding tissue. It has been reported that the bat wing develops not
only from the forelimb bud, but also the trunk and hindlimb bud. The receptors
mediating sensorimotor integration in mammalian flight and their developmental origins,
however, are unknown.
We hypothesize that sensorimotor circuits develop as a result of the wing’s unusual
ontogeny. We used neuronal tracing to identify sensory neurons and motor pools that
innervate Eptesicus fuscus wings. These data reveal that wing-skin sensory innervation
arises from cervical dorsal root ganglia (DRG) that innervate other mammalian
forelimbs, but also from mid- and lower thoracic DRGs. By contrast, all motor pools
were above upper thoracic levels, leading to a mismatch with sensory ganglia. These
data suggest a developmental basis for the bat’s unusual forelimb maps in
somatosensory brain regions.
To analyze the structure and function of wing touch receptors, we used
immunohistochemistry of wing skin and in vivo recordings from primary somatosensory
cortex. We found a novel repertoire of somatosensory receptors in wing skin.
Information theoretic analysis of airflow cortical responses indicated the importance of
fast signaling to guide wing kinematics. Our findings reveal that evolutionary pressures
enabling powered flight in bats have also given rise to somatosensory specializations of
its hand-wing.
*Equally contributing authors
Poster 18
A comparative analysis of gene regulation during the maternal-to-zygotic
transition in zebrafish and medaka
Miler T. Lee, Ashley R. Bonneau, Valeria Yartseva, Carter M. Takacs, Antonio J.
Giraldez
Department of Genetics, Yale University
In animals, early embryogenesis is guided first by factors provided maternally, and
subsequently by factors generated de novo in the zygote. This transfer of
developmental control, called the maternal-to-zygotic transition (MZT), encompasses
two major activities: (i) activation of the zygotic genome; and (ii) regulated clearance of
maternal RNAs and proteins (maternal clearance). Currently, our understanding of the
conserved mechanisms underlying the MZT is incomplete. Previously, we used highthroughput analyses to show that widespread zygotic gene activation in zebrafish is coregulated by the core transcription factors Nanog, Pou5f1 (Oct4) and Sox19b. Here, we
apply a similar approach to study the MZT in medaka (Oryzias latipes), a fish with
similar ontogeny to zebrafish despite ~330 million years of separation. We generated an
mRNA-Seq time course spanning the medaka MZT, and compared the expression
dynamics to zebrafish. We observe high divergence in the timing and levels of genes
expressed; however, some expression patterns were conserved. First, early activation
of the microRNA miR-430 is common to both species, suggesting shared mechanisms
of transcriptional activation likely involving Nanog. Second, miR-430's role in mediating
maternal clearance is also conserved, which we confirm using knockdown of medaka
miR-430. Finally, a subset of orthologous maternal RNAs are cleared in both species in
a manner not depending on microRNAs, suggesting deep conservation of the
mechanisms regulating maternal clearance. Together, our results help to elucidate the
conserved components of the zygotic transcriptional network, which is key to
understanding the mechanisms that drive totipotency and differentiation.
Poster 19
Development of a technique to extract posterior midgut cells from Drosophila
embryos for the purpose of transcriptome analysis
Piccininni, Jenna (New York University School of Medicine, New York, NY, USA)
The differentiation of the endoderm into two distinct cell types, those that remain
epithelial and those that become mesenchymal via the epithelial to mesenchymal
transition (EMT), is a crucial step in the development of the midgut.1 This process also
is relevant to neoplastic metastatic disease and thus more knowledge about the genes
involved may be beneficial in developing therapies to prevent metastasis.2 We have
developed a technique to extract posterior midgut (PMG) cells from mid-stage 10
embryos using micropipette dissection. We then verified the accuracy of this technique
via immunofluorescent staining. With this established, in the future we plan to use this
technique to analyze the transcriptomes of the mesenchymal and epithelial cells of the
posterior midgut.
Poster 20
The role of a highly conserved messenger RNA modifying enzyme in metazoan
development
Hongay, Cintia (Clarkson University, Potsdam, NY, USA)
Without the proper signals, a growing mass of cells simply becomes a larger mass of
identical cells. Genes for cellular coordination such as Notch, a key regulator of cell
identity, proliferation, differentiation and apoptosis, have evolved along with
multicellularity about a billion years ago to allow for the vast diversity of metazoan life
forms. Given the broad range of phenotypes that could be obtained by modifying levels
of gene expression, we have acknowledged that fine tuning of gene expression must be
crucial when two identical cells adopt their specialized fates. However, how this
delicate balance is accomplished is not fully understood. The focus of our lab is to
investigate the role of the evolutionarily conserved mRNA (m6A) modifying enzyme
IME4 (Inducer of Meiosis 4) in metazoan development. First identified and studied in S.
cerevisiae, IME4 is responsible for the switch between non-sporulating and sporulating
diploid cells, thus enabling gametogenesis. IME4 is conserved from yeast to humans
but its role in multicellular eukaryotes is still being deciphered. Our working hypothesis
is that IME4 expression is developmentally regulated and whether it is "ON" or "OFF"
(or fully expressed or not) will impact the expression profile of its target genes and
consequently the overall developmental fate of the cell. We have shown IME4
requirement for Notch signaling during follicle cell differentiation in oogenesis, and our
preliminary data indicate an essential role in both fruit flies and zebrafish embryonic
development. Our main goal is to understand IME4's role in developmental processes
and to establish a conceptual basis for studying its function in humans.
Poster 21
A role for FGF8a in neurovascualr signaling in developing zebrafish
Wysolmerski, Erin (USA); Santiago-Mangual, Kathyana (University of Vermont,
Burlington, VT, USA); Ebert, Alicia (University of Vermont, Burlington, VT, USA)
Fibroblast growth factors (FGFs) are critical in many aspects of embryonic development
and other cellular functions including apoptosis, cell adhesion, and proliferation. We
identified mRNA expression of FGF8a in the retinal ganglion cells (RGCs) and its
receptor FGFR1b in surrounding retinal vasculature of 2 day-old zebrafish. Antisense
morpholino knockdown of FGF8a resulted in a significant reduction in the number of
RGCs and also a reduction in the corresponding tectal innervation. In addition, FGF8a
morphant embryos have mispatterned retinal vasculature, suggesting a role in
neurovascular signaling. It has previously been reported that zebrafish survive and
develop normally for 7 days without blood flow as it receives nutrients by simple
diffusion. To rule out hypoxia, we utilized the silent heart mutant, which lacks cardiac
troponin t resulting in embryos without blood flow, as heart contractility does not initiate.
Cell counts from these fish have however, shown a loss in RGC numbers. Therefore,
using immunohistochemistry, we looked to see if loss of RGCs was due to lack of
proliferating cells using pHH3 or increased cell death using active caspase 3 in both
silent heart and FGF8a mutant fish. We hypothesize that the reduced cell numbers will
be due to a lack of proliferating cells and not cell death. To further our understanding of
this intricate developmental system we intend to look closer into the connection
between the RGCs and the developing vasculature.
Poster 22
Live imaging of regulated epithelial cell death during tissue remodeling
Mesa, Kailin (Yale University, New Haven, CT, USA); Greco, Valentina (Yale University,
New Haven, CT, USA)
Cell death is an essential component of tissue development and maintenance.
Physiological cell death programs promote the rapid removal of excess or dysfunctional
cells without eliciting harmful effects on surrounding healthy tissue. Studying this
process under physiological conditions in vivo has been difficult due to the rapid rate by
which dead cells are normally removed as well as the counterbalancing effects of
proliferation within the same tissue. This has limited our understanding of the signals
and cells involved in the regulation of physiological cell death and tissue remodeling in
vivo.
Recent technological advances have been made in our lab to visualize dynamic cellular
behaviors non-invasively in vivo in the skin of live mice over time by two-photon live
microscopy (Rompolas et al., 2012; Rompolas, Mesa & Greco, 2013). The mammalian
skin hair follicle cycles through stereotypic rounds of growth, programmed cell death,
and quiescence which are morphologically well-defined, making it an ideal model to
study tissue dynamics. Combining these unique features with tools to genetically
manipulate, label and track cells in vivo, we have a temporal approach to uncover the
mechanisms that regulate physiological cell death and tissue remodeling as they
contribute to tissue homeostasis, which has broad implications for the therapeutic
treatment of neoplastic and degenerative diseases. The uncovered cellular and
signaling mechanisms regulating homeostatic cell death and tissue remodeling will be
discussed at the poster.
Poster 23
Mechanisms of stem cell fate in the hair follicle niche
Rompolas, Panteleimon; Mesa, Kailin; Greco, Valentina
Yale University School of Medicine, Department of Genetics, New Haven, CT, USA
In adult tissues, stem cell niches constitute a spatially distinct microenvironment,
including neighboring cells, signals and extracellular material, which is crucial for
regulating stem cell behavior and function. Anatomical and molecular heterogeneity
appears to be a common feature between mammalian stem cell niches across different
tissues; however, it is not clear whether the specific location that a stem cell occupies
within the niche also determines its function. To address this question we utilize the
highly compartmentalized hair follicle stem cell niche because of its stereotypic
regeneration and accessibility for direct microscopic observation in vivo. Thus, we
devised a novel approach by combining intravital microscopy with genetic lineage
tracing tools, which enables us to first, mark single stem cells in different positions
within the niche and second, re-visit the same cells and monitor their lineages
throughout regeneration over the period of several weeks to months. Using this method
we show directly that the location of a stem cell within the hair follicle niche determines
its fate. The hair follicle niche is spatially organized so that we can identify three
functionally distinct compartments; the upper and lower bulge and the hair germ.
Depending where a stem cell resided at the onset of regeneration reproducibly
determined its fate during hair growth. Furthermore, we uses laser-induced cell ablation
to test whether hair follicle stem cells are required for hair regeneration and to address
how injury-induced cell mobility between different niches affects their fate. We found
that when a hair stem cell compartment is ablated, the niche is able to recover the lost
population and proceed with hair regeneration. Surprisingly, epithelial populations that
do not normally participate in hair growth are mobilized to enter the hair follicle niche.
These new cells contribute to both re-establish the lost stem cell pool and sustain hair
regeneration for several months. Furthermore, we provide evidence towards a) the
origin of the hair germ, b) the cellular mechanisms utilized for stem cell clone expansion
during hair growth and c) functional independence of the stem cell niche compartments
during hair growth. This study provides a general paradigm for niche-induced fate
determination in adult tissues.
Poster 24
Investigating a role for Maskin, a TACC3 orthologue, in neural crest cell migration
Bearce, Elizabeth (Boston College, Brighton, MA, USA); Nwagbara, Belinda (Boston
College, Chestnut Hill, MA, USA); Farris, Anna (Boston College, Chestnut Hill, MA,
USA); Evans, Matthew (Boston College, Chestnut Hill, MA, USA); Ebbert, Patrick
(Boston College, Chestnut Hill, MA, USA); Lowery, Laura Anne (Boston College,
Chestnut Hill, MA, USA)
Neural Crest Cells (NCCs) are multipotent cells born along the neural plate; they will
later comprise the peripheral nervous system and connective tissue of the face, neck
and heart. NCCs are migratory, and undergo epithelial-mesenchymal transition (EMT)
to pervade surrounding tissue. This motility is predominantly actin-directed, but it has
become clear that microtubules (MTs) play an extensive role in migration. MTs heavily
infiltrate the leading edge of NCCs, and are thought to mediate cell polarization. These
and all MTs exhibit dynamic instability, and are regulated by a family of ‘plus-endtracking proteins (+TIPs), which accumulate at MT plus-ends to control MT behaviors.
TACC3, a member of the transforming acidic coiled-coil family, is one such protein, and
it has recently come to light as a putative player in EMT. In vitro studies found that overexpressing TACC3 increased motility and markers of EMT. Separately, TACC3 was
shown to influence MT growth parameters, and unpublished data from neurons confirms
that Maskin, a TACC3 orthologue in Xenopus laevis, localizes and interacts with other
+TIPs. Yet no research has sought to explicitly characterize TACC3’s role in cell
migration. This project integrates high resolution visualization and computational
analysis to elucidate whether TACC3 regulates MT dynamics in NCC migration.
Xenopus laevis embryos were injected with mRNA cocktail for fluorescent proteins EB1,
which binds growing MT plus ends, and Maskin. NCCs were harvested and cultured on
coverslips. NCCs began to migrate away from explants, and live images were captured
via spinning disc confocal microscopy. Images were analyzed with plusTipTracker, a
MATLAB-based software designed to monitor +TIP comets.
Poster 25
Delineation of ESDN-Dependent Signaling Mechanisms Required for Zebrafish
Eye Development
Joy, Ryan (University of Vermont, Burlington, VT, USA); Wysolmerski, Erin (University
of Vermont, Burlington, VT, USA); Ballif, Bryan (University of Vermont, Burlington, VT,
USA); Ebert, Alicia (University of Vermont, Burlington, VT, USA)
Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is an orphan
transmembrane receptor, originally found in a screen to identify proteins with signal
peptides expressed in vascular endothelial cells. We identified ESDN in two large-scale
phosphoproteomic analyses. The first found ESDN tyrosine phosphorylated from murine
embryonic brain. The second was a functional proteomics screen to identify substrates
of Src family tyrosine kinases that when phosphorylated would lead to their interaction
with the signaling adapter Crk-Like (CrkL). Given ESDN’s extracellular domain is
reminiscent of neuropilins, which play important roles in the migration of both vascular
endothelial cells and neurons, we examined the expression pattern of Esdn across
zebrafish development by in situ hybridization. Esdn expression is strongly neuronal,
including in the retina, and greatly overlaps with Crkl expression. Morpholino-based
knockdown of Esdn in zebrafish resulted in incomplete development of the retina and
reduced numbers of retinal ganglion cells (RGCs). The morphant phenotype was
rescued RNA encoding full-length ESDN in a dose-dependent fashion. To assess
phenotypes, we scored brain innervation by RGCs in whole-mount embryos. We also
examined RGC cell counts in sectioned embryos during the first three days of
development. These data support a role for ESDN in neuronal development, specifically
in RGCs.
Poster 26
Elucidation of PlexinA2 Signaling Mechanisms Critical for Zebrafish Eye
Development
Weir, Marion; Bassett, Rachael; D'Elia, Kristen; Ballif, Bryan; Ebert, Alicia
University of Vermont, Burlington, VT, USA
Neuronal migration in response to guidance cues is essential for proper patterning of
the central nervous system. Semaphorins (Semas) are a class of guidance cues that
have long been known to govern neuronal positioning. Recently we identified Sema6APlexinA2 (PlxnA2) signaling to be essential for proper development of the zebrafish eye.
However, relatively little is known about the molecular mechanics of Sema-PlxnA signal
transduction. Crystal structure studies suggest that Semas cluster PlxnA receptors.
Furthermore, Sasaki et al. (2002) showed that the Src family tyrosine kinase Fyn both
phosphorylates and binds constitutively to PlxnA receptors. This leads to the hypothesis
that Sema-dependent receptor clustering induces Fyn-dependent phosphorylation of
PlxnA receptors which in turn effectuates PlxnA signal transduction. However, the
specific Fyn-dependent PlxnA phosphorylation sites have yet to be identified. Using
SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture)-based quantitative
mass spectrometry we have identified a novel PlxnA2 phosphorylation site induced by
Fyn. The implications of this phosphorylation in Sema-PlxnA signaling, and its role in
zebrafish eye development are discussed.
Poster 27
Toddler - a novel embryonic signal hidden in non-coding RNA
Pauli, Andrea (USA); Norris, Megan (Cambridge, USA); Valen, Eivind (Cambridge,
USA); Chew, Guo-Liang (Cambridge, USA); Gagnon, James (Cambridge, USA);
Zimmerman, Steven (Cambridge, USA); Mitchell, Andrew (Cambridge, USA); Ma, Jiao
(Cambridge, USA); Dubrulle, Julien (Cambridge, USA); Reyon, Deepak (Charlestown,
USA); Tsai, Shengdar (Charlestown, USA); Joung, Keith (Charlestown, USA);
Saghatelian, Alan (Cambridge, USA); Schier, Alexander (Cambridge, USA)
It has been assumed that most if not all signals regulating early development have been
identified. Contrary to this expectation, we identified more than 300 non-annotated
protein-coding genes, including 28 candidate signaling proteins expressed during
zebrafish embryogenesis.
To determine if any of these putative novel signals might have a function, we focused
on one of them that we call Toddler. Toddler had previously been annotated as a noncoding RNA, but it encodes a short, conserved, and secreted peptide. Both absence
and over-production of Toddler reduce the movement of mesendodermal cells during
zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell
movement, suggesting that Toddler is neither an attractant nor a repellent but acts
globally as a motogen. Toddler drives internalization of G-protein-coupled APJ/Apelin
receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results
indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes
gastrulation movements, and might be the first in a series of uncharacterized
developmental signals.
Poster 28
May factors inducing echinoplutei cloning be more widespread than previously
believed?
Carrier, Tyler (Orono, ME, USA); Arellano, Shawn (Anacortes, WA, USA)
Larval cloning, a form of developmental plasticity, is widespread amongst echinoderms
and has been demonstrated in each class except crinoids. To date, mucus of visual
predators, ocean acidification, and abrupt changes in food concentration have been
demonstrated to stimulate larval cloning in echinoids. Field observations show that the
ctenophore Pleurobrachia bachei and Dendraster excentricus echinoplutei abundances
in East Sound, Orcas Island, WA are inversely correlated. A primary grazing experiment
revealed that the presence of P. bachei mucus induces larval cloning. Here, we
investigate whether mucus concentration or larval density is the controlling factor in the
observed cloning. Filtered seawater previously exposed to 1, 2, 4, or 8 P. bachei had no
significant effect on echinoplutei cloning at constant larval density. When exposed to a
single P. bachei, the likelihood of cloning significantly increased by 1.3-fold with each
larvae added and significantly decreased by 5.2-fold without the presence of P. bachei.
It is suggested that a mucopolysaccharide produced by specialized colloblasts and
mucus sensory cells of P. bachei causes physiological stress. This stress may increase
defensive chemicals produced by echinoplutei, sensed within an aggregation, and
increase likelihood of cloning.
Poster 29
The contribution of P granules to the germline expression profile in C. elegans
Campbell, Anne; Updike, Dustin
MDI Biological Laboratory, Salsbury Cove, ME, USA
Germ granules, called P granules in C. elegans, are conserved ribonucleoprotein
aggregates found in diverse species such as worms, flies, and humans. They provide
a microenvironment for post-transcriptional regulation events specific to the germline,
but their effect on the accumulations of individual transcripts in the germline is not yet
known. To address this we performed mRNA-sequencing on dissected germlines from
worms with normal P granules and compared them to germlines where P-granule
assembly was impaired by depleting 4 core P-granule components with RNAi. Our
results suggest that P granules function, in part, to regulate the onset of gametogenesis,
while also acting as part of a larger mechanism to maintain germ cell totipotency.
Poster 30
Chromatin diminution in the nematode Strongyloides papillosus
Kulkarni, Arpita; Streit, Adrian
Max Planck Institute for Developmental Biology, Tuebingen, Germany
In many taxa, sex determining systems evolve rapidly. Strongyloides spp. nematodes
form facultative, sexually reproducing free-living generations in between
parthenogenetically reproducing parasitic generations. Depending on the species,
different modes of sex determination exist. S.stercoralis (in man) and S.ratti (in rats)
employ environmentally controlled XX/XO sex determination (2n=6 in females). In
S.papillosus (in sheep) females have only two pairs of chromosomes (2n=4). In males a
sex specific chromatin diminution event eliminates an internal portion of one of the
homologous larger chromosomes resulting in a hemizygous region. This region is
evolutionarily related to the X chromosome in S. ratti. We show that Parastrongyloides
trichosuri, a facultative parasite of Australian possums and representative of a closely
related sister genus, employs XX/XO sex determination. This strongly suggests that
XX/XO sex determination is ancestral within the genus Strongyloides. The larger
chromosome in S. papillosus is likely the result of a chromosome fusion and in males
chromatin diminution functionally restores the ancestral XX/XO system.
We used a combination of quantitative genome re-sequencing and genetic analysis to
determine the genomic regions undergoing chromatin diminution in S. papillosus males.
In particular, we identified the boundaries of this event on the chromosome. Similar to
the situation in Ascaris sp., where chromatin diminution creates a difference between
the soma and the germline, the break points in S. papillosus do not show any obvious
sequence features that might be involved in the process. We are in the process of
characterizing the nature of the new chromosome ends created by chromatin
diminution.
Poster 31
Evolution of Maturin suggests a role for ubiquitination during primary
neurogenesis
Martinez-De Luna, Reyna I. (SUNY Upstate Medical University, Syracuse, NY, USA);
Freeman, Jr., Robert M. (Harvard Medical School, Boston, MA, USA); Zuber, Michael
(USA);
Maturin is a novel protein required for the proneural pathway to regulate neural
differentiation, however the molecular mechanism(s) by which it functions are unknown.
Maturin has no identifiable functional or structural motif, yet has been highly conserved
during vertebrate evolution. To infer possible molecular mechanism(s), we identified
Maturin orthologs from more evolutionarily distant organisms to identify conserved
regions and generate phylogenetic trees. In situ hybridization was used to determine
and compare the expression patterns of Maturin in select species. More than 100
vertebrate Maturin orthologs were identified. Vertebrate predicted proteins range in size
from 129 (pufferfish) to 141 (lamprey) residues. Although multiple small regions of
similarity were observed, the most highly conserved region was the Maturin Domain, a
29-residue sequence that was invariant in most vertebrates. Invertebrate Maturins were
also identified, including orthologs from cephalochordates, hemichordates, and marine
polychaetes. All identified invertebrate Maturins contained an extended C-terminus with
sequence homology to the E2 family of ubiquitin-conjugating enzymes (UBE2).
Phylogenetic analysis indicates the C-terminus extensions are most similar to the UBE2
Q subfamily. X. laevis UBE2 QL1 and Q2 are co-expressed with Maturin during neural
development and Maturin•UBE2Q transcripts were detected in the hemichordate
Saccoglossus kowalevskii during embryonic development. Our observations suggest a
model in which a single Maturin•UBE2Q gene evolved into independent Maturin and
UBE2Q genes, as well as a possible molecular mechanism by which Maturin may have
functioned in neural development for more than 500 million years.
Poster 32
And now for something not completely different: The Drosophila melanogaster
septin Sep2 is redundant with Sep5 for imaginal cell proliferation, but is required
for oogenesis.
O'Neill, Ryan; Clark, Denise
University of New Brunswick, Fredericton, NB, CAN
Septins are a family of cytoskeletal proteins that form hetero-oligomeric
complexes. Septins function in many processes including cytokinesis. Mammalian
septins are classified into subgroups based on phylogeny and interchangeability within
septin hetero-oligomeric complexes. Mammalian septin complexes always contain
septins from the SEPT2, SEPT6, and SEPT7 subgroups. Drosophila melanogaster has
five septin genes, where Sep2 and Sep5 are the only members of the SEPT6 subgroup.
Therefore, D. melanogaster septin complexes could have either Sep2 or Sep5, or both,
in septin complexes. Sep5 arose via retrotransposition of Sep2 ~62 Myr ago in the
lineage leading to D. melanogaster. We are exploring the functional diversification of
this Sep2/Sep5 duplication. Sep5 mutants appear wild type, whereas Sep2 mutant
females are semisterile. In Drosophila oogenesis, a stem cell gives rise to a cystoblast
which undergoes four rounds of division, forming a cyst with 15 nurse cells and one
oocyte. Sep2 mutant females have egg chambers with fewer or greater than 15 nurse
cells, suggesting a defect in cystoblast divisions. Expression of a Sep2 cDNA or
presence of a Sep2-GFP fusion transgene rescues this Sep2 mutant egg chamber
phenotype. However, a Sep5 cDNA only partially rescues this phenotype, showing that
Sep2 and Sep5 have diverged in protein function. Sep2 Sep5 double mutants have
early pupal lethality and lack imaginal discs, which give rise to adult structures, showing
that Sep2 and Sep5 share a redundant function in imaginal cell proliferation. We are
using the Sep2-GFP transgene to further characterize the function of Sep2 in
oogenesis, and we are exploring the possible redundant function of Sep5 during
oogenesis.
Poster 33
Luteinizing hormone signaling decreases cyclic GMP production in rat ovarian
follicles by rapidly dephosphorylating the NPR2 guanylyl cyclase through a PPP
family phosphatase
Egbert, Jeremy (University of Connecticut Health Center, Farmington, CT, USA);
Shuhaibar, Leia C. (University of Connecticut Health Center, Farmington, CT, USA);
Edmund, Aaron B. (University of Minnesota, Minneapolis, MN, USA); Robinson, Jerid
W. (University of Minnesota, Minneapolis, MN, USA); Van Helden, Dusty (University of
Minnesota, Minneapolis, MN, USA); Uliasz, Tracy F. (University of Connecticut Health
Center, Farmington, CT, USA); Baena, Valentina (University of Connecticut Health
Center, Farmington, CT, USA); Potter, Lincoln R. (University of Minnesota, Minneapolis,
MN, USA); Jaffe, Laurinda A. (University of Connecticut Health Center, Farmington, CT,
USA)
In mammals, fully-grown oocytes are maintained in meiotic arrest by the diffusion of
cGMP from the surrounding granulosa cells, where it is produced by the natriuretic
peptide binding guanylyl cyclase receptor 2 (NPR2) under the influence of the agonist
C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP levels
fall rapidly in both the granulosa cells and the oocyte, resulting in meiotic resumption.
Here we show that LH signaling rapidly dephosphorylates NPR2, reducing its guanylyl
cyclase activity. NPR2 was immunoprecipitated from rat follicle membranes and
electrophoresed on gels containing Phos-tag-acrylamide, which retards the migration of
phosphorylated proteins. NPR2 from control follicles was visualized by western blotting
as several distinct bands, indicating multiple phosphorylated species. When follicles
were exposed to LH for 10 minutes, most of the NPR2 signal shifted to a predominant
lower band, consistent with receptor dephosphorylation. These changes remained
stable for at least 4 hours after LH exposure. In parallel assays, LH reduced NPR2
guanylyl cyclase activity by ~50% with a time course similar to its dephosphorylation.
Importantly, incubation of follicles with phosphatase inhibitors specific to the PPP family
prevented both NPR2 dephosphorylation and inactivation. Follicle CNP content
remained constant during the first 2 hours after LH exposure, but decreased to ~50% by
4 hours, which would further reduce guanylyl cyclase activity. In conclusion, these
sequential events result in the decrease in intracellular cGMP that induces meiosis to
resume in the oocyte.
Poster 34
How are +TIPs and Microtubule dynamics regulated within the growth cone?
Erdogan, Burcu; Enzenbacher, Tiffany; Lowery, Laura Anne
Boston College, Chestnut Hill, MA, United States
During development, a neuron surveys the environment with the growth cone, located at
the tip of its axon. Accuracy of its path finding is essential in locating the correct target
and eventually building the functional neuronal network. By interpreting guidance cues,
the growth cone is constantly restructured by actin filaments and microtubules (MTs).
MTs are highly dynamic polymers that are continuously switching between growth and
shrinkage and are regulated by microtubule associated proteins (MAPs). Plus end
tracking proteins, +TIPs, are MAPs that localize to plus ends of MTs and they have
been shown to regulate MT dynamics, and as a result, growth cone directionality.
Our goal is to understand how +TIPs alter their behavioral and functional outputs to
affect MT regulation in response to guidance cues. We address this question by
measuring the localization dynamics of candidate +TIPs when the growth cone
encounters guidance cues. Here we conducted an in vitro stripe assay in which neural
tube explants from Xenopus laevis embryos were cultured in a sample dish in the
presence of repulsive axon guidance cue, ephrin. The relative changes in the MT
dynamics and the localization behavior of the +TIPs as the growth cones navigate
through the guidance signals were evaluated quantitatively following high-resolution live
imaging of tagged +TIPs. The behavior of the studied +TIPs was assessed by
comparison to EB1, a +TIP that binds all growing MT plus ends, behavior and used as a
MT growth tracker. In sum, our findings reveal the role of +TIPs localization in MT
dynamics as it relates to growth cone directionality due to guidance cue signals.
Poster 35
Tbx3 is required for normal eye formation in Xenopus laevis
Motahari, Zahra
SUNY Upstate Medical University, USA
Six eye field transcription factors or EFTFs (tbx3, rax, pax6, nr2e1, six3, and six6) and
otx2 are expressed in a dynamic and overlapping pattern in the eye field during its
specification. Inactivation of six of these genes results in animals with abnormal or no
eyes. The exception is tbx3, for which a null phenotype has not been reported. Although
tbx3 can regulate the expression of other EFTFs and its misexpression can alter
dorsoventral patterning of the retina at optic vesicle stages, it is not known if tbx3 is
required at early neural plate stages for eye formation. Here we present evidence, that
in the pseudotetraploid Xenopus laevis, both homeologs of tbx3 (tbx3a & tbx3b) are
expressed in the eye field. We found tbx3 is the only EFTFs sufficient to reprogram
pluripotent cells to retinal cells in vivo with high efficiency. To determine if tbx3 is
required for normal eye formation, we designed antisense morpholino oligonucleotides
to X. laevis tbx3a and tbx3b. We found that simultaneous knockdown of tbx3a and
tbx3b result in abnormal eye formation. Eye phenotype were not observed if only one of
the two tbx3 homeologs was knocked down. The BMP inhibitor Noggin can induce the
expression of the EFTFs and mimics their ability to convert pluripotent cells to retinal
cells. Interestingly, Tbx3 morpholinos are able to block the ability of Noggin to induce
retina tissue. Finally, donor eye fields from Tbx3 morpholino-injected embryos fail to
form retina when transplanted to host embryo eye fields. These results support the
conclusion that Tbx3 is required for normal eye field formation. The precise cellular and
molecular mechanisms by which Tbx3 is required for eye field and eye formation is
currently being investigated.
Poster 36
Progressive degeneration alters the regenerative capacity of the Xenopus laevis
retina
Ku, Ray Yueh (Upstate Medical University SUNY, USA); Choi, Rene; Zuber, Michael
(Syracuse, United States)
The ability to regenerate retina (the light detecting tissue of the eye) was lost during
evolution. Planarians, fish and amphibians can regenerate all retinal cell types following
damage. In contrast, chickens regenerate only a subset of cell types and only at
embryonic stages, while mammals do not regenerate retina at all. Xenopus laevis
(African clawed frog) can regenerate the entire retina in as little as 30 days after partial
or even complete retinectomy, both before and after metamorphosis. In a previous
study we demonstrated that X. laevis tadpoles regenerate rod photoreceptors following
rod-specific ablation. Given the ability of X. laevis to regenerate an entire retina, we
reasoned that the regenerative capacity of the Xenopus retina should be unaffected by
even extensive degeneration. To test this hypothesis, we maintained tadpoles from 10
to 90 days under conditions in which rod photoreceptors could not regenerated, then
allowed recovery for as long as 90 days. Cell type specific markers were used to
determine the fate and morphology of cells during both the degenerative and
regenerative phases. With prolonged degeneration, the Xenopus retina progressed
through degenerative stages similar to those observed in the mammalian retina.
Inconsistent with our hypothesis, we found that the regeneration of rod photoreceptors
was incomplete in tadpoles with extensive degeneration. Even after 90 days of
recovery, large regions of the retina still lacked rod photoreceptors. Our results suggest
that despite the ability of Xenopus to regenerate an entire retina following retinectomy,
progressive degeneration results in a condition in which regeneration cannot take place.
Poster 37
Identifying Regulatory Cofactors of the Caenorhabditis elegans Cellular Fusogen,
EFF-1, and Their Effects on Epithelial Cell Fusion
Spica, Patrick; Klempic, Emra; Shinn-Thomas, Jessica
Utica College, Utica, NY, USA
Cell-cell fusion is the process by which multinucleated cells called syncytia form from
plasma membrane fusion of cells developmentally programmed to fuse. Cell fusion is a
vital process that occurs in a wide variety of organisms. Specific mechanisms and
essential proteins for cell fusion are currently under investigation with limited
understanding of a common cell-cell fusion mechanism. Caenorhabditis elegans (C.
elegans), a free-living and self-fertilizing nematode, is an ideal model system for
studying cell-cell fusion because one-third of their somatic cells fuse to form syncytia in
an invariant and predictable spatiotemporal manner. The only currently known cellular
fusogens, proteins that fuse membranes, of non-viral origin have been identified in C.
elegans. The cellular fusogen EFF-1 in C. elegans is essential for epithelial cell fusion
but molecules that regulate it are largely unknown.
We hypothesize that EFF-1 requires cofactors for its expression, localization, and
function. This study aims to search for, define, and characterize these regulatory
molecules. We performed an enhancer modifier genetic screen using a C. elegans
strain (eff-1(oj55)) that expresses an allele of eff-1 with an intermediate loss-of-function
phenotype. We induced mutations within eff-1(oj55) animals using the mutagen, ethyl
methanesulphonate. We then screened for the “dumpy” strong loss-of-function
phenotype that can result from an increase in reduced cell fusions. Finally, we
confirmed that isolated mutants showed a decrease in cell fusions with fluorescence
imaging of epithelial cell membranes in epithelial syncytial tissues like the hypodermis.
We aim to identify and characterize these intragenic or extragenic mutations.
Poster 38
How will downgrading the SCC4 and SYN4 gene expression during meiosis of
Arabidopsis plants effect their development?
Collier, Alexis
Lincoln University, Lincoln University, PA, USA
Both Mitosis and Meiosis are fundamental biological processes crucial to the growth
and development of organisms. Mitosis is a means of growth and repair, while meiosis
is the production of gametes in sexually reproductive organisms. However, both are
processes in which cells divide to yield daughter cells. The determining factor in the
proper execution of cell division is sister chromatid cohesion, which dictates whether or
not chromosomes separate correctly into daughter cells (Ulhmann ,2004).Cohesins are
protein complexes that regulate sister chromatid cohesion during meiosis and mitosis
and are made up of four proteins, SMC1, SMC3, SCC3 and an a-kleisin protein (Liu and
Makaroff.,2006). ?-Kleisins are core components of meiotic and mitotic cohesin
complexes. Arabidopsis contains four genes that encode ?-kleisin proteins: SYN1,
SYN2, SYN3 and SYN4 (Yuan L, et al 2012). Binding of the cohesin complex is aided
by another complex that contains two proteins, SCC2 and SCC4. The objective of this
project is to analyze the effect that the downgrading of the SCC4 and SYN4 genes
during meiosis has on the growth and development of Arabidopsis plants. These results
will be compared and contrasted with results of the downgrading of the SCC4 and
SYN4 genes in Arabidopsis plants during mitosis, (These plants were grown by
previous undergraduate researchers). In addition, both the 35s plants (genes were
downgraded during mitosis) and the DMC1 plants (genes were downgraded during
meiosis) will be compared to the Arabidopsis wild type plants for their specific
phenotypes. The method used to downgrade these genes is called RNAi (RNA
interference). The goals accomplished at the end of this project were the creation of two
DMC1 plasmids, the transformation of those DMC1 plasmid into wild type via
Agrobacterium, the genotyping of the 35s plants (Because we did not transform the 35s
plasmids into the wild type plants ourselves, it was necessary to verify that they actually
contained the 35s plasmid along with two inserts of the SCC4 or SYN4 gene.), and the
comparison of the 35s plants to the wild type plants.
Poster 39
Aplip1 contributes to Kinesin- and Dynein-dependent myonuclear movement
Auld, Alexander (Boston College, Brighton, MA, USA); Eric Folker
Department of Biology, Boston College, Chestnut Hill, MA
Nuclear movement is a tightly regulated process that is conserved amongst cell types
and throughout evolution. In muscle, mispositioned nuclei can impair muscle function
and are prevalent in individuals with muscle disease. Therefore, identifying the
mechanisms of nuclear movement and positioning is crucial to understanding both
muscle development and disease. We study nuclear movement in Drosophila in which
muscle structure is similar at the cellular level to that of mammals. In Drosophila, nuclei
translocate towards the muscle ends. During this translocation, the nuclei move
directionally and are polarized with molecularly distinct leading and lagging edges. Both
the polarity of the nucleus, and the directionality of its movement require the
coordinated actions of the microtubule motor proteins Kinesin and Dynein. However, it
remains unclear how the activities of these proteins are regulated and spatially
segregated on the nuclear surface. Jnk Interacting protein 1 (JIP1) binds and regulates
the activity of both Kinesin and Dynein in neurons. We therefore tested whether Aplip1
(the Drosophila version of JIP1) regulates Kinesin and Dynein activity during nuclear
movement in muscle we found that Aplip1 is necessary for nuclear movement in
muscle. Furthermore, embryos doubly-heterozygous for Aplip1 mutations and either
Dhc64C or Khc mutations failed to properly position their myonuclei. These data
indicate that Aplip1 interacts with both Dynein and Kinesin to regulate myonuclear
position. Future experiments will determine how Aplip1 contributes to Dynein and
Kinesin dependent nuclear movement in muscle.
Poster 40
Sema6a-PlxnA2 Signaling Negatively Regulates rasl11b to Maintain Proliferation
of Retinal Precursor Cells during Zebrafish Eye Development
St. Clair, Riley; Waldron, Ashle; Ballif, Bryan; Ebert, Alicia
University of Vermont, Burlington, VT, USA
Eye development in vertebrates is a complex process involving many developmental
cues and signal transduction pathways. We have uncovered an important role for
Semaphorin6a-PlexinA2 (Sema6a-PlxnA2) signaling in maintaining cellular cohesion
and proliferation in developing zebrafish eye fields. However, the molecular
mechanisms underlying these phenotypes are unknown. Microarray analysis found that
upon morpholino-based knock-down of Sema6a or PlexinA2, there was a three-fold
increase in the expression levels of the poorly-characterized small GTPase RasL11b.
The present study used established cell lines and transgenic zebrafish to test the
hypothesis that overexpressed RasL11b has a dominant-inhibitory effect on Ras-MAPK
signaling and thereby contributes to the reduced retinal precursor cell proliferation
observed in Sema6a or PlxnA2 morphant eye fields.
Poster 41
The Role of Dual-Specific Phosphatases in Modulating Fgf Signaling in the
Developing Hindbrain
Maurer, Jennifer; Sagerstrom, Charles
UMass Medical School, Worcester, MA, USA
Early in development, the vertebrate hindbrain is transiently divided into distinct
segments called rhombomeres. Each rhombomere will give rise to a unique set of
differentiated neurons. Many genes that are expressed in rhombomere-specific patterns
have important roles in patterning the early hindbrain region. Dusp2 is a dual-specific
phosphatase expressed exclusively in rhombomere 4 (r4) between 10 and 14 hours
post-fertilization (hpf). Dusp2 targets and de-phosphorylates ERK MAP kinases, but its
only known role is in the context of the immune system. At this early time in
development, the primary source of ERK activation in r4 is the Fgf signaling pathway.
Fgf signaling is necessary for proper hindbrain patterning, as inhibition of the pathway
leads to loss of r5 and r6; however, r4 patterning is not affected. This suggests that Fgf
signaling is regulated differently in r4. We hypothesize that Dusp2, along with another
phosphatase Dusp6, regulates Fgf signaling in r4 by de-phosphorylating ERK proteins.
Using injections of morpholino oligos, we have knocked-down Dusp2 and Dusp6 in
zebrafish embryos. We then assayed for various hindbrain patterning and neuronal
markers, and found that knock-down of Dusp2 and of Dusp6 causes loss of one or both
of the Mauthner neurons. Using CRISPR-Cas9 genome editing technology, we have
created knock-out zebrafish lines for Dusp2, Dusp6, and another Fgf regulator Spry1.
We have currently identified positive founders, and hope to confirm the phenotypes
seen with the morpholino in these mutant lines. Determining how Dusp2 and Dusp6
interact with the Fgf signaling pathway will provide a deeper understanding of the
mechanisms controlling hindbrain development.
Poster 42
Roles of Histone H3K27me3 demethylases in epigenetic dynamics
Kamikawa, Yasunao; Donohoe, Mary E.
Burke Medical Research Institute/ Weill Cornell Medical College, White Plains, NY, USA
The post-translational modifications of histones mediate a variety of genomic functions
including DNA replication and RNA transcription. The tri-methylation of histone H3
lysine27 (H3K27me3) is a mark for repressed genomic loci and has pivotal roles for
normal development, especially in the maintenance of cell identities, by preventing
ectopic gene expression. Jmjd3 and Utx are the H3K27 specific demethylases and are
required for broad biological events including inflammatory responses, early
development, and inducing pluripotent state. The H3K27me3 modification is best
observed in X-chromosome inactivation (XCI), a crucial developmental process that
balances the gene dosage in mammals between XX females and XY males by
epigenetically silencing one of the two female X chromosomes. Interestingly, XCI is
coupled with the loss of pluripotency. The female embryo and embryonic stem cells
establish XCI during cellular differentiation. Once XCI is established, the entire silenced
X-chromosome is highly enriched with H3K27me3. In contrast, X-chromosome
reactivation (XCR) occurs with the removal of H3K27me3 from the inactive Xchromosome when the somatic cells are reprogrammed into pluripotent cells. These
facts suggest that H3K27me3 demethylases have pivotal role for XCI and/or XCR. We
will present our on-going studies that link the dynamic activities of the H3K27
demethylases with XCI and cellular differentiation together with XCR and pluripotency.
Poster 43
Characterizing Shh and ciliary signaling in the hop-sterile mouse
Xin, Daisy
Yale University, New Haven, CT, USA
The primary cilium is a key organelle involved in a variety of developmental processes,
including left-right specification, organogenesis, and tissue patterning. In the past
decade, work from multiple labs has identified and elucidated the function of a handful
of ciliary proteins, however many remain unknown or uncharacterized.
The hop-sterile (hop) mouse exhibits characteristics of cilia defects such as preaxial
polydactyly and male sterility. Sonic hedgehog (Shh) signaling is required to pattern the
developing neural tube and limb and this pathway is transduced through the cilium in
vertebrates. In the embryonic limb bud, we find a disruption of Shh signaling in hop
mutants. Additionally, Shh is reduced in the neural tube, resulting in a loss of ventral
neuronal progenitors. Together, these phenotypes suggested that the hop mutation
affects a key cilia gene.
Using exome sequencing, we identified the affected gene as Tetratricopeptide repeat
protein 26 (Ttc26), a known cilia protein that is prematurely truncated in hop mutants.
We find that Ttc26 no longer localizes to hop cilia despite normal localization of other
cilia markers. Ttc26 has been shown to interact with IFT88, a protein involved in ciliary
trafficking, but we see a loss of this interaction in hop that likely explains why Ttc26 is
absent from mutant cilia. Surprisingly, unlike many cilia mutants, we did not find
reduced numbers of cilia in hop. However, we saw abnormal microtubule architecture
suggesting impaired ciliogenesis. Altogether, the hop mutant is a unique and viable
mouse model that will provide insight into how Ttc26 affects cilia formation and function,
as well as overall vertebrate development.
Poster 44
Retinal cells die in the small-eyed zebrafish mutant, good effort, at the time of
neuronal birth.
Bailey, Travis; Hyde, David
University of Notre Dame, Notre Dame, IN, USA
The good effort (gef) mutant was found in a genetic screen to identify ENU-induced
mutations resulting in eye defects. The retina of gef embryos successfully develops the
optic cup and the retinoblast layer as well as few early differentiating retinal ganglion
cells, but the retinoblast layer quickly degenerates. Meiotic mapping localized the
mutation to an interval of many genes on Chromosome 9. Clones of candidate gene
mRNA transcripts were sequenced and compared to wild type. The mRNA transcripts of
candidate gene, chromosome assembly factor 1 b (chaf1b), were found to lack an early
exon. Genomic sequencing uncovered a 3 bp intronic deletion disrupting a splice donor
site was predicted to result in exon skipping and a truncated protein, the result of a
premature stop codon. Morpholino-mediated gene knock down phenocopied the gef cell
death phenotype (assayed by acridine orange or by TUNEL labeling at 2 days post
fertilization)
and
retinal
neuron
specification
defects
(assayed
by
immunohistochemistry). Transcripts of proposed downstream genes of chaf1b loss-offunction, tumor suppressor tp53 and its target gene, bax, were found to be upregulated
in gef mutant embryos compared with wild-type embryos by qRT-PCR analysis. To test
whether tp53 was required for neuronal death in the gef mutant embryos, gef mutants
were injected with tp53 morpholino at the 1-cell stage. The gef mutant and tp53
morphant embryos displayed significant cell death similar to gef mutant embryos
injected with control morpholinos at 2 days post fertilization. These data suggest that
the small eye gef mutant phenotype is due to a mutant allele of the chaf1b gene, which
results retinal cell death independent of p53.
Poster 45
The Structure and Function of Maskin/TACC3 in Axon Outgrowth
Evans MF, Faris AE, Nwagbara BU, Lowery LA
Boston College, Brighton, MA, USA
The growing axon tip of a developing neuron is called a growth cone. In order to form
the exact synaptic connection required for proper neurological function, the growth cone
hosts an orchestra of hundreds of different genes and proteins interacting with
extracellular cues to steer growth in the right direction. Mutations in genes required for
this process are associated with many neurodevelopmental disorders and deficits in
damage repair. The goal of our current research is to study one of the components of
this pathway, known as the Maskin protein in the Xenopus model organism we work
with, and homologous to TACC3 in humans. Our preliminary data shows that knocking
down this protein changes microtubule polymerization dynamics, altering the rate of
growth and steering of the axon. Maskin is also involved in synaptic LTP/LTD through
regulation of specific translation of mRNAs within the growth cone. Additionally, our
lab’s unpublished work has shown it to be one of a family known as +TIPs, protein
complexes that bind the (+) end of microtubules to regulate their behavior. Therefore we
believe Maskin/TACC3 is a key player in multiple pathways involving neural
development. Our lab is examining its presence within growing axons through the
effects of selectively knocking down and restoring its expression. Here, we present the
beginnings of a detailed structure/function analysis of Maskin in regards to its binding
and activity with other proteins in the growth cone. We are examining which domains
and specific amino acid residues mediate Maskin’s function in regulation of cytoskeletal
growth and steering in developing neurons.
Poster 46
Characterization of Maskins Role in the Microtubule Dynamics of Xenopus Laevis
Growth Cones
Ebbert, Patrick (Boston College, Chestnut Hill, MA, USA); Nwagbara, Belinda (Chestnut
Hill, USA); Faris, Anna (Chestnut Hill, USA); Lowery, Laura Anne (Chestnut Hill, USA)
The transforming acidic coiled-coil family protein maskin has been identified as an
important microtubule-associated protein, in particular an important microtubule plusend tracking protein (+TIP), in Xenopus laevis axons. We investigate the relationship
between maskin and other known +TIPs using a variety of experimental methods both
in vitro and ex vivo in order to gain a further understanding of maskin's role in the
growth cone, the site at which developing axons obtain local guidance cues to orient
outgrowth in a particular direction. Differential co-immunoprecipitation of maskin with
another known +TIP, XMAP215, seeks to reveal potential interaction between these two
proteins at various stages of neural development. Live imaging of fluorescently labeled
maskin co-localizing with various other +TIPs as well as with other cytoskeletal
elements in the growth cone hopes to provide a visual mechanism that reveals maskin's
context with these other known components. Finally, computational analysis of various
microtubule dynamics affected by maskin knockdown and over-expression seek to
determine the phenotypic role of maskin in the regulation of microtubule dynamics and
provide quantitative measures of maskin's influence in these regards. It is our
expectation that full characterization of the maskin protein will provide great insights into
the field of developmental neurobiology and allow for a greater understanding of the
mechanisms which underlie the axon guidance pathways involved in
neurodevelopmental defects and diseases.
Poster 47
BMP signaling during early development of the annelid Capitella teleta
Corbet, Michele (Worcester, MA, USA)
Bone morphogenic proteins (BMPs) are a family of signaling molecules that specify cell
fate in a concentration-dependent manner. BMPs and their antagonists function in
dorsal-ventral (D-V) axis specification and neural fate specification in vertebrates and
Drosophila. In hemichordates application of exogenous BMPs during development
dorsalizes the animals, but does not repress neural fate specification, suggesting that
BMP signaling may have played an ancestral role D-V axis specification. There is little
data on D-V axis and neural fate specification from lophotrochozoans (one of two major
protostome clades). However, in the lophotrochozoan annelid Helobdella, short-range
BMP5-8 and the antagonist Gremlin pattern the D-V axis in the prospective segmented
ectoderm. BMPs also pattern the D-V axis in the annelid Platynereis dumerilii but do not
appear to repress central nervous system development. The annelid Capitella teleta has
a clear D-V axis, with an anterior brain and a ventral nerve cord. Brain development
begins during gastrulation with the ingression of single cells from localized areas of
anterior ectoderm. Ventral nerve cord development is less well characterized, but
begins shortly after gastrulation. We assessed the role of BMPs at early cleavage
stages and during gastrulation in C. teleta. After applying exogenous recombinant
BMP4 protein at multiple time windows, we did not see clear evidence of abnormal D-V
patterning. However, recombinant BMP4 clearly affected neural development, including
formation of the brain and eyes. These findings contrast what has been observed in
other lophotrochozoans, and raise questions about the ancestral function of BMP
signaling.
Poster 48
Toddler regulates germ layer migration during vertebrate gastrulation
Norris, Megan; Pauli, Andrea; Schier, Alex
Harvard University, Cambridge, MA, USA
Gastrulation specifies and shapes the germ layers resulting in generation of the body
axis and proper organization of organ precursor cells. While germ layer induction and
later convergence and extension movements have been studied in detail, the
mechanisms that link these two processes to promote internalization and migration of
mesendoderm remain unclear. We discovered a novel peptide, Toddler/Apela, which is
highly conserved throughout vertebrates and essential in zebrafish. Using in situ
hybridization we show that reduced or excess Toddler causes defects in internalization
and subsequent migration of endodermal and mesodermal germ layers. Toddler
mutants resemble loss of the GPCR Apelin Receptors (Aplnr) and expression of Apelin,
the only previously known ligand for Aplnr, rescues toddler mutants. Our results suggest
that Toddler signals through Aplnr to promote internalization and cell migration after
germ layer induction and before dorsal convergence and extension.
Poster 49
mel-28, a key regulator of early embryonic development in C. elegans
unexpectedly links disparate cellular processes
Fernandez, Anita (Fairfield University, Fairfield, CT, USA); Mis, Emily (NYU Center for
Genomics and Systems Biology, New York, NY, USA); Lai, Allison (Fairfield University,
Fairfield, CT, USA); Mauro, Michael (Fairfield University, Fairfield, CT, USA); Quental,
Angela (Fairfield University, Fairfield, USA); Bock, Carly (Fairfield University, Fairfield,
CT, USA); Piano, Fabio (NYU Center for Genomics and Systems Biology, New York,
NY, USA)
Early embryonic development requires tight coordination of many cellular processes
including DNA replication, chromosome segregation, and the breakdown and
reformation of the nuclear envelope. In C. elegans, the ubiquitous MEL-28 protein
shuttles between the nuclear envelope and the kinetochore during the cell cycle and
has crucial roles in nuclear envelope function and chromosome segregation. Embryos
completely lacking the mel-28 gene product are inviable. Strikingly, mel-28 homozygous
mutants derived from heterozygous mothers (zygotic mutants) are rescued by maternal
protein contribution and are indistinguishable from wild-type animals. Thus MEL-28 is
required for fundamental cellular processes in the embryo but apparently dispensable
for post-embryonic development.
We hypothesized that the post-embryonic roles of mel-28 could be redundantly supplied
by other genes. To test this we performed an RNAi-based genetic interaction screen in
the mel-28 zygotic mutant background, individually disrupting ~85% of the genes in the
C. elegans genome. We found 65 genes that cause 100% sterility in mel-28 animals but
not in the wild type. In addition to expected candidates, like nuclear pore components,
we found genes with surprising roles, including dynein/dynactin function, protein sorting,
and vesicle trafficking. This study has shown that mel-28 function works in concert with
other functions to promote the functionality of the germ line in C. elegans.
Poster 50
Instructive polarization of early embryonic cells by the cadherin-catenin complex
and the RhoGAP PAC-1
Klompstra, Diana; Anderson, Dorian; Nance, Jeremy
NYU School of Medicine, New York, NY, USA
Early embryonic cells in many species polarize radially by distinguishing their contacted
and contact-free surfaces. In C. elegans, radial polarity begins at the four-cell stage,
when cell contacts restrict the PAR polarity proteins to contact-free surfaces. We
previously identified the RhoGAP PAC-1 as an upstream regulator that is required to
exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 is
recruited specifically to sites of cell contact and directs PAR protein asymmetries by
inhibiting the Rho GTPase CDC-42. How PAC-1 is able to sense where contacts are
located and localize to these sites is unknown. We identified an N-terminal fragment of
PAC-1 that is sufficient for localization to cell contacts and showed that its localization
depends on HMR-1/E-cadherin. HMP-1/α-catenin and JAC-1/p120-catenin, which
interact with the HMR-1 cytoplasmic tail, function redundantly to recruit the PAC-1 Nterminus. We identified a conserved adaptor protein that physically links the PAC-1 Nterminus to the cadherin-catenin complex. E-cadherin has a conserved role in
promoting contact-induced cell polarization, but there has been debate as to whether it
functions instructively (defining contact sites) or permissively (promoting sufficient
adhesion for other polarity regulators to operate). We show that ectopically localizing
the intracellular domain of HMR-1 to contact-free surfaces of cells recruits full-length
PAC-1 and depolarizes cells, demonstrating that HMR-1 plays an instructive role in
polarization by recruiting a symmetry-breaking polarity regulator to cell contacts.
Poster 51
In vivo characterization of genes affecting human brain development using
CRISPR/Cas9 mediated genome engineering in zebrafish
Juan P. Fernandez1, Miguel A. Moreno-Mateos1, Murat Gunel2, Antonio J. Giraldez1
1 Genetics Department, Yale University School of Medicine. New Haven, CT, USA
2 Neurosurgery, Neurobiology and Genetics Departments, Yale University School of
Medicine. New Haven, CT, USA
Primary autosomal recessive microcephaly (MCPH) is a rare neurodevelopmental
disorder in children that results in decreases in cognitive abilities and a significant
reduction in brain size, affecting 1 in 100,000 in consanguineous populations and 1 in 1
million in non-consanguineous populations. Despite considerable interest in MCPH as a
model disorder for brain development, the underlying mechanism has not been
completely established and only a few genes mutations have been reported. Using
exome capture the Gunel Laboratory has identified mutations in genes affected in
patients with MCPH, however, many of these are only present in individual families,
thus requiring independent validation of their role in brain development. In this work, we
focus our analysis in the validation of this novel genes, to determine whether the
CRISPR/Cas9 system provides a robust tool to test the function of these genes in an F0
screening. As a proof of principle, we showed that CRISPR/Cas9 mediated
mutagenesis in known developmental gene (sox32, s1pr2, ntla, tbx6, ndr and cdh1)
phenocopy the described mutants, indicating that this system can be used for F0
screening. Extending our analysis to brain-specific genes we were able to recapitulate
brain specific phenotype such as sleepy (lamc1), snakehead (atp1a1a.1) and mind
bomb (mib) directly in injected embryos. Furthermore, we observed consistent
morphological abnormalities within the brain structures in 3 out of 12 screened genes at
28 hpf. Our results lead us to conclude that CRISPR/Cas9 mediated genome
engineering efficiently targets both alleles for the genes analyzed in most cells,
providing an effective system to test gene function in vivo.
Poster 52
PAH activation of the aryl-hydrocarbon receptor and its effects on neural crest
cell development in Zebrafish
Chen, Diane; Deadwyler, Michelle C; Cho, Gina; Kesich, Lydia-Rose; Barresi, Michael
JF
Smith College, Northampton, MA, USA
On April 20th 2010, the Deepwater Horizon oil platform sank, triggering the release of
4.93 million barrels of oil into the Gulf of Mexico from 5000ft below. Concerns have
been raised about the effects of crude oil on marine flora and fauna in the Gulf
especially those exposed to such toxin during embryonic stages. Examination of native
Gulf species is difficult, but the Zebrafish provides a tractable model system to directly
test the teratogenic effects of crude oil toxins. We investigated the effects of the water
accommodated fraction (WAF) of crude oil on zebrafish embryogenesis, and found
several defects in treated embryos that included cardiovascular and craniofacial
malformation that we postulate could be the result of impaired cranial neural crest cell
development. Analysis of the DWH oil showed the presence of polycyclic aromatic
hydrocarbons (PAHs) and BTEX compounds (benzene, toluene, ethylbenzene, and
xylene). Using a candidate approach, we exposed Zebrafish to the 16 PAHs designated
priority pollutants by the EPA, and all yielded a similar disappearance of the fifth
pharyngeal arch. Previous studies have shown that PAHs activate the aryl hydrocarbon
receptor signaling pathway (AhR), which upregulates expression of xenobiotic
metabolizing enzymes. We are currently investigating whether cross talk between the
AhR pathway and canonical Wnt signaling functions as the molecular mechanism to
mediate potential neural crest malformations following exposure to crude oil toxicants.
Understanding the molecular mechanisms that mediate interactions between the
environment and embryo will provide insight into both the regulation of developmental
plasticity as well as the risks present for native fish species in the Gulf.
Poster 53
The diencephalic glial bridge represents a heterogeneous population of astroglial
cells during commissure formation in zebrafish forebrain.
Schneider, Caitlin; Sinha, Risha
Smith College, Northampton, MA, USA
Neuronal connections between the two halves of the nervous system are established by
midline crossing axons forming commissures. The embryonic zebrafish forebrain
represents a simple system to dissect the biochemical, molecular, and cellular
processes involved in commissural development. Previously, we showed that astroglial
cells establish a bridge-like structure across the forebrain midline, which may promote
axonal pathfinding in that region. Astroglial cells express Glial fibrillary acidic protein
(GFAP), an intermediate filament found in cells displaying radial glial, mesenchymal,
and multi-branching cell morphologies. In addition, the zebrafish radial fiber antibodies
(Zrf1-4) demarcate distinct domains within the diencephalic glial bridge. While Zrf1
recognizes GFAP, it is unknown what proteins Zrf 2, 3, and 4 bind. Elucidating the
antigens that the Zrf antibodies recognize could reveal important insight into astroglial
development and the construction of the brain. Currently, we are using
immunoprecipitation, Western Blot, and liquid chromatography mass spectrometry
(LCMS) to identify the proteins recognized by Zrf2-4. Lastly, we are confirming the
correlation between Zrf expression patterns and cell morphology using a combination of
astroglial
marking
transgenics,
cell
transplantation,
and
multi-labeling
immunocytochemistry. With these techniques, we are attempting to build a threedimensional map of the diencephalon. We conclude that the diencephalic glial bridge is
made of at least three different astroglial cell morphologies that exhibit differential Zrf
expression.
Poster 54
Cilia are essential for programmed cell death in limb interdigit
Shylo, Natalia; Andrade, Stephanie; Pan, Xinghua; Tanaka, Yoshiaki; Weissman,
Sherman; Weatherbee, Scott
Yale University, New Haven, CT, USA
A primary cilium boasts a diversity of roles in a developing organism. With its
involvement in left-right polarity determination, organogenesis and a number of
signaling pathways, this organelle is becoming an increasingly important target of
research. Cilia and Sonic Hedgehog (Shh) signaling are important for limb patterning
and digit identity specification. In order to separate Shh signaling from alternate
functions of the cilium, we removed cilia from the posterior region of the limb after Shhmediated patterning had been completed. Surprisingly, the interdigit tissue lacking cilia
did not regress as normal, resulting in syndactyly of posterior digits. This is the first time
that cilia have been implicated in interdigit cell death, independent of early signaling
events. Key pathways responsible for interdigit cell death include BMP, FGF and
Retinoic acid (RA) signaling. BMP and FGF targets, as well RA receptor RAR-Beta
showed no changes in expression patterns. However, MMP11, a direct target of RA in
vitro, was decreased in the affected interdigit. A closer examination of RA signaling by a
reporter gene revealed normal signal distribution, suggesting that regulation of MMP11
in the limb requires cilia, but is RA independent. We then took an unbiased approach
and performed mRNA sequencing of mutant and control interdigit tissue. We verified the
lack of changes in the BMP, FGF and RA pathways, but uncovered minor changes in
the expression of other signaling components, including the MAP kinase pathway. Our
continued research will identify the machinery that links cilia to interdigit cell death and
regulation of MMP11.
Poster 55
Endocrine disrupting alkylphenolic pollutants have juvenile hormone activity in
lobsters and bind nuclear receptors
Laufer, Hans and Chen, Ming
University of Connecticut, Storrs, CT, USA and The Marine Biological Laboratory,
Woods Hole, MA, USA
Millions of tons of bisphenol A (BPA) and other alkylphenols are produced annually as
detergents, antioxidants, or plastics, which degrade, 60% end up polluting marine
environments. They have juvenile hormone (JH) activity interfering with larval survival,
molting and metamorphosis of lobsters. Methyl farnesoate (MF), a crustacean JH also
affects reproduction. Signaling pathways for MF include nuclear receptors such as the
JH retinoid x receptor (RXR/RXR) and ecdysone receptor (EcR/RXR). Lobster RXR and
EcR proteins were expressed using in vitro transcription and translation. The RXR
complex binds to MF in a dose-dependent manner with an equilibrium dissociation
constant ( Kd ) of 2.707X10-9 M while it binds to BPA at a Kd of 10.37X10-9 M.
Competitive binding assays determined the 50% effective concentration of competitor
that binds H3- MF by 50% (EC50). Assays used RXR and/or EcR proteins, H³-MF, and
unlabeled MF, bisphenol A, and ecdysone competitors. MF and BPA bind to RXR/RXR,
the ecdysteroid receptor (ECR/RXR), and control (empty vector) proteins. BPA is more
effective in binding RXR/RXR dimer (EC50=9.6X10-8M) compared to MF
(EC50=2.9X10-7M). BPA and MF binding with control proteins were significantly lower
than receptors. BPA was more effective binding ECR/RXR heterodimer (EC50=9.5X108M) compared to MF (EC50=7.3X10-7M) and edysone (EC50=2.8X10-6M) competed
with MF when binding to ECR/RXR heterodimer. When using recombinant RXRs, H3BPA, unlabeled BPA, MF, 4-cumylphenol, BPA was more effective in binding RXR
proteins (EC50=1.158X10-8M) compared to MF (EC50=5.519X10-8M) and 4cumylphenol (EC50=3.614X10-8M). These experiments provide evidence for MF acting
through the RXR/RXR signaling pathway. They also establish a molecular basis for
developmental and reproductive signal disruption by environmental alkylphenols.
Poster 56
Defining mechanisms of imprinted expression at Igf2r/Airn during mouse
gastrulation
Marcho, Chelsea; Bevilacqua, Ariana; Veeramani, Swarna; Mager, Jesse
University of Massachusetts-Amherst, Amherst, MA, USA
Genomic imprinting is an epigenetic phenomenon resulting in differential mono-allelic
gene expression in a parent of origin manner. In the mouse genome, there are
approximately 140 imprinted genes, many of which occur in imprinted gene clusters.
DNA methylation and histone modifications have been shown to be allele specific at
imprinted loci, corresponding with imprinted expression. At the Igf2r/Airn cluster the
paternally expression non-coding RNA Airn is responsible for the silencing the paternal
alleles of Igf2r, Slc22a2, and Slc22a3 resulting in reciprocal imprinting at the locus.
Here we document changes in imprinted genes expression in a tissue-specific and timedependent manner during gastrulation. We show that in embryonic tissue prior to
gastrulation, Igf2r is biallelic and Airn is not expressed. Once gastrulation commences,
Igf2r and Airn become reciprocally expressed and imprinted.
To examine the epigenetic mechanism involved in the establishment of Igf2r/Airn
imprinting, we characterized changes in DNA methylation by bisulfite sequencing of two
differentially methylated regions (DMR) at the locus. Consistent with ES cell
differentiation models, we found spreading of DNA methylation at DMR2 during the start
of gastrulation, corresponding to the changes in expression. Additionally, expression
and binding of CTCF, a factor involved in imprinted regulation at other imprinted loci,
suggests CTCF may also play a role in imprinting Igf2r/Airn.
Our data suggests a model similar to H19/Igf2 in which DNA methylation changes
during the onset of gastrulation, blocking the binding of the methylation sensitive factor
CTCF, which allows paternal Airn expression and silencing of paternal Igf2r.
Poster 57
Kinase regulation of ADAM13 promotes cranial neural crest cell migration
Abbruzzese, Genevieve (Amherst, USA); Cousin, Helene (Amherst, USA); Alfandari,
Dominique (Amherst, USA);
The cranial neural crest (CNC) is a group of highly migratory cells that forms the face
and jaw of all vertebrates. We has previously shown that the cell surface
metalloprotease ADAM13 is essential for CNC cell migration and that one of its critical
activities is to cleave the cell adhesion molecule Cadherin-11 to release its adhesive
extracellular domain. In addition, we have also discovered a novel and essential role for
ADAM13 in gene regulation. We have shown that the ADAM13 cytoplasmic domain
(C13) is cleaved off from the membrane-bound protease and translocates into the
nucleus where it regulates the expression of multiple genes. Controlling the level of at
least one of these target genes, Calpain8-a, is critical to promote CNC cell migration. To
better understand the mechanism by which C13 functions, we have generated multiple
truncations and point mutations within the ADAM13 cytoplasmic domain in order to
assess the role of various predicted phosphorylation sites and protein interaction
domains. Our results suggest that the kinase GSK3 primes C13 for subsequent
phosphorylation by a Polo-like kinase, and that these events are required for ADAM13
function in CNC migration. These phosphorylation events are not required for controlling
the proteolytic activity of ADAM13, but instead appear to be important for the activity of
C13 in the nucleus. We have identified several interesting binding partners for ADAM13
that may be important for regulating Calpain8-a expression and will determine whether
phosphorylation mediates these binding interactions.
Poster 58
Zebrafish second heart field development relies on progenitor specification in
anterior lateral plate mesoderm and nkx2.5 function
Guner-Ataman, Burcu (Massachusetts General Hospital, Harvard Medical School,
Charlestown, MA, USA); Paffett-Lugassy, Noelle (Massachusetts General Hospital,
Harvard Medical School, Charlestown, MA, USA); Adams, Meghan S. (Massachusetts
General Hospital, Harvard Medical School, Charlestown, MA, USA); Nevis, Kathleen R.
(Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA);
Jahangiri, Leila (Massachusetts General Hospital, Harvard Medical School,
Charlestown, MA, USA); Obregon, Pablo (Massachusetts General Hospital, Harvard
Medical School, Charlestown, MA, USA); Kikuchi, Kazu (Duke University, Durham, NC,
USA); Poss, Kenneth D. (Duke University, Durham, NC, USA); Burns, Caroline E.
(Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA);
Burns, C. Geoffrey (Massachusetts General Hospital, Harvard Medical School,
Charlestown, MA, USA)
Second heart field (SHF) progenitors perform essential functions during mammalian
cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in
zebrafish expressing latent TGF-binding protein 3 (ltbp3) that exhibits several defining
characteristics of the anterior SHF in mammals. However, ltbp3transcripts are
conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF
progenitors are specified in higher vertebrates. Instead, ltbp3expression initiates at the
arterial pole of the developing heart tube. Because the mechanisms of cardiac
development are conserved evolutionarily, we hypothesized that zebrafish SHF
specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage
traced gata4+and nkx2.5+ALPM populations predicted to contain SHF progenitors,
based on evolutionary conservation of ALPM patterning. Traced cells were identified in
SHF- derived distal ventricular myocardium and in three lineages in the outflow tract
(OFT). We confirmed the extent of contributions made by ALPM nkx2.5+cells using
Kaede photoconversion. Taken together, these data demonstrate that, as in higher
vertebrates, zebrafish SHF progenitors are specified within the ALPM and express
nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and
essential role during zebrafish SHF development. Embryos injected with an
nkx2.5morpholino exhibited SHF phenotypes caused by compromised progenitor cell
proliferation. Co-injecting low doses of nkx2.5and ltbp3morpholinos revealed a genetic
interaction between these factors. Taken together, our data highlight two conserved
features of zebrafish SHF development, reveal a novel genetic relationship between
nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF
biology.
Poster 59
Differential protein expression in ascl1a -/- zebrafish intestine
Wallace, Kenneth (USA); Baral, Reshica (Clarkson University, Postdam, NY, USA); Li,
Xuanwen (University of Pennsylvania, Philadelphia, PA, USA); Grosser, Tilo (University
of Pennsylvania, Philadelphia, PA, USA)
The acheate-scute complex-like 1a (acsl1a) gene is expressed within the intestinal
epithelium prior to differentiation of secretory cells from the enterocytes. In ascl1a
mutants, we find a complete loss of all secretory cells. While we do not find expression
of ascl1a enteric neuronal precursors during their migration through the intestine, there
is also only 10% of the WT level of differentiated enteric neurons at the end of
embryogenesis. Within ascl1a mutants, the enteric neuronal precursors migrate in the
correct position and numbers up to the third day but are absent on the fourth day of
embryogenesis. To identify changes between ascl1a and mutant embryos, 5dpf ascl1a
mutant and WT intestines were dissected, and subjected to LC-MS/MS mass
specrotoscopy. Spectral counts were used to identify unique and differentially
expressed proteins. Preliminary evaluation of differentially expressed proteins suggests
cytoskeletal changes and increased oxidative stress.
Poster 60
Assessing the Role of Yin-Yang 1 in Mammalian Granulosa Cells
Haversat, Jocelyn, Chelsea Marcho and Jesse Mager
University of Massachusetts, Amherst, MA, USA
The DNA binding zinc-finger protein Yin-Yang 1 (YY1) is involved in both activating and
silencing a variety of genes through diverse mechanisms including transcription factor
interactions and histone modifications. YY1 has been previously shown to have an
essential role in the oocyte for oocyte-granulosa cell communication and expansion. In
order to determine if YY1 is also required to receive paracrine signals in the developing
follicle, we used Amhr2-Cre to conditionally delete YY1 specifically in granulosa cells.
Adult YY1 cKO mutant females are infertile and have a striking reduction in overall size
of the reproductive track (Ovaries, oviduct and uterus). No follicles (or oocytes) are
present in 3 month old cKO females, suggesting that YY1 expression in the granulosa
cells is required for initial follicle formation. Examination of newborn ovaries indicates a
high prevalence of multi-oocyte follicles, suggesting a failure of the mechanism by which
germ cell nests break down early in development. Additionally, newborn cKO ovaries
have a higher number of total oocytes but reduced numbers of primordial follicles.
These results suggest that YY1 is required on both sides (oocyte and granulosa cell) of
the paracrine signaling that occurs in the mammalian follicle.
Poster 61
Myocardin-related transcription factors mediate
mesenchymal transition during development
epicardial
epithelial-to-
Trembley, Michael1, Lissette S. Velasquez2, Corey M. Hoffman1, Karen L. de Mesy
Bentley3, and Eric M. Small1,2
Departments of 1Pharmacology and Physiology, 2Medicine, and 3Pathology and
Laboratory Medicine, Aab Cardiovascular Research Institute, University of Rochester
School of Medicine and Dentistry, Rochester, NY, USA
The epicardium is a source of multi-potent progenitor cells that envelop the heart and
contribute to various cardiac lineages through the process of epithelial-to-mesenchymal
transition (EMT). Although known upstream cues promote epicardial EMT via changes
in gene expression and actin cytoskeletal dynamics, the link between these signals and
progenitor cell motility remain unclear. Myocardin-related transcription factors (MRTF)
are primarily retained in the cytoplasm through interactions with G-actin. However, upon
cytoskeletal reorganization and G-actin depletion, nuclear MRTFs associate with serum
response factor (SRF) to drive cellular motility and contractility gene programs
reminiscent of EMT. Here, we identify the SRF/MRTF gene regulatory axis as a key
mediator of epicardial EMT. We found significant enrichment of MRTF-A and -B in the
epicardium prior to EMT. MRTFs were later expressed in a spatial and temporal
manner concurrent with EMT and epicardial-derived cell (EPDC) differentiation.
Furthermore, MRTF deletion attenuates contractile gene expression in epicardial
explants and impairs migration of EPDCs into subjacent cell layers using ex vivo
assays. Epicardial EMT and EPDC differentiation are necessary for proper coronary
vessel formation. We found that genetic ablation of MRTFs results in disrupted
coronary plexus formation, endothelial cell dysfunction, and sub-epicardial
hemorrhage. The vascular phenotype observed in MRTF-A/BepiDKO mice results in
part from the depletion of epicardial-derived coronary pericytes. These data suggest a
critical role for MRTFs in coronary vessel formation by regulating epicardial EMT and
mobilizing EPDCs.
Poster 62
DBL-1 Target Gene Regulation By SMA2, SMA-3, and SMA-4.
Madaan, Uday
Queens College, CUNY, Westbury, NY, USA
DBL-1 Target Gene Regulation By SMA-2, SMA-3, and SMA-4. Uday Madaan,
Jianghua Yin, Edlira Yzeiraj, Cathy Savage-Dunn. Department of Biology, Queens
College,
and
the
Graduate
Center,
CUNY,
Flushing,
NY
11367.
The DBL-1 ligand, a TGF-β homolog in C. elegans, is necessary for body size
regulation, mesodermal patterning, innate immunity, aging, reproductive life span, and
male tail sensory ray identity. Transcriptional response to DBL-1 relies on Smads SMA2, SMA-3, and SMA-4. Although the initial steps of the DBL-1 pathway are known, the
mechanisms of DBL-1 target gene regulation are still relatively unknown. Previously
experiments in our lab demonstrated that the hypodermis is the main tissue responsible
for body size regulation via the DBL-1 pathway. In addition, at least three cuticle
collagen genes (col-41, rol-6 and col-141) were shown to be targets of the DBL-1
pathway involved in body size regulation. We tested whether col-41 is a direct or
indirect target of the DBL-1 pathway. Via Electrophoretic mobility shift assay (EMSA) we
illustrated that col-41 is not a direct target of Smads. This was further confirmed by
SMA-3 ChIP-seq data obtained recently (M Kudron and V. Reinke, pers. comm.). In
contrast, SMA-3 binding is detected downstream of col-141. Future experiments will test
whether
col-141
is
a
direct
or
indirect
target
of
Smads.
A second set of experiments conducted to test target gene regulation by Smads led to
an interesting observation. Upon QRT-PCR analysis of dbl-1 and sma-3 mutants, we
observed that targets genes of the pathway are largely regulated in opposite directions
between the two mutants. This could have two possible implications: 1) In absence of
DBL-1, SMA-3 is regulating genes in an opposite manner; 2) DBL-1 can regulate target
genes in a SMA-3-independent manner. Further experiments need to be conducted to
confirm any speculation beginning with construction of the sma-3;dbl-1 double mutant.
Poster 63
ADAM13 Signaling in Cranial Neural Crest Migration
Abbruzzese, Genevieve; Alfandari, Dominique; Mathavan, Ketan
University of Massachusetts, Amherst, MA, USA
Formation of craniofacial structures is dependent on proper migration of cranial neural
crest (CNC) cells in the developing embryo. Cell surface disintegrin metalloproteinases
are important for this movement. Our lab has shown that knockdown of ADAM13 in
Xenopus embryos impairs CNC migration. However, this phenotype can be rescued by
the extracellular fragment of Cadherin-11, a known ADAM13 substrate. Our aim is to
elucidate the receptor and pathway utilized by this fragment (EC1-3) to stimulate CNC
migration. Preliminary in vivo results show an increase in phosphorylated Akt levels in
the CNC of embryos overexpressing EC1-3. Because Akt converges on several
signaling pathways, we performed in vitro experiments to test if EC1-3 is able to bind to
growth factor receptors, including FGF and EGF/ErbB receptors. Early data shows the
fragment binding to FGFR1 and ErbB4 homodimers. We will explore if these
interactions occur in vivo and if they are responsible for an increase in phospho-Akt and
the promigratory behavior of CNC cells.
In addition to its proteolytic activity, ADAM13 is able to regulate gene expression via its
C-terminus. The cytosolic tail of ADAM13 (C13) can be cleaved from its transmembrane
counterpart and translocate to the nucleus. During this time, C13 may interact with a
number of proteins to control gene expression. A proteomic study of ADAM13
completed by our lab has revealed a number of potential binding partners. Of these is
PA2G4, a cytosolic protein that is capable of transcriptional and translational regulation.
Confirming our proteomics data, we have shown that C13 as well as the cytoplasmic
tails of ADAM9 and ADAM19 are able to bind PA2G4. We will confirm these interactions
in vivo and will determine the functional relevance of these interactions. Exploring the
interactions of PA2G4 and EC1-3 with ADAM13 and cell receptors, respectively, will
provide us with a deeper understanding of how ADAM13 is involved in CNC migration.
Poster 64
Identification of two distinct liver bud populations that differentially contribute to
the early liver lobes.
Rhee, Siyeon; Wang, Jikui (Amherst, USA); Palaria, Amrita; Ortiz-Pineda, Pablo;
Tremblay, Kimberly
University of Massachusetts, Amherst, MA, USA
Understanding how distinct cell types arise from multipotent progenitor cells is a major
question in stem cell biology. During murine liver development, the liver bud arises at
E8.5 from the foregut and invades the septum transversum beginning at E9.0. Here we
present detailed morphological analysis of liver development between E9.0-10.5. The
histological analysis 1) reveals two distinct migratory population from the liver bud, an
anterior liver bud and a posterior liver bud that will form the lower and upper liver lobes
respectively and 2) suggests that there may be two different inducing sources.
Interestingly, recent experimental data from our lab supports the hypothesis that these
two migratory populations are induced by different mesenchymal sources. Embryos
treated with FGF inhibitors primarily affects the anterior liver bud, while embryos treated
with a BMP inhibitor primarily affects the posterior liver bud. The data presented herein
identifies key morphogenetic events that can now be used to further explore mutant
phenotypes and suggests novel information that can be used to better understand the
endogenous processes governing normal liver bud development and growth.
Poster 65
RNAseq of the two endogenous liver precursor populations reveals verifiable
differences in gene expression
Ortiz-Pineda, Pablo A.; Ray, Abigail; Mager; Tremblay, Kimberly
University of Massachusetts, Amherst, MA, USA
The E8.25 murine definitive endoderm is a single cell epithelial layer that comprises the
precursor cells of the entire gut tube and associated digestive and respiratory organs.
The liver bud arises at ~E8.75 from the union of three discrete cell populations that have
been identified within the E8.25 endodermal sheet: two bilaterally symmetric “lateral
precursors” (LP) and a small cluster of cells in the ventral midline of the endoderm lip
(VMEL). The LP produces the bulk of the liver bud while the VMEL contributes to the
anterior-most portion of the liver bud. We reasoned that the differences in liver/gut
contribution exhibited by these two populations are supported by molecular differences.
To test the hypothesis that these two liver progenitors are functionally discrete and to
better understand the molecular mechanisms supporting liver specification, we created
transcriptional profiles from cells isolated from each progenitor population. We pooled
cDNA from validated single cells from each of the two liver precursors and used them to
obtain a high-resolution map of the full transcriptome. As expected, we found that over
75% of the most abundant genes are similarly expressed in each population under a
threshold of absolute score >5. By comparison, the remaining 24% (880 genes) were
differential expressed (LP=504; VMEL=376). We have validated more than 90 genes
from the ~300 displaying the most differential expression using RT & qPCR and got
over 91% correlation with the RNAseq data. In addition to uncovering the functional
differences between the two liver precursor populations a goal of this study is to isolate
a gene or genes that can be used to genetically identify the two (or three) liver precursor
populations.
Poster 66
Coordinating adhesion and signaling: Regulation of β1 integrin and β-catenin by
ADAM13 and MDC13 metalloproteases
Advani, Siddheshwari and Alfandari, Dominique
University of Massachusetts, Amherst, MA, USA
Adhesion and signaling are precisely and simultaneously controlled during
morphogenesis to produce tissues and organs with a shape and organization
compatible with their function. This process is critical for embryo development and
inefficient control can result in cancer. Thus understanding such proteins that control
adhesion and signaling is doubly important. One such family of proteins is the ADAMs
(A Disintegrin And Metalloprotease), a family of transmembrane zinc metalloproteases.
They have been implicated in fertilization, neurogenesis, myogenesis, and neural crest
cell migration. The disintegrin domain of ADAMs permit binding to integrins to increase
adhesion, while the metalloprotease activity induce cleavage of cell adhesion and
signaling molecules. ADAM13 is essential for the migration of cranial neural crest (CNC)
cells in Xenopus laevis. ADAM13-mediated cleavage of cell-cell adhesion molecules
such as Cadherin-11, paraxial protocadherin, and extracellular matrix proteins such as
Fibronectin facilitates invasion and migration of the CNC to give rise to craniofacial
structures. The loss of ADAM13 and its paralogue, MDC13 leads to a significant
developmental delay prior to CNC migration. These embryos show a decrease in β1
integrin, the major fibronectin receptor, and β-catenin, the downstream effector of the
Wnt signaling pathway. The decrease in β-catenin leads to decreased transcription of
downstream target genes. Moreover, β-catenin is decreased in the dorsal blastopore lip,
a key embryonic signaling center. We hypothesize that the loss of ADAM13 and its
paralogue MDC13 leads to delays in migration due to the lack of coordination between
adhesion and signaling in the embryos.
Poster 67
The Role of TGF-beta and Insulin Signaling Pathways in Lipid Metabolism
Clark, James; Almonte, Vanessa, Savage-Dunn, Cathy
Queens College, CUNY, Flushing, NY, USA
Transforming Growth Factor -beta (TGF-β) is a large family of peptides that control cell
functions such as differentiation, proliferation, and regulation of the immune system.
Misregulation of TGF-β has been implicated with a number of diseases. Understanding
the pathways that lead to such disorders is integral to finding potential targets for drug
therapy. Research has shown that DBL-1, a ligand in the TGF-β super family, plays a
major role in body size regulation in C. elegans. A microarray analysis of genes
regulated by the DBL-1 pathway has also identified a number of genes related to insulin
signaling and fat metabolism. Insulin involvement in fat metabolism is well documented.
However, a function of insulin in growth regulation of C. elegans has not been
elucidated. Our goal is to explore the cross talk between TGF-β and Insulin in growth
regulation. Two target genes from the microarray, fat-6 and fat-7, Δ-9 fatty acid
desaturases, were chosen for study, in addition to dbl-1, lon-2, an inhibitor of dbl-1, and
daf-2, an insulin receptor. Oil Red-O, a lipophilic dye, was used to observe the level of
lipid storage in the organisms. It was observed that the single mutants, dbl-1, fat-5, fat6, and fat-7, and the double mutants, fat-5;fat-6 and fat-5;fat-7, have less fat than
N2. We then determined mitochondrial DNA content by qPCR. Mitochondrial genes in
the sma-3 and fat-6;fat-7 mutants were upregulated 2.8- and 2.3-fold, respectively. We
hypothesize that decreased fat storage could be due to increased β-oxidation in
mitochondria. We will next use cold shock to determine how the pathways affect
mitochondrial activity under stress. Finally, body size was measured during
development to understand effects on overall growth. The fat-6 and fat-7 strains were
paradoxically observed to be longer than N2, but this may be due to compensating
increases in expression of the unmutated homolog. The goal is to understand how
alterations in these signaling pathways affect an organism’s development and growth.
Poster 68
Connexin43 phosphorylation changes during lactation
Norris, Rachael (Fred Hutchinson Cancer Res Ctr, WA, USA); Lampe, Paul (Seattle,
WA, United States)
During lactation, cellular junctions between the epithelial cells of the mammary gland
help coordinate the secretion and ejection of milk. Connexin43 (Cx43), a gap junction
protein, plays a crucial role in lactation, based on evidence that different connexin
isoforms cannot fully replace its function. The C-terminal tail of Cx43 is likely important
for this process because it contains several regulatory phosphorylation sites that affect
gap junction turnover, pore permeability, and protein interactions. In support of this idea,
the phosphorylation pattern of Cx43 in mammary tissue changes within hours after
parturition; however, it is unknown which phosphorylation sites are modulated.
To gain a better understanding of how Cx43 helps coordinate milk ejection, we used
phospho-specific antibodies to investigate which regulatory sites are changed during
lactation. We found that phosphorylation increases on casein kinase 1 regulated sites
S325/328/330, which are involved in gap junction assembly. Additionally, while Cx43
has been reported to be expressed primarily in myoepithelial cells, we found that it is
also expressed in both alveolar and ductal luminal cells. In fact, Cx43 gap junctions are
adjacent to, but separate from Cx26 junctions, and a subset of Cx43 in alveolar cells is
phosphorylated on S365 and co-localizes with the scaffolding protein ZO-1. Based on
these findings, we have developed a model to explain how phosphorylation of Cx43 and
its expression in both mammary epithelial cell types can account for its role in lactation.
Poster 69
Identify the role of P-domain in autoinhibition of Drosophila E(spl)-M8
Bandyopadhyay, Mohna
West Virginia University, Morgantown, USA
The Notch signaling pathway plays an important role in the stereotypical patterning of
the Drosophila sensory organs. In the developing retina, inhibitory Notch signaling is
required for specification of founding R8 cells. During this process Atonal activity is
antagonized by E(spl)-M8 repressor. The ability of E(spl)-M8 to antagonize Atonal
during R8 patterning requires phosphorylation by protein kinase CK2. An autoinhibition
model suggests that the C-terminal domain (CtD) of M8 mediates a ‘cis’ interaction with
the residues of bHLH through Orange domain, a region that is required for interaction
with Atonal. Phosphorylation by CK2 appears necessary but not sufficient to displace
this ‘cis’ interaction and turn M8 into an active repressor. The presence of highly
conserved consensus site for MAPK proximal to the CK2 site raises the possibility that
multi-site phosphorylation controls M8 activity. Here we provide some preliminary
results that suggest that these stretch of specific Ser residues are included in the
phosphorylation domain (P-domain) of the CtD. Our studies with deletion mutants
would reveal the role of the P-domain in mediating auto-inhibition and will provide new
insight into the phosphorylation dependent regulation of the bHLH repressors, which are
conserved across taxa.
Poster 70
Modulation of FOXD3 Activity in Human Embryonic Stem Cells Directs
Pluripotency and Paraxial Mesoderm Fates
Arduini, Brigitte (Rensselaer Polytechnic Institute, Troy, NY, USA); Brivanlou, Ali (The
Rockefeller University, New York, NY, USA);
Transcription factor Foxd3 has been described as a key member of the pluripotency
network in mice as well as being involved in the formation of many critical vertebrate cell
types in vivo. Yet comparatively little is known about the roles of FOXD3 in human
development. We describe FOXD3 expression in human embryonic stem cells (hESCs)
and in a subset of mouse paraxial mesoderm derivatives. Further, using transgenic
hESC lines, we find that increasing FOXD3 activity in hESCs is sufficient for rapid and
specific generation of mesenchymal cell types of the paraxial mesoderm, even under
pluripotency maintenance conditions. Gene expression diagnostic of chondroblasts,
skeletal myoblasts, osteoblasts and adipoblasts is observed within 48 hours of FOXD3
induction, as are morphological and genetic hallmarks of epithelial-to-mesenchymal
transition. FOXD3-overexpressing cells can be maintained for several passages, while
downregulation of the transgene leads to further differentiation. Preliminary data
suggest that while changing the exogenous signaling environment does not alter the
experimental outcome, the duration of FOXD3 overexpression has a significant impact
on the range of cell fates generated. Finally, FOXD3 loss-of-function also leads to
differentiation, toward endoderm and mesoderm. Taken together, these data indicate
that a balance of FOXD3 activity is required to maintain pluripotency.
Poster 71
Wnt and FGF regulate epithelial to mesenchymal transition during mesoderm
formation in the tailbud
Goto, Hana and Martin, Benjamin
Stony Brook University, Stony Brook, NY, USA
Gastrulation is a process in which cells within an embryo undergo dramatic cellular
reorganization to establish three primary germ layers (ectoderm, mesoderm, and
endoderm), which will later differentiate into various tissues in the course of
development. During mesoderm formation, cells of the epiblast undergo changes in
their cellular state from epithelial to mesenchymal in nature as these cells join the
deeper hypoblast layer. This change in cellular state is known as epithelialmesenchymal transition (EMT). EMT has been shown to be a requirement for
mesoderm establishment during gastrulation. Recent mouse and zebrafish studies have
revealed the presence of axial stem cell population in the growing posterior end of an
embryo, known as the tailbud. This group of stem cells continues to make cellular fate
decisions whether to join the neural ectoderm or paraxial mesoderm even after
gastrulation has concluded. As the embryo grows from anterior to posterior, stem cells
that become committed to join the paraxial mesoderm ultimately give rise to specialized
tissues such as the cartilage and the skeletal muscle. To determine if EMT is involved in
the formation of paraxial mesoderm from stem cells residing in the tailbud, we looked for
expressions of genes that are known to be involved in the EMT process. Our analyses
of gene expressions, which included two newly identified vimentin-like genes, suggest
that EMT does occur during mesoderm formation in the tailbud. In addition, we show
Wnt signaling is responsible for initiating the EMT event, while FGF signaling has a role
in the termination of the event for mesoderm formation.
Poster 72
A web of genes necessary for the identity or specification of the gonadal sheath
Vallier, Laura
Hofstra University, Hempstead, NY, USA
The development of a tissue requires the expression of a complex of genes in the
correct temporal and spatial manner. In the model organism, Caenorhabditis elegans,
the gonadal sheath is a tissue composed of five pairs of cells, which surrounds the
proximal two-thirds of each hermaphrodite gonad arm. The gonadal sheath has four
crucial functions important for fertility: 1) maintenance of the continually dividing mitotic
stem cell pool, 2) exit from pachytene, 3) ovulation into the spermatheca and 4)
promoting male germ cell fate. Gonadal sheath pair 1 (Sh1) is located distally and Sh5
is located proximally, abutted to the spermatheca. Sh3, 4, and 5 are necessary for
ovulation into the spermatheca and have a more dense filament network than do Sh1
and 2; Sh1 is necessary for the establishment of the continually dividing stem cell
pool. Complete loss of the sheath results in sterility. Approximately 200 genes have
been localized to the gonadal sheath; however, few of these have give clues as to the
signals necessary or important for the establishment of identity or maintenance of these
cells.
We are using the gonadal sheath to identify gene products that either give rise to the
sheath or establish and maintain its identity. Using the tnIs6 [lim-7::GFP] transgene (gift
of David Greenstein) that marks the gonadal sheath, we are utilizing an RNAi feeding
library to search for genes whose products are necessary for the presence of the
sheath. Candidate genes are those that, after feeding the dsRNA corresponding to that
gene, result in hermaphrodites that do not fluoresce green (no sheath) and that are
sterile or have only a few progeny. After screening chromosomes I and II in the library,
we have identified 40 candidate genes, of which the largest class comprises those in
metabolism, transcription and translation processes; among other broad categories
recovered were genes necessary for signaling and transport. The usefulness of this
approach in uncovering new and relevant candidate genes lies in the fact that of these
40 candidate genes, only smgl-1 overlaps with previously defined genes related to the
gonadal sheath. In addition approximately one half of the remaining 39 candidate
genes are predicted to interact with genes that are known to localize to the gonadal
sheath.
Poster 73
Slit1a positively promotes post-optic commissure formation in zebrafish
Antoine, Abigail and Park, Jin Sook
Smith College, Northampton, MA, USA
Communication between the two sides of the body requires properly positioned
commissures that cross the midline of the central nervous system. Slit-Roundabout
(Robo) signaling is an important guidance mechanism in commissure formation. In most
contexts, Slit-Robo signaling functions to repel pathfinding axons to prevent growth
cone movement into inappropriate regions. However in the zebrafish forebrain, we
propose that Slit1a functions in a novel way to positively promote axon-astroglial
interactions supporting postoptic commissure (POC) formation.
We show that the cells of the “diencephalic glial bridge” express slit1a and global
misexpression of Slit1a causes ectopic wandering POC axons. We and others have
shown that POC formation is greatly reduced in the you-too (Gli2DR) mutant, which is in
part due to the expanded midline expression of the known repellents slit2 and slit3.
Global misexpression of slit1a in the you-too mutant can rescue POC midline crossing.
We have uniquely applied the methods of Geographical Information Systems to quantify
the phenotypes associated with axonal and astroglial cell positioning. Our results
suggest that slit1a functions distinctly from slit2/slit3 to positively influence midline
crossing of POC axons. Importantly, local misexpression of Slit1a is capable of causing
POC axon wandering off of the glial bridge and toward Slit1a expressing cells. this
Slit1a function.We propose a working model in which a balance of Slit1a positive
guidance verse Slit2/3 mediated repulsion regulates midline crossing of POC axons. We
hypothesize that Slit1a-Robo signaling may provide a system of contact attraction
between axon and astroglial cell.
Poster 74
Introductory Biology Research in a High School Setting
Fields, Melanie
Sidwell Friends School, Washington, DC, USA
At Sidwell Friends School, student research is encouraged through the time that they
are in high school. Rising 9th graders have a choice between taking Biology 1 or an
accelerated course called Biology 1A. In addition, students will be given the opportunity
for further research through a neuroscience class in the next academic year. In Bio 1A,
the students spend a significant portion of their academic year planning, designing and
conducting their Independent Research Projects (IRP’s). These IRPs provide students
the opportunity to pursue a practical approach to investigative learning. Students read
the scientific literature, consult with scientists and design a protocol for their IRP. By
working with scientists, students are able to refine and polish their experiments
extensively.This year, students conducted various experiments on the development and
behaviors of zebrafish (Danio rerio). Such experiments range from testing the effects of
nicotine on their learning abilities to a study of electromagnetic fields on young and adult
zebrafish. One group is testing to see shoaling preferences with respect to numbers of
fish, and another is observing the effects of a colored environment on the pigmentation
of zebrafish. Another group is testing the effects of acetone on embryonic development.
The opportunity for young scientists to design and conduct research is an incredible
learning tool that helps bring their biology education to the next level.
Poster 75
Extracurricular Biological Research through Independent Investigations
Fields, Melanie
Sidwell Friends School, Washington, DC, USA
At Sidwell Friends School, high school biology research beyond 9th grade projects is
facilitated through the BRAIN (Biological Research and Investigations in Neuroscience)
Club. The club provides upperclassman with the opportunity to conduct extra-curricular
experiments and promote their interest in neuroscience. Club members are given the
chance to conduct research both in and outside of our lab at Sidwell, as well as through
collaborations with the Mayo Clinic, Georgetown University, and the National Institutes
of Health. These collaborations in past years have led to projects such as zebrafish
shoaling behavior studies, an experiment on the regeneration of hair cells in zebrafish, a
study on the addictive effects of methamphetamines in rats. The opportunity to work
with professional scientists allows club members the opportunity to further refine their
experiments and improve their science education. The BRAIN Club has also created a
program called Sci-Fins, which brings elementary school students from the Cesar
Chavez Public Charter Schools in DC to Sidwell every Tuesday to learn basic concepts
in biology.In addition, BRAIN Club students mentor ninth graders throughout their
research projects. Students will be given the opportunity for further research through a
neuroscience class starting up in the next academic year.
Poster 76
Autophagy, Melanoma and Zebrafish
Fields, Melanie
Sidwell Friends School, Washington, DC, USA
Melanoma is a fatal form of skin cancer with increasing incidence around the world.
Because of its aggressive nature and lack of effective treatments, research into the
molecular mechanisms is vital. New therapeutic strategies need to be developed. This
research used tumors isolated from genetically-engineered zebrafish that produce the
mutated form of human BRAF gene and are deficient in the p53 gene. Then we used a
cell line derived from human melanomas. Testing across species is valuable in
determining whether autophagy is an important phenomenon in melanoma. Autophagy,
a mechanism that allows digestion of unwanted organelles and proteins, can be
triggered when cancer cells employ it as a stress survival mechanism, or when exposed
to cancer drugs. This study used chloroquine (CQ), an inexpensive anti-malarial drug
that can inhibit autophagy, to determine if there is a role for autophagy in the survival of
melanoma cells. Initially, we extracted proteins from melanoma tumors from the
BRAF/p53 zebrafish, and ran a Western blot. The control was incubated in primary
antibody (AB) actin; the experimental was incubated in AB LC3. By calculating the
density of the LC3 signal of wild-type samples and melanoma samples, normalized to
the actin control, we found that the melanoma samples exhibit more autophagy than the
wild-type samples (insert ratio here). We then treated a human melanoma-derived cell
line, Mel501, with CQ, to determine if it has an effect on the process of autophagy. We
ran a Western blot with proteins from Mel501, and incubated in LC3 and actin. If
successful, this could lead to a new therapeutic strategy for melanoma by suppressing
autophagy.
Poster 77
Nucleoporin 188 affects left-right patterning in Xenopus tropicalis
del Viso, Florencia, Jordan Myers, Martina Brueckner, Patrick Lusk and Mustafa
Khokha
Yale School of Medicine, New Haven, CT, USA
Congenital Heart Disease (CHD) is the most common major birth defect in children,
affecting approximately 1 in 130 births. Heterotaxy (Htx) is a severe form of this
disease, characterized by abnormal formation of the embryonic left-right (LR) axis. In
patients with Htx we have identified several gene mutations that are totally novel to leftright patterning (1). One of these is the nucleoporin 188 (Nup188) which is part of the
second major structural unit of the nuclear pore complex (NPC). Recently, it was
demonstrated that Nup188 controls passage of membrane proteins across the nuclear
pore in vitro (2). Mutations in this gene in human patients are a possible cause of Htx.
To understand how this gene affects LR patterning we knockdown Nup188 using an
antisense morpholino (MO). Loss of function of Nup188 specifically affected LR
patterning and cell signaling. A better understanding of the mechanisms regulating LR
patterning will help to explain the causes of Htx, a severe disease of children.
1. Fakhro KA et al. Rare copy number variations in congenital heart disease patients identify unique
genes in left-right patterning. Proc Natl Acad Sci USA. 108(7):2915-20 (2011).
2. Theerthagiri G et al. The nucleoporin Nup188 controls passage of membrane proteins across the
nuclear pore complex. J. Cell Biol. 189(7):1129-42 (2010).
Poster 78
Wnt signaling and Sox2 regulate the contribution of midline progenitors to the
notochord and floor plate of zebrafish embryos
Row, Richard and Martin, Benjamin
Stony Brook University, Stony Brook, NY, USA
The notochord and floor plate are crucial signaling centers during early embryogenesis.
In vertebrate embryos the notochord runs through the center of the trunk and tail and
provides structural support, in addition to producing the morphogen Sonic Hedgehog.
The floor plate of the neural tube consists of the ventral-most cells of that structure and
participates in neural tube patterning and axonal pathfinding. As the body elongates
during segmentation stages a pool of midline progenitor cells contributes to both of
these tissues; these progenitors are maintained immediately dorsal to the posterior end
of the notochord through the end of segmentation. Previous studies have shown the
effects of extracellular signals on these progenitors during gastrulation but it is unknown
whether they retain plasticity after gastrulation completes. We use transgenic zebrafish
to cell autonomously activate or block different signaling genes, including Wnt signaling
and sox2. The power of this approach lies in the fact that the transgenes are inducible
by heat shock, so embryogenesis can proceed normally until the stage of interest. We
also take advantage of the optical transparency of zebrafish embryos and the ability to
generate chimeric embryos by cell transplantation. Transgenic cells can be transplanted
into wild-type embryos such that they contribute to the midline progenitor pool, and
signaling pathways can subsequently be manipulated by inducing expression of the
transgene. Wnt signaling has already been shown to be crucial in patterning a different
population of progenitor cells in the extreme posterior of zebrafish embryos. When Wnt
signals are blocked in those cells they adopt only neural fates instead of contributing to
both neural and somitic tissues. We demonstrate that midline progenitors retain the
ability to adopt either fate through the end of segmentation, and that Wnt signaling is
required for the notochord fate. We find that a crucial function of Wnt signals is to
repress production of the transcription factor Sox2, which we show specifies the neural
(floor plate) fate in these cells.
Poster 79
Lineage Allocation in the Mammalian Embryo: Defining a transcriptional circuitry
Donohoe, Mary and Pinto, Hugo
Burke Medical Research Institute, White Plains, NY, USA
Regenerative medicine holds great promise for replacing or regenerating damaged
tissues or organs. Embryonic stem cells (ESC) and induced pluripotent stem cells (IPS)
have the potential to differentiate into all the cell types of an adult and thus have great
potential for replacement cells in disease. A major quest in regenerative medicine is
elucidating the mechanisms directing cellular differentiation and de-differentiation
(epigenetic reprogramming). A common transcriptional circuitry orchestrates these
cellular decisions in human and mouse systems. Here we investigate the transcriptional
circuitry underlying a fundamental binary choice cells need to make: namely, the
decision to attain pluripotency versus one to differentiate. Understanding the
mechanisms of cell lineage specification is imperative to utilize the unlimited potential of
these stem cells. The knowledge of how lineage allocation takes place can serve as a
reference to unravel general mechanisms of similar processes during development as
well as mechanisms of cell transformation and cancer.
Poster 80
Elucidating the role of S6-Kinase in cell fate specification using the C. elegans
germ line
Roy, Debasmita and Hubbard, E. Jane
NYU School of Medicine, New York, NY, USA
Proper balance of proliferation and differentiation of stem/progenitor cells is crucial for
normal development as well as tissue homeostasis. We are using development of the
C. elegans germ line as a model system to study this balance.
We (Korta et al. 2012) established that the C. elegans ortholog of the Target of
Rapamycin (TOR) pathway components: TOR, p-70-S6-Kinase (S6K), and eukaryotic
initiation factor-4E (eIF4E) promote proper accumulation of germline progenitors during
larval development. Concomitant loss of S6K and eIF4E reduces the number of
progenitors more severely than either single mutant, and this reduction is comparable to
depletion of TOR. In addition to regulating the accumulation of germline progenitors,
loss of S6K results in cell cycle as well as cell fate defects. The activity of a Notch
signaling pathway promotes the proliferative cell fate and/or inhibits differentiation of C.
elegans germ cells. We showed that the loss of S6K both enhances differentiation
defects associated with reduction of Notch (Notch(rf)) and suppresses defects
associated with elevated Notch.
We are further investigating the role of S6K in cell fate specification. First, we
determined that similar to S6K’s role as a regulator of germ cell accumulation, it also
acts germline-autonomously and requires phosphorylation by TOR to specify cell fate.
Second, we show that unlike Notch(rf) S6K(null), Notch(rf) eIF4E(null) retains a
progenitor pool, albeit smaller than either single mutant. Similar results are seen when
Notch(rf) animals are subject to germline-restricted TOR RNAi. These data are
consistent with the canonical model that TOR signaling promotes cell cycle progression
but suggests that S6K has an additional role in preventing differentiation in conjunction
with Notch.
To determine how S6K prevents differentiation, we tested other known enhancers of
Notch(rf): Cyclin-E and MAPK (Fox et al. 2011, Lee et al. 2007). To investigate if either
of these acts in a simple linear pathway with S6K to regulate cell fate we used germlinerestricted RNAi to deplete them in Notch(rf) S6K(null) mutants. Our preliminary data
suggest that neither Cyclin-E nor MAPK acts linearly with S6K to regulate cell fate.
Furthermore, we are taking both unbiased forward genetic and biochemical approaches
identify the mechanism by which S6K regulates germ cell fate.
Poster 81
Role of Ubiquitin in Self vs. Non-Self Reactions in M. prolifera
Degani, Rinat and Faszewski, Ellen
Mount Holyoke College, South Hadley, MA, USA
Microciona prolifera, the red beard sponge, is able to distinguish self vs non-self when
an individual comes into contact with a non-self entity (allograft). When this occurs, a
zone of contact (ZOC) forms between the two individuals. Previous research using
caspase-3, a marker of programmed cell death (apoptosis), has identified the role of
apoptosis in this immune response. The purpose of this project was to examine if the
localization of another apoptotic marker, ubiquitin, was present in the sponge during this
immune response. To do this, we prepared cross-sections from sponge allografts fixed
at 2hr, 4hr, 6 hr and 8hr periods and stained for the presence of ubiquitin. Preliminary
results indicate that while although ubiquitin staining was observed in some cells in the
sponge, no marked reactions were seen in the ZOC. These results indicate that while
ubiquitin is present in M. prolifera, its limited presence in the ZOC suggests a minimal
role of ubiquitin in self vs. nonself recognition.
Poster 82
Novel endosomal mechanisms in human axonal growth mediated by Christianson
syndrome protein NHE6
Morrow, Eric
Brown University, Providence, RI, USA
The endosomal Na+/H+ exchanger 6 (NHE6) and NHE9 have general significance to
autism because genes for these exchangers are misregulated in postmortem autism
brain. NHE6 is downregulated and NHE9 is upregulated in postmortem autism brain
(Schwede et al., 2013). Also loss-of-function mutations are found in NHE6 in a novel,
neurologic disorder called Christianson syndrome (CS) which is among the most
common X-linked developmental brain disorder. In mouse, we have also demonstrated
that NHE6 null neurons demonstrate attenuated endosomal TrkB signaling, and defects
in neuronal arborization and synapse development (Ouyang et al., 2013). Objective: We
hypothesize that NHE6-associated early endosomes contribute to human axonal
growth, and that reduced NHE6 levels (as associated with autism or null mutations in
CS) contribute to abnormalities in long-range axonal growth and development. Methods
and Results: We are studying axonal growth in neurons differentiated from induced
pluripotent stem cells (iPSCs) derived from patients with mutations in NHE6. We have
established iPSC lines from CS patients with six distinct mutations in NHE6 and we are
using lines from the patients' unaffected brothers as controls. Interestingly, as in
postmortem autism brain, in iPSCs wherein NHE6 gene expression is reduced, NHE9 is
upregulated. This observation indicates that the in vitro stem cell model recapitulates
processes observed in neurologic disease in vivo. We have developed protocols to
differentiate these iPSCs into cells with properties similar to deep-layer, frontal cortical
pyramidal projection neurons, that stain positive for both Tbr1 and CTIP2. These human
neurons differentiated from iPSCs demonstrate that NHE6-associated vesicles stain for
Rab5, consistent with NHE6 localization on early endosomes. Using live-cell imaging in
these human neurons, we observe that the lumen of early endosomes is over-acidified
as compared to control (pH=6.3 in control; and pH=5.3 in NHE6 mutant lines,
p<0.0038). NHE6 mutant neurons also show deficiency in axon growth. After 27 days of
neuronal induction, axon length in NHE6 mutant pyramidal neurons is 81.1um as
compared to 122.2um in control (p<0.02). Conclusions: Our study is novel as we are
studying axon growth in human cells with properties of cortical projection neurons
derived from patients. We demonstrate that neurons with genetic mutations in NHE6
derived from patients with CS have reduced axon length. These data therefore support
a model wherein CS and other autism-related disorders may result in part from
abnormalities in long-range connectivity.
Ouyang, Q., Lizarraga, S.B., Schmidt, M., Yang, U., Gong, J., Ellisor, D., Kauer, J.A., and Morrow, E.M. (2013).
Christianson Syndrome Protein NHE6 Modulates TrkB Endosomal Signaling Required for Neuronal Circuit
Development. Neuron 80, 97-112.
Schwede, M., Garbett, K., Mirnics, K., Geschwind, D.H., and Morrow, E.M. (2013). Genes for endosomal NHE6 and
NHE9 are misregulated in autism brains. Mol Psychiatry.
Poster 83
New approaches to the investigation of matrix remodeling during vertebrate
development.
Crawford, Bryan
University of New Brunswick, Canada.
The extracellular matrix (ECM) comprises the majority of most animal tissues by mass,
and it provides structural integrity and other important mechanical characteristics, as
well as essential biochemical functions in cell adhesion, signal transduction, and other
molecular mechanisms. But in order to grow and change shape during embryonic
development, regeneration or wound-healing, the ECM must be broken down and
remodeled by a large and complex family of zinc-dependent proteases, known as the
Matrix Metalloproteinases (MMPs). The regulation of MMP activity is therefore of crucial
importance to the mechanisms of morphogenesis as well as many other physiological
and pathological processes. Unfortunately, MMP activity is regulated at largely posttranslational levels, rendering many of our most powerful assays of gene expression
less useful. Furthermore, most of what has been learned regarding the modulation of
MMP activity has emerged from in vitro studies, and it is not obvious how these
mechanisms give rise to the regulated remodeling of the ECM in living tissues. These
challenges necessitate the development of novel approaches to the analysis of MMP
regulation that elucidate the activation and biologically relevant activity of these
proteases in vivo. Our lab has developed several new assays that can be applied to the
analysis of ECM remodeling activity in the living zebrafish embryo; I will discuss these
techniques and the insights they have provided into matrix remodeling occurring during
the first 72 hours of zebrafish development.
Poster 84
Comparing lumen size and vascular development between diploid and triploid
zebrafish.
Small, Christopher; Benfey, Tillmann; Crawford, Bryan.
University of New Brunswick, Canada.
Triploidy can be induced in many species of fish by preventing the expulsion of the
second polar body using heat or pressure shock shortly after fertilization. This technique
produces a sterile fish with some evidence suggesting more rapid somatic growth; both
traits highly desired by the aquaculture industry. However, triploid fish are less robust
than diploid conspecifics, likely due to a reduced ability to deliver oxygen to
metabolically active tissues. The mechanism underlying this limitation is largely
unexplored. Triploid fish have 3 sets of chromosomes and the correlation between DNA
content, nucleus size, and cell size is maintained so that each cell is larger than its
diploid counterpart. One hypothesis suggests that oxygen delivery is impaired due to
blood flow limitations in the microvascular system, as capillary lumen diameter is less
than the width of an erythrocyte, forcing the cell to undergo deformation when traversing
a capillary – a larger triploid cell may have more difficulty with this process. This
hypothesis is based on the prediction that lumen diameter, like other morphometric
characteristics, is consistent between diploids and triploids. To test this prediction, I
have crossed Tg(Flk1:GFP) and nacre zebrafish to generate a pigment-less fish with
GFP in the vasculature allowing for in vivo imaging of lumen diameter. This will also
provide an opportunity to explore the effect of cell size and triploidy on vascular
patterning in a developing embryo.
Poster 85
Directing Traffic during RNA Localization in the Oocyte Cytoplasm
Gagnon, J.A., Kreiling, J.A., Powrie, E.A., Wood, T.R., Mowry, K.L.
Brown University, Providence, RI, USA
In many organisms, localization of maternal mRNAs provides the basis for
developmental polarity. Among vertebrates, Vg1 mRNA is a prominent example of a
localized mRNA that plays a role in embryonic patterning. Vg1 mRNA encodes a
peptide growth factor whose restricted expression is required for proper patterning
during Xenopus embryogenesis. Vg1 mRNA is localized during oogenesis to the
vegetal cortex of the oocyte and our recent work has focused on the motors responsible
for this process. Our experiments measuring directionality of transport in live oocytes
have revealed discrete domains of unidirectional and bidirectional transport that are
dependent on distinct molecular motors. Specifically, both dynein and kinesin motors
mediate steps in the vegetal RNA transport pathway, with dynein acting prior to kinesin.
Moreover, dynein, but not kinesin-1, promotes unidirectional transport of RNA towards
the vegetal cortex. Thus, vegetal RNA transport occurs through a multi-step pathway
with a dynein-dependent cue to control directionality of polarized RNA transport in the
oocyte.
Poster 86
List of Participants
First Name
Genevieve
Yusuff
Joshua
Last Name
Abbruzzese
Abdu
Abrams
Susan
Siddheshwari
Dominique
Karen
Abigail
Brigitte
Paola
Spyros
Mary
Alexander
Travis
Bryan
Mohna
Reshica
Michael
Mary
Elizabeth
souhila
Magdalena
Ashley
Antoine
Samara
Hannes
C. Geoffrey
Caroline
Anne
Tyler
Allan
Erin
Diane
Nicole
Lionel
James
Ethan
Alexis
Michele
Helene
Ackerman
Advani
Alfandari
Alim
Antoine
Arduini
Arlotta
Artavanis-Tsakonas
Astumian
Auld
Bailey
Ballif
Bandyopadhyay
Baral
Barresi
Baylies
Bearce
bentaya
Bezanilla
Bonneau
Borensztejn
Brown
Buelow
Burns
Burns
Campbell
Carrier
Carrillo-Baltodano
Carter
Chen
Chilingaryan
Christiaen
Clark
Cohen
Collier
Corbet
Cousin
Bryan
Chitra
Rinat
Florencia
Elizabeth
Crawford
Dahia
Degani
del Viso
Deschene Jacox
Mary
Donohoe
Matthew
Patrick
Alicia
Jeremy
Burcu
Cassandra
Anita
Juan
Pooja
Eric
James
Dunn
Ebbert
Ebert
Egbert
Erdogan
Extavour
Fernandez
Fernandez
Flora
Folker
Gagnon
Affiliation
UMass
NYU School of Medicine
NYU School of Medicine - Skirball
Institute
The Jackson Laboratory/ HHMI
UMass Amherst
Umass amherst
Harvard University
Smith College
Rensselaer Polytechnic Institute
Harvard university
Harvard Medical School
University of Maine
Boston College
SUNY Geneseo
University of Vermont
West Virginia University
Calrkson University
Smith College
Sloan Kettering Institute
Boston College
Hospital for Special Surgery
University of MA - Amherst
Yale University
Brown University
Albert Einstein College of Medicine
Massachusetts General Hospital
Massachusetts General Hospital
MDI Biological Laboratory
University of Maine
Clark University
University of Maine GSBSE
Smith College
SUNY New Paltz
New York University
The Graduate Center, CUNY
University of Rochester SMD
Lincoln Univeristy of Pennsylvania
Clark University
UMass Amherst, vet & animal science
dept
University of New Brunswick
Hospital for Special Surgery
Mount Holyoke College
Yale School of Medicine
Yale University, School of Medicine,
Genetic Department
Burke Med Research Institute/Weill
Cornell Medical College
Stony Brook University
Boston College
University of Vermont
University of Connecticut Health Center
Boston College
Harvard University
Fairfield University
Yale University
University at Albany, SUNY
Boston College
Harvard University
E-Mail Address
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Priyanjali
Valentina
Burcu
Ghosh
Greco
Guner-Ataman
Whitney
Kat
Jocelyn
Natalie
Clarissa
Cintia
Vivian
Leila
Blair
Andreas
Kimberly
Ruth
Tony
Ryan
Celina
Yasunao
Hable
Hadjantonakis
Haversat
Heer
Henry
Hongay
Irish
Jahangiri
Jenkins
Jenny
Johnson
Johnson
Joudi
Joy
Juliano
Kamikawa
Dionna
Emra
Ray Yueh
Arpita
Kasper
Klempic
Ku
Kulkarni
Hans
Miler
Kate
Sarah
Laura
Uday
jesse
Craig
Susan
Chelsea
Kara
Adam
Benjamin
Reyna
Spencer
Ketan
Jennifer
Marie
Daniel
Gretchen
Laufer
Lee
Lewis
Loh
Lowery
Madaan
mager
Magie
Mango
Marcho
Marshall
Martin
Martin
Martinez
Mass
Mathavan
Maurer
McGovern
McIntyre
McLinden
Kailin
Neva
Hannah
Izabela
Tessa
Chelsea
zahra
Charles
Tin
Megan
Rachael
Mesa
Meyer
Miller
Mlynarska
Montague
Moriarty
Motahari
Nelson
Nguyen
Norris
Norris
Belinda
Ryan
Pablo
Christopher
Nwagbara
O'Neill
Ortiz-Pineda
Owen
UMASS Medical School
Yale University
Massachusetts General Hospital,
Harvard Medical School
University of Massachusetts Dartmouth
Sloan-Kettering Institute
UMASS Amherst
Massachusetts Institute of Technology
University of Maine
Clarkson University
Harvard Medical School
Columbia University
Albert Einstein College of Medicine
University of Massachusetts Amherst
Wesleyan University
Boston University
University of Vermont
Yale University
Burke Medical Research Institute/ Weill
Cornell Medical College
Yale University School of Medicine
Utica College
SUNY Upstate Medical University
Max Planck Institute for Developmental
Biology
University of Connecticut
Yale University, Genetics Dept.
Syracuse University
Hospital for Special Surgery
Boston College
Queens College, CUNY
university of Massachusetts
Quinnipiac University
Harvard University
University of Massachusetts- Amherst
Columbia University
MIT
Stony Brook University
Upstate Medical University
SUNY New Paltz
University of Massachusetts - Amherst
UMass Medical School
Kingsborough CC
NYU Medical Center
UMass Amherst, vet and animal science
dpt
Yale University
Clark University
SUNY New Paltz
SUNY New Paltz
Harvard University
Smith College
SUNY Upstate Medical University
UMass Medical School
Utica College
Harvard University
Fred Hutchinson Cancer Research
Center
Boston College
University of New Brunswick
University of Massachusetts
Massachusetts General Hospital
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Mary
Rebeka
Vaibhav
Amrita
Jin Sook
Sangbum
Andrea
Jenna
Owen
Pack
Pai
Palaria
Park
Park
Pauli
Piccininni
Hugo
Prashanth
Siyeon
Katherine
Panteleimon
Richard
Debasmita
Elizabeth
Cathy
Caitlin
Natalia
Risha
Christopher
Eric
Joel
Michelle
Patrick
Akash
Riley
Hermann
Marlon
Jessica
Kimberly
Michael
Pinto
Rangan
Rhee
Rogers
Rompolas
Row
Roy
Salvino
Savage-Dunn
Schneider
Shylo
Sinha
Small
Small
Smith
Smith
Spica
Srivastava
St. Clair
Steller
Stoeckius
Thomas
Tremblay
Trembley
Dustin
Updike
Eivind
Laura
Antonia
Gayatri
Victoria
Kenneth
Scott
Marion
Kristi
Tao
Christopher
Erin
Daisy
Valeria
Ozge
Denise
Michael
Valen
Vallier
van den Elzen
Venkiteswaran
von Saucken
Wallace
Weatherbee
Weir
Wharton
Wu
Wylie
Wysolmerski
Xin
Yartseva
Yildizy
Zannino
Zuber
Simmons College
UMass Amherst
Tufts University
University of Massachusetts
Smith College
Yale University
Harvard University
New York University School of
Medicine/The Skirball Institute of
Biomolecular Medicine
Burke Medical Research Institute
University at Albany/RNA Institute
University of Massachusetts
Harvard University
Yale University School of Medicine
Stony Brook University
NYU-School of Medicine
SUNY New Paltz
Queesn College, CUNY
Smith College
MCGD
Smith College
University of New Brunswick
University of Rochester
MBL
University of Maine
Utica College
University of Vermont
The Rockefeller University
Yale University
Utica College
University of Massachusetts
University of Rochester School of
Medicine and Dentistry
Mount Desert Island Biological
Laboratory
Harvard University
Hofstra University
Yale University
New York University School of Medicine
Smith College
Clarkson University
Yale University
University of Vermont
Brown University
Burke Medical Research Institute
Cincinnati Children's Hospital
University of Vermont
MCGD
Yale University
UMass Medical School
Umass Medical School
SUNY Upstate Medical University
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Venues:
Accommodations, posters and meals: Swope (15)
Talks: Lille (6)