1. health and fitness
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Developmental Cell, Volume 31
Supplemental Information
The PI3K Class III Complex Promotes Axon Pruning
by Downregulating a Ptc-Derived Signal
via Endosome-Lysosomal Degradation
Noa Issman-Zecharya and Oren Schuldiner
Figure S1. Issman-Zecharya et al.
A
201Y>UAS-UVRAG-FLAG
Axons
Cell bodies
B
7h APF
Flag
A'
B'
GFP
Figure S1. Transgenic UVRAG is expressed in axons and cell bodies, related to Figure 1
(A-B) Confocal Z-projections of 7 h after puparium formation (APF) brains expressing
a UVRAG-Flag-tagged transgene driven by the MB specific driver, 201Y-GAL4,
strengthened by the presence of a UAS-Gal4 transgene. Flag staining (A, B) is evident in
both axons (A) and cell bodies (B) that are also labeled with membrane bound
mCD8::GFP (GFP; A’, B’). Scale bar is 20µm
Figure S2. Issman-Zecharya et al.
endoBEY00696
A
dor8
B
FasII
201Y>CD8
car∆146
C
Atg600096
D
Figure S2. endoB, dor and car are not required for MB γ axon pruning, related to Figure 2
(A-D) Confocal Z-projections of adult endoBEY00696 (A), dor8 (B), car∆146 (C), and Atg600096
(D) MB γ neuroblast MARCM clones. All the mutants prune normally.
Scale bar is 20µm
Figure S3. Issman-Zecharya et al.
201Y>UAS-GFP-2xFYVE
WT
A
B
C
GFP
L3 APF
0h APF
6h APF
D
E
F
18h APF
24h APF
Adult
WT
G
Atg61
Vps155
H
I
H'
I'
GFP
5h APF
MB
G'
CB
Figure S3. GFP-2XFYVE time course analysis, and the PI3K-cIII is required for PI(3)P
formation, related to Figure 3
(A-F) Confocal Z-projections of WT MB MARCM clones over-expressing the PI(3)P
reporter, GFP-2XFYVE, driven by 201Y-GAL4 at the indicated developmental time points.
Puncta staining is produced in the cell bodies and axons throughout development.
(G-I) Confocal Z-projections of WT (G), Atg61 (H) or Vps155 (I) MARCM clones labeled by
the GFP-2XFYVE reporter at 5h APF. While in WT brains, FYVE puncta are abundant in
axons and cell bodies, they are absent from Atg61 and Vps155 mutant neurons. Dashed
color-coded squares demarcate the regions of the cell bodies (CB) shown in insets G’-I’.
Scale bar: 20um
Figure S4. Issman-Zecharya et al.
201Y>UAS-Shits1-kitamoto
A
29˚c
FasII
201Y>CD8
Figure S4. The Shits1-kitamoto prune normally at the restrictive temperature, related to Figure
4
(A) MB neurons expressing Shits1-kitamoto driven by the γ neurons specific driver, 201YGAL4, prune normally at the restrictive temperature.
Scale bar is 20µm
ure S5. Issman-Zecharya et al.
Snx16∆2
A
FasII
201Y>CD8
Figure S5. Snx16 is not required for axon pruning, related to Figure 5
(A) Confocal Z-projections of adult Snx16∆2 (A) MB γ neuron MARCM clones prune
normally.
Scale bar is 20µm
UVRAGLL03097 Stat92E85c9
A
fasIIeB112 UVRAGLL03097
B
UVRAGLL03097 tepII116
C
FasII
201Y>CD8
UVRAGLL03097 Dl RevF10 SerRX82
D
Nnd-1 UVRAGLL03097
E
Atg61
F
Over exposed
ptc16 Atg61
G
H
FasII
GMR16A06>CD8
UVRAGLL03097
I
UVRAGLL03097 UAS-Ptc
J
K
FasII
GMR71G10>CD8
Figure S6. Double mutants of UVRAG and different candidate genes do not suppress
pruning defect, and overexpression of Ptc within UVRAG MARCM clones exacerbates the
pruning defect, related to Figure 6
(A-E) Confocal Z-projections of MB neuroblast MARCM clones of the indicated genotype.
Double mutant of UVRAGLL03097 with stat85c9 (A), fasIIeB112 (B), tepII116 (C), DlRevF10 SerRX82
(D), and Nnd-1 (E) all exhibit a pruning defect that is comparable to UVRAGLL03097 alone.
(F-G) Overexposed confocal Z-projections of Atg61 (F) or ptc16; Atg61 double mutants (G)
MB neuroblast MARCM clones. These are images are the modified versions from Figure
6C and 6D.
(H) Quantification of pruning defects of the phenotypes in overexposed images as in G
and F. Z-projections from the two genotypes were blindly ranked for the severity of the
pruning defect. Wilcoxon two sample test was performed to determine significance: ***,
p<0.001.
(I-J) Confocal Z-projections of UVRAGLL03097 MB neuroblast MARCM clones (I) or
UVRAGLL03097 also additionally expressing Ptc (J). MARCM clones are labeled with
GMR71G10-Gal4.
Scale bar is 20µm
(K) Quantification of pruning defects of the phenotypes in I and J. Z-projections from the
two genotypes were blindly ranked for the severity of the pruning defect. Wilcoxon two
sample test was performed to determine significance: ***, p<0.001.
Figure S7. Issman-Zecharya et al.
A
A'
FasII
GMR16A06>CD8 Control
Control
Rank 0
B
Rank 2
UAS-Ptc
C
Rank 3
UAS-Ptc
D
Rank 5
UAS-Ptc
E
F
F'
F''
ptc>CD8
6h APF
hh-lacZ
ptc>CD8
hh-lacZ
G
G'
G''
Repo-Gal4>hh-RNAi
H
C155-Gal4>hh-RNAi
I
201Y-Gal4>hh-RNAi
J
FasII
Figure S7. Ptc overexpression analysis in another Gal4 driver and Hh does not play a role
in axon pruning, relates to Figure 7
(A-D) Confocal Z-projection representing examples of brains that were ranked in Figure 7F
as ranking severity #0 (A, A’), #2 (B), #3 (C), and #5 (D). In this experiment various
transgenes were driven in MB neurons by the GMR16A06-Gal4.
(E) Quantification of pruning defects of flies expressing CD8-GFP or additionally UASPtcS2 or UAS-Ptc driven by the GMR71G10-Gal4. Z-stacks were blindly ranked for the
severity of the pruning defect. Kruskal-Wallis test was performed to determine significance.
***, p<0.001. Note that some control GMR71G10-Gal4 brains were ranked as exhibiting
mild pruning defects. Further studies are required to determine whether these are truly
unpruned. Nonetheless, as all Z-stacks were ranked blindly and in an identical manner,
even if there is some low background level of defect, the overexpression of ptc or ptcS2
dramatically exacerbates this.
(F-G) Confocal Z-projection of 6h APF brain (F-F’’) and imaginal eye disc (G-G’’)
expressing membrane bound GFP (mCD8::GFP) driven by ptc-Gal4 (F-G) and hh-nuclear
lacZ (F’-G’).
While mCD8::GFP is evident in both MB axons and cell bodies (F) and in imaginal eye
disc (G), hh-nuclear lacZ completely lacks expression in the brain region (F’) while is
evident in the imaginal eye disc region (G’).
(H-J) FasII staining of confocal Z-projection of MB neurons expressing Hh-RNAi in glia
(driven by Repo-Gal4; H) all postmitotic neurons (C155-Gal4; I) and MB neurons (201YGal4; J) exhibiting a WT phenotype.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Supplemental Drosophila strains
The following stocks were kindly provided by other groups: Snx16∆2 (A. Rodal); dor8 and
car∆146 (H. Kramer); Stat92E85c9 (L. Gilboa); fasIIeB112 (F. Tokunaga); hh-lacZ, endoBEY00696
, Atg600096, tepII116, DlRevF10 SerRX82, Nnd-1,UAS-Shits1-kitamoto , ptc-Gal4, and hh-RNAi
(Bloomington Drosophila Stock Center). Nnd-1 and fasIIeB112 were recombined onto FRT19A
containing chromosomes. FRT40A was added to UAS-Shits1-kitamoto and UAS-GFP-2XFYVE transgenes, and tepII116 was recombined onto FRT40A containing chromosome.
endoBEY00696 and Snx16∆2 alleles were recombined onto FRTG13 containing
chromosomes. Atg600096 was recombined onto FRT82B containing chromosome.
Allele description
A complete description of the origin of the alleles described in this study as well as the
nature of the mutation is detailed in the following table (ordered by appearance in the text):
Allele
UVRAG
LL03097
UVRAG
B21
Atg6
1
5
vps15
∆m22
Vps34
∆3d
Atg1
Atg7
d77
Atg7
d14
UAS-Shi
sara
12
Snx16
∆1
ts1-pJFRC99
Mutation
piggyBac transposone inserted within the first coding
exon
Imprecise excision of P{SUPor-P}Uvrag[KG04163] that
deletes the first exon and most of the second exon of
Uvrag
Ends-out homologous recombination has resulted in the
entire Atg6 open reading frame being replaced by a w
marker
Amino acid replacement: Q144@
Imprecise excision. The entire coding region is removed.
966bp deletion including start codon
Imprecise excision of the P{XP}Atg7[d06996] insertion,
resulting in a deletion that removes exons 5 and 6 and
most of exon 4 of Atg7
Imprecise excision of the P{EPgy2}Atg7[EY10058]
insertion, resulting in a deletion that removes portions of
Atg7
Increased expression of the temperature sensitve
1
dominant negative version of Shiribre (Shi ) by
translational enhancers
EY04602
Imprecise excision of the P{EPgy2}Sdc
insertion,
resulting in a 5.8kb deletion that removes the complete
open reading frame of Sara
Imprecise excision of the progenitor insertion, resulting
in a 3068bp deletion that removes the entire Snx16
coding region
Reference
This study
(Lee et al., 2011)
(Shravage et al., 2013)
(Wu et al., 2007)
(Juhasz et al., 2008)
(Scott et al., 2004)
(Juhasz et al., 2007)
(Juhasz et al., 2007)
(Pfeiffer et al., 2012)
(Bokel et al., 2006)
(Rodal et al., 2011)
Rab11
Hrs
∆FRT
D28
Stam
2896
UAS-Shrub
ptc
DN
16
Smo
hh
Ac
hh
ts2
3
endoB
dor
car
EY00696
8
∆146
Atg6
00096
Deletion that removes the promoter and first two exons
of Rab11
Amino acid replacement: Q270@
EMS mutant - Amino acid replacement: Q6@ as well as
A505T
UAS-Shrub-GFP acts as a dominant negative
A premature stop codon (location not precisely known)
which is presumabley located before the first
transmembrane domain.
Point mutation G1783A
P30
Deletion of sequences beginning in the P{PZ}hh
insertion and extending to between position +0.8 to +8.6
P30
(coordinate 0 is the P{lacZ} insertion of hh ), removing
the hh promoter and part of the coding region.
Temperature sensitive mutation generated by EMS.
P{EPgy2} insertion
Insertion of DNA at least 4.3kb in length
Imprecise excision. Sequences encoding amino acid
residues 1-584 out of 617 residues are removed
P-element insertion about 100 bp upstream of the Atg6
1
UAS-Shi
Snx16
∆2
Stat92E
fasII
Dl
N
85c9
eB112
RevF10
Ser
RX82
nd-1
ts-1-kitamoto
UAS transgene that drives the expression of the shi
coding region which encodes a temprature sensitve and
dominant negative form of Shibire (Nucleotide
substitution G to A)
Imprecise excision of the progenitor insertion, resulting
in a 2298bp deletion that removes the entire Snx16
coding region
Amino acid replacement: R442P
Fas2Imprecise excision of the P-element from Ecol\lacZ
A31
. 1.7kb deletion of Fas2 genomic sequences,
including 5' untranslated sequences
Revertant of a P-element insertion located between -20
and -30 upstream of the Dl transcription start site.
Deletion of approximately 760bp removing most of the
first exon and the proximal part of the promoter
(including the TATA box)
Deletion of 0.5kb
Insertion of 3 bases between Q2567 and Q2568
encoding an additional Q residue in addition to a 41
nucleotide deletion at the 3' end of the 8th exon
(Bogard et al., 2007)
(Lloyd et al., 2002)
(Chanut-Delalande et al.,
2007)
(Sweeney et al., 2006)
(Strutt et al., 2001)
(Chen and Struhl, 1998)
(Lee et al., 1992)
(Ma et al., 1993)
Gene distruption project
2001
(Shestopal et al., 1997)
(Akbar et al., 2009)
Berkeley Drosophila
Genome Proj
(Kitamoto, 2001)
(Rodal et al., 2011)
(Silver and Montell, 2001)
(Grenningloh et al., 1991)
(Haenlin et al., 1990)
(Thomas et al., 1991)
(Lyman and Young, 1993)
Supplemental Drosophila genotypes
Figure S1.
(A-B’) 201Y,CD8::GFP/+;UAS-UVRAG-Flag/UAS-GAL4
Figure S2.
(A) hsflp,CD8::GFP/+;G13,201Y,CD8::GFP, endoBEY00696/ G13,Gal80
(B) dor8,19A/Gal80,19A,hsflp,CD8::GFP;G13,201Y,CD8::GFP/+
(C) car∆146,19A/Gal80,19A,hsflp,CD8::GFP;G13,201Y,CD8::GFP/+
(D) hsflp,CD8::GFP/+;201Y,CD8::GFP /+;82B,Atg600096/82B ,Gal80
Figure S3.
(A-G) hsflp/+;40A,UAS-GFP-2xFYVE/Gal80,40A;UAS-CD8/+
(H) hsflp/+;UAS-GFP-2xFYVE/201Y;82B,Atg61/82B,Gal80
(I) hsflp/+;UAS-GFP-2xFYVE/201Y;82B,vps155/82B,Gal80
Figure S4.
(A) 201Y,CD8::GFP/+;UAS-Shits1-kitamoto
Figure S5.
(A) hsflp,CD8::GFP/+;G13,Snx16∆2,201Y,CD8::GFP/G13,Gal80
Figure S6.
(A) hsflp,CD8::GFP;UVRAGLL03097,40A/G80,40A,201Y,CD8::GFP;
Stat92E85c9,82B/Gal80,82B
(B) fasIIeB112,19A/Gal80,19A,hsflp,CD8::GFP;UVRAGLL03097,40A/
Gal80,40A,201Y,CD8::GFP
(C) hsflp,CD8::GFP;UVRAGLL03097,tepII116,40A/Gal80,40A,201Y,CD8::GFP
(D) hsflp,CD8::GFP;UVRAGLL03097,40A/Gal80,40A,201Y,CD8::GFP;
DlRevF10,SerRX82,82B/Gal80,82B
(E) Nnd-1,19A/Gal80,19A,hsflp,CD8::GFP;UVRAGLL03097,40A/Gal80,40A,201Y,CD8::GFP
(F) hsflp,CD8::GFP;;82B,Atg61/ GMR16A06-Gal4,82B,Gal80
(G) hsflp,CD8::GFP;42D,ptc16/42D,Gal80;82B,Atg61/GMR16A06-Gal4,82B,Gal80
(I) hsflp,CD8::GFP/+;UVRAG LL03097,40A/Gal80,40A; GMR71G10-Gal4/+
(J) hsflp,CD8::GFP/+;UVRAG LL03097,40A/Gal80,40A;UAS-Ptc/GMR71G10-Gal4
Fig S7.
(A) 10xUAS-IVS-CD8::GFP/+;GMR16A06-Gal4/+
(B-D) 10xUAS-IVS-CD8::GFP/+;GMR16A06-Gal4/UAS-Ptc
(E)
1. 10xUAS-IVS-CD8::GFP/+;GMR71G10-Gal4/+
2. 10xUAS-IVS-CD8::GFP/+;UAS-PtcS2/GMR71G10-Gal4
3. 10xUAS-IVS-CD8::GFP/+;UAS-Ptc/GMR71G10-Gal4
(F-G) ptc-Gal4/CD8::GFP;hh-lacZ/+
(H) Repo-Gal4/+;hh-RNAi32489/+
(I) c155-Gal4/+;;hh-RNAi32489/+
(J) 201Y/+;hh-RNAi25794/+
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