BIPN 144 Midterm 2
Review
Spring 2015
Localized Determinants and
Neuronal Fate Specification
Central Nervous System
Figure 5.6
Localized Determinants –
Factor that is localized to a
region of the cell and then
gets partitioned into only
one of the two daughter
cells following cell division
Pros (TF) - turns off
genes that determine NB
identity
Peripheral Nervous System: Sensory Organ Precursor (SOPs)
• equivalent to the neuroblast in CNS without the self regeneration (SOPs do not make
more SOPs)
Figure 5.7
I. Multiple Dendrite (Touch
and Pain Receptor)
• from single SOP
cut (TF): necessary and
sufficient to determine
ES cell identity
II.
III.
●
Figure 5.8
●
IIa and IIb cells form an equivalence group
○ IIa has the same potential to become
a IIb cell and make a neuron
Numb bias Notch signaling
○ Numb partitioned into IIb cell
○ Numb binds to ICD of Notch →
decrease Notch signaling
Eye Development/ Cellular
Interactions
Eye Development in Flies
1.
Fig. 4-5
2.
3.
4.
5.
6.
Invagination of the
epidermis to form the eye
primordium/ eye imaginal
disc
Growth of imaginal disc
Hh activates Dpp in anterior
cells
Dpp activates Hh
Propagation of
morphogenetic furrow from
the posterior to anterior of
imaginal disc
Begin photoreceptor
formation posterior of
furrow
• Total of 8
photoreceptors
• R7 – sensitive to UV
light
Mosaic Analysis
X-ray
+/-
Recombination
during mitosis and
cell division
+/- +/+
-/-
• pigment mutation allows cells that
are sev -/- to be colored
Fly eye
What cells must be wild type for normal development to occur? /
What cells can be mutant for normal development to occur?
• Normal development = complete facet (containing R1-R8) with cells functioning normally
• Sevenless (Receptor Tyrosine Kinase)
• Result: R7 cannot be mutant for sevenless for a normal facet to form
• Significance: Sevenless required for R7 only
•Boss (ligand to Sevenless receptor)
•Result: R8 cannot be mutant for boss for a normal facet to form
•Facets with R8 cells mutant for boss did not form R7 cells
•Significance: Boss required for R8 only
•Boss is a ligand for sevenless
Boss - ligand expressed on R8 cells
Sevenless - Receptor Tyrosine Kinase (RTK)
Yan - Repressor of Phyl
Pnt - Activator of Phyl
Phylopod (Phyl) - determines R7 cell fate
Fig. 6a
Eye Development in
Vertebrates
1.
2.
3.
4.
5.
6.
7.
Fig. 6-5
Neural tube forms outpockets,
which turns into optic vesicles
signaling between the vesicle and
epidermis → epidermis starts
thickening to form future lens
future lens pushes against future
retina to form optic cup
lens splits from epidermis
lens signals to epidermis to form
cornea
pigment cells and photoreceptors
in back of retina
ganglion cells form along luminal
surface and sends bundled axons
(optic nerve) to brain
Midterm 2 Review Session, Part 2 of 3 - Pax6, Neural
Differentiation, CNS Pathfinding
Pax6/Eyeless - the Master Gene
IN FAVOR
1.
2.
3.
Pax6 gene, in all
species, is
expressed early in
the eye
primordium
Reduction of Pax6
gene function
leads to small or
absent eyes in
flies, mice, and
people
Overexpression of
Pax6 in flies leads
to ectopic eye
formation
AGAINST
1.
2.
3.
4.
Pax6 is expressed
more broadly than just
the eye
Elimination of function
results in more severe
brain defects than just
having no eyes
Pax6 can only induce
eye formation in some
tissue when
misexpressed
There are other genes
that act like Pax6
Neuronal Differentiation - Neural Crest cells
1.
Neural crest cells are migrating cells and have the potential to develop into different cell types
a. Anterior cells will become cholinergic ganglia (these are in the parasympathetic system)
b. Posterior cells will become adrenergic ganglia (which are in the sympathetic system)
i. Experiment - Chick and Quail cell transplants
Cell Migration/Axon Pathfinding
1.
Cells have intrinsic characteristics and therefore have an initial direction of
migration.
a. Gold plate experiment
2.
3.
A few critical choice points are present in navigation
Many checkpoints have redundant cues.
Process Extension
1.
2.
3.
4.
5.
Lamellipodium extends and retracts filopodia
When the filopodia is attracted to something, it adheres to the signal.
All other filopodia retract.
The lamellipodium pulls the filopodia back, and the adhered filopodia stays
where it is. Therefore, the growth cone moves towards the substrate.
Axon extension continues.
Pathfinding In the Central Nervous System
CNS Pathfinding
1.
Pioneer axon - the first axon to navigate a path due to intrinsic cues and
interactions with guidepost cells.
2.
Secondary axons/follower axons - fasiculate with the pioneer axons to get to
their target
Guideposts - physical places or things that guide the axon in its journey to the
target
a. Experiment - grasshopper limb bud
3.
Midline Axon Guidance
1.
2.
3.
4.
5.
6.
Netrin (Unc6) - Secreted protein produced at the
ventral midline which acts as an attractant and as a
repellant
Unc5 - a receptor that causes repulsion from Netrin
Unc40 - a receptor that causes attraction to Netrin
Slit - Repellent signal from the ventral midline
Robo1/Robo2/Robo3 - Receptors for Slit
Comm - downregulated Robo receptors in axons
before they cross the ventral midline
Motor Neuron Axon Guidance (Drosophila)
·
·
SNb axons innervate ventral muscles
SNa axons innervate lateral muscles
Fasciclins= homophilic adhesion molecules (self-adhesive)
FasII is involved in holding SNb, SNa together.
mutants
Beaten path-= FasII is not downregulated so SNb is not able to
defasciculate from SNa and cannot branch out
o Similar to overexpression of FasII in a normal embryo
·
Short stop-= FasII is downregulated so SNb can branch from SNa but
gets stalled at the entrance to the muscle group
·
Stranded-= SNb is able to defasciculate and enter the muscle group but
does not get very far (cannot reach targets)
·
Walkabout (clueless)-= SNb is able to defasciculate and enter further
into muscle group but cannot recognize its target & cannot form synapse
·
Pathfinding in the Visual System
SPERRY EYE ROTATION EXPERIMENT (VERTEBRATES)
Sperry removed an eye from a frog and rotated it 180° before replacing it.
Connections were reformed but they noticed that the tongue of the frog moved
to opposite directions from its prey.
- The eye must have a coded way to map to the tectum in order to reconnect its
axons to the same cell targets.
Chemoaffinity model
Each retinal axon had a molecular tag that corresponded to a specific tag in the
tectum. This would explain why the axons were able to map to the same
location in the tectum.
- If this were true, that means there would have been a specific tag for
EACH axon found in the eye (that’s a lot of tags-not the most efficient
system).
There is an alternative explanation!
Anterior/Posterior tectal membrane experiment
Nasal axons grew where they wanted to (on both anterior and posterior
membranes)
· Temporal axons grew only along anterior membranes
·
Drawing on the blackboard.
N = nasal
T = temporal
A = anterior
P = posterior
Half-Tectum experiment
All of the axons were able to squeeze into the half-tectum and position
themselves relative to one another (as they would have in the full tectum). The
gradients were still present and in effect, but the axons were able to change
their threshold for the ligand to ensure every axon forms a connection in the
limited space of the half-tectum.
Half-Eye experiment
Early: the axons map to their relative positions towards one
half of the tectum
Later: the axons spread out their connections (in their
relative positions) to occupy the full tectum.
à The axons are competing for targets in the tectum & they
can communicate with each other to spread out according
to their relative sensitivity to the Eph ligand.
Fish eye
Eye and tectum continue to grow and axons from the new grown eye inhabit
new regions of the tectum.
DSCAM
-
DSCAM is a homophilic adhesion molecule that is also a signaling receptor expressed on the
surface of photoreceptor axons.
-
DSCAM is a self-adhesive molecule but its signaling induces a repulsive interaction. This
interaction is involved in branching of axons, as only those with the same isoform can bind and
repel each other. Different axons express different forms of DSCAM (which cannot bind and
cannot repel itself), allowing them to bundle together while forming branches.
-
if all axons only had one form of DSCAM, the branches cannot fasciculate and would all repel
each other
DSCAM
Alternative splicing- splicing out and selecting only certain exons result in
38000 different possible forms of DSCAM.
-
Branching therefore is highly specific.
DSCAM -> PAK (kinase) -> Rho/Rac/CDC42 (members of Rho GTPase family)
-> navigation of axon using actin.
APOPTOSIS
a “POP” tosis.
(In worms) Egl1—| Ced9—| Ced4
Ced3
(In vertebrates) Bax—| Bcl2—| Apaf1
Apoptosis
Caspase 3
Apoptosis
-
The order of the pathway was discovered by studying double mutants
-
The gene whose phenotype is expressed, means it is downstream (functions after the
other gene)
·
Egl1-/Ced9- double mutant= INCREASED cell death
(same phenotype as a Ced9- single mutant, this means Ced9 functions downstream of Egl1)
·
Ced3- (or Ced4-)/Ced9- double mutant= DECREASED cell death
(same phenotype as a Ced3- or Ced4- single mutant, this means Ced3 and Ced4 function
downstream of Ced9)
STEPS
1.
Ced9/Bcl2 in the mitochondrial membrane helps stabilize the
membrane to keep contents inside
2.
EgGl1/Bax does not have this stabilizing effect so increased expression
of EgGl1/Bax displaces Ced9/Bcl2 & Cytochrome C leaks out of the
mitochondria
3.
Cytochrome C binds Ced4/Apaf activates Ced3/Caspase 3
4.
Ced3/Caspase 3 cleaves ICAD (inhibitor of CAD)
5.
CAD (caspase activated DNase) will be active and cuts up DNA into
smaller pieces to be packaged for other cells to use
Good luck on the MIDTERM 2!!!