Development 142: doi:10.1242/dev.124370: Supplementary information
Supplementary Materials and Methods
Drosophila stocks
The following stocks are described elsewhere and were obtained from the
Bloomington Drosophila Stock Center (Indiana University, IN): UAS-Src64, UASsrcEGFP,
UAS-Src64myr-Tomato
(UAS-IVS-myr-tdTomato,
#32221),
UAS-
LifeactGFP, Nanos-Gal4, bamΔ86, abl1, abl4, SCARΔ37, Arpc1Q25sd, Btk29e482, Rac1J11
Rac2Δ FRT2A mtl/+, FRT2A control and FRT40A control. bamΔPEST (gift from Dennis
McKearin, Howard Hughes Medical Institute, Chevy Chase, MD), bam-GAL4 (gift
from Julie Brill, The Hospital for Sick Children, Toronto, Canada), Src64Δ17 and
Src64KO (gifts from Alana O’Reilly, Fox Chase Cancer Center, Philadelphia, PA),
Abl-GFP (gift from Jennifer Zallen, Howard Hughes Medical Institute and
Developmental Biology Program, Sloan Kettering Institute, New York, NY),
Cortactin
D4
(gift from Pernille Rørth, Institute of Molecular and Cell Biology,
Singapore), MARCM FRT40A (yw hsflp tub-GAL4 UAS-GFP.nls/FM7; tub-GAL80
FRT40A/CyO) (gift from Thomas Vaccari, IFOM - FIRC Institute of Molecular
Oncology, Milan, Italy) and hsflp;;Ubi-GFP FRT2A (gifts from Alana O’Reilly and
Antibodies
Antibodies used were rabbit anti-Cindr (1:1000) and guinea pig anti-Anillin (1:50)
(Haglund et al., 2010), mouse anti-α-spectrin (1:20, 3A9), mouse anti-HtsF (1:50,
1B1), mouse anti-Bam (1:10), mouse anti-SCAR (1:10), rat anti-DE-cadherin (1:50,
DCAD2) and mouse anti-Peanut (1:10, 4C9H4, all DSHB), rabbit anti-pTyr (WB
1:1000, IF 1:300, T1325, Sigma), mouse anti-pTyr (1:300, sc-508, Santa Cruz),
mouse anti-tubulin (WB, 1:10 000, T9026, Sigma), rabbit anti-pSrc-Y418 (WB,
Development • Supplementary information
Ji-Long Liu, University of Oxford, Oxford, United Kingdom.).
Development 142: doi:10.1242/dev.124370: Supplementary information
1:1000, 44-660G, Invitrogen), mouse anti-GFP (1:200, Invitrogen) and GFP-Booster
(1:200, gba488, Clontech). The rabbit Src64-CT antibody was a gift from Alana
O’Reilly (Fox Chase Cancer Center, Philadelphia, PA). Rabbit anti-pTyr conjugated
to agarose beads, 16-101, were from Millipore. Phalloidin-Alexa488 or Rhodaminephalloidin (1:400, Molecular Probes) were used to visualize F-actin. Hoechst dye was
used at 1 g/ml (33342, Invitrogen). Secondary antibodies were conjugated to Alexa488, DyLight-549 (1:500, Molecular Probes), HRP, Cy2, Cy3 or Cy5 (1:500, Jackson
Immunoresearch). Click-IT EdU labeling was performed according to manufacturer’s
protocol (C10338, Invitrogen).
Western blotting and immunoprecipitation
Due to smaller size of Src64KO mutant testes, we dissected fewer wild type males to
equalize protein levels. Testes from 15 wild type males and 30 Src64KO males were
dissected lysed in a HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10% glycerol, 1% Tx-100, 25 mM NaF and 10 μM ZnCl2,
adjusted to pH 7.5) supplemented with fresh protease (Sigma-Aldrich) and
phosphatase (PhosSTOP, Roche Diagnostics) inhibitors. Lysate was mixed with
before loading onto a 4–20% gradient SDS-PAGE gel (BioRad), transferred onto a
PVDF membrane and probed with a pTyr antibody. To assess the levels of tyrosinephosphorylated Abl in testes of wild type and Src64KO, pTyr-proteins were
immunoprecipitated using a hot lysis-protocol where all protein-interactions are
destroyed upon boiling the lysates in 1% SDS. Specifically, this determines the
phosphorylation of the particular protein of interest without interference of associated
proteins which are co-sedimented in regular immunoprecipiation protocols. To this
Development • Supplementary information
Laemmli buffer (BioRad) containing a final concentration of 0.1 M DTT and boiled
Development 142: doi:10.1242/dev.124370: Supplementary information
end 12 wild type males and 28 Src64KO males were dissected, and testes were lysed in
1% SDS and boiled for 10 minutes. The lysates were cleared using a QiaShreddar
column (Qiagen), diluted 1:1 with 2x IP buffer (2% Triton X-100, 0.5% sodium
deoxycholate, 1% BSA, 2mM EDTA, 40 mM sodium fluoride) supplemented with
Phosphatase inhibitor cocktail (Sigma), Complete EDTA-free protease inhibitor
cocktail (Roche) and 2 mM N-ethylmaleimide (Sigma) and incubated with 30 μl antipTyr agarose beads (Millipore) at constant rotation (over night, 4°C). The beads were
rinsed three times in 1X IP buffer, boiled (5 minutes) in Laemmli sample buffer (BioRad) containing 0.1 M DTT and subjected to SDS-PAGE. The Western blot was
performed with an anti-pSrc-Y418 antibody. Western blots of tissue lysates using
anti-pSrc-Y418, anti-Tubulin or anti-actin antibodies were performed as described
above.
Confocal microscopy
Confocal images were acquired using a Zeiss LSM 780 confocal microscope
equipped with a 63x NA 1.4 oil DIC III (Plan-Apochromat) objective. Z-stacks were
obtained and maximum projections were generated using the Zen 2010 software.
schematics, respectively. Images were adjusted for clarity. Imaris (Bitplane) was used
to make a volume rendering and the image shown in Fig. 7K from a confocal z-stack.
Phase contrast microscopy was performed according to (Insco et al., 2009).
Structured illumination microscopy
We performed 3D structured illumination microscopy (SIM) on an OMX V4 system
(DeltaVision OMX microscope, Applied Precision, GE Healthcare, Issaquah, WA)
Development • Supplementary information
Adobe Photoshop and Adobe Illustrator were used to assemble figures and make
Development 142: doi:10.1242/dev.124370: Supplementary information
equipped with two cooled sCMOS cameras and 405, 488, 568 and 642 nm laser lines.
A small area of the testes tip was illuminated with a grid pattern and for each image
plane, 15 images were collected (5 phases for 3 rotations of the illumination pattern).
To ensure proper reconstruction, at least 7 image planes, with z-spacing of 125 nm
were collected from each RC. Super resolution images were reconstructed from the
raw image files using Softworx software (Applied Precision, GE Healthcare,
Issaquah, WA) and processed in ImageJ for presentation.
Electron microscopy
Tissues were dissected and immediately immersion fixed in 2% glutaraldehyde in 0.1
M cacodylate buffer. Samples were further postfixed in 1% OsO4 and 1.5% KFeCN,
en bloc stained in 1% uranyl acetate and dehydrated in graded ethanol series. After
embedding in Epon resin the samples were sectioned on an Ultracut (Leica)
microtome and 60-100 nm sections collected on carbon/formvar coated grids.
Samples were observed in a JEOL-JEM 1230 microscope at 80 kV and images
recorded with a Morada camera (Olympus) and further processing was done with the
NanoLC-mass spectrometry and protein identification
1000 wild type males were dissected and lysed in a HEPES buffer (50 mM HEPES,
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Tx-100, 25 mM NaF
and 10 μM ZnCl2, adjusted to pH 7.5) supplemented with fresh protease (SigmaAldrich) and phosphatase (PhosSTOP, Roche Diagnostics) inhibitors, and subjected
to immunoprecipitation with an agarose-conjugated phosphotyrosine antibody (4G10,
Millipore) according to manufacturer’s instructions. Immunoprecipitated proteins
Development • Supplementary information
Adobe Photoshop software.
Development 142: doi:10.1242/dev.124370: Supplementary information
were separated on an SDS-PAGE gel, stained with Coomassie G-250 and in-gel
digested using 0.1 µg of trypsin in 25 µl of 50 mM ammonium bicarbonate, pH 7.8.
After micorpurification using µ-C18 ZipTips (Millipore, Oslo, Norway), the peptides
were dried in a SpeedVac and dissolved in 10 µl 1% formic acid, 2.5% acetonitrile in
water. Half of the volume was injected into a nano-UHPLC system (Ultimate 3000
RSLC, Dionex, Sunnyvale, CA, USA) coupled to an ESI-ion trap/Orbitrap (LTQ
Orbitrap XL, Thermo Scientific, Bremen, Germany) mass spectrometer. For
separation of peptides, an Acclaim PepMap 100 column (50 cm x 75 μm) packed with
100 Å C18 3 µm particles (Dionex) was used with a flow rate of 300 nL/min and a
solvent gradient of 3 % B to 35 % B in 110 minutes. Solvent A was 0.1% formic acid
and solvent B was 0.1% formic acid/90% acetonitrile. The mass spectrometer was
operated in the data-dependent mode to automatically switch between Orbitrap-MS
and LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2000)
were acquired in the Orbitrap with the resolution R = 60,000 at m/z 400 after
accumulation to a target of 1,000 000 charges in the LTQ. The method used allowed
sequential isolation of up to the six most intense ions for fragmentation on the linear
ion trap using collision induced dissociation (CID) at a target value of 10,000 charges.
accurate mass measurements, the lock mass option was enabled. Other instrument
parameters were set as previously described (Koehler et al., 2009). Data were
acquired using Xcalibur v2.5.5 and raw files were processed to generate peak list in
Mascot generic format (*.mgf) using ProteoWizard release version 3.0.331. Database
searches were performed using Mascot in-house version 2.3. to search from SwissProt selected for Drosophila (11.2011, 5,437 entries) assuming the digestion enzyme
trypsin, at maximum one missed cleavage, fragment ion mass tolerance of 0.60 Da,
Development • Supplementary information
Target ions already selected for MS/MS were dynamically excluded for 60 sec. For
Development 142: doi:10.1242/dev.124370: Supplementary information
parent ion tolerance of 10 ppm and oxidation of methionines, acrylamide adducts of
cysteines, and phosphorylation of tyrosines as variable modifications.
Supplementary References
Haglund, K., Nezis, I. P., Lemus, D., Grabbe, C., Wesche, J., Liestol, K., Dikic, I., Palmer, R. and
Stenmark, H. (2010). Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster.
Curr Biol 20, 944-950.
Insco, M. L., Leon, A., Tam, C. H., McKearin, D. M. and Fuller, M. T. (2009). Accumulation of a
differentiation regulator specifies transit amplifying division number in an adult stem cell lineage. Proc
Natl Acad Sci U S A 106, 22311-22316.
Development • Supplementary information
Koehler, C. J., Strozynski, M., Kozielski, F., Treumann, A. and Thiede, B. (2009). Isobaric peptide
termini labeling for MS/MS-based quantitative proteomics. Journal of proteome research 8, 43334341.
Figure S1, Related to Figure 1. Relative pTyr intensities in 4-, 8-, 16- and maturing 16-cell cysts.
(A, C and E) Representative images of 4, 8, and 16-cell cysts (CCs) from a wild type testis stained with antibodies against pTyr
(green), Anillin (red) and -Spectrin (blue). Scale bar, 1 m. (B, D and F) Graphs showing the relative intensities of pTyr on M1
to M4 RCs in cysts as indicated (n=16, 22 and 17 cysts quantified respectively). Error bars represent standard deviation. (G and
H) Representative images and graph of maturing primary spermatocyte 16-CCs (m16-CCs), stained and quantified as in (E and
F). 14 cysts were quantified. Error bars represent standard deviation. Note that RC diameters and relative pTyr intensities are
more similar, with less distinct differences between the non-M1 RCs. (I) Graph showing the relative RC diameters in 4-, 8-, 16and m16-CCs (n=16, 22, 17 and 14 cysts quantified respectively). Error bars represent standard deviation.
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Figure S2, Related to Figure 1. Relative pTyr intensities upon changes in the timing of differentiation.
(A and B) A mitotic (A) and a maturing (B) 8-CC from a Bam∆PEST male stained with antibodies against pTyr (green), Anillin
(red) and -Spectrin (blue). (C) Graph showing the relative pTyr intenstites on RCs in mitotic and maturing 8-CCs in a Bam∆PEST
testis (n=8 and 9 cysts, respectively). Error bars represent standard deviation. (D) A cell with 5 RCs connected to it in a cyst from
the Bam∆86 heterozygous males. (E) Graph showing the quantification of the relative pTyr levels on the 5 RCs in a 32-CC, as in
(D) (n=4 cysts). Error bars represent standard deviation. (F) Two 32-CCs found in the Bam∆86/+ males, stained as in (A). The left
cyst is still in its mitotic phase, while the cyst to the right is starting spermatocyte maturation. Note the difference in pTyr
distribution. All scale bars, 1 m.
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Figure S3, Related to Figure 2. Src64, but not Btk29, is required for RC phosphorylation in male germline cysts.
(A to F) Confocal images of testes of indicated genotypes stained with antibodies against pTyr (green and gray) and -Spectrin
(red). Hoechst marks nuclei in blue. Scale bar, 20 m. (G and H) GFP-positive control and Btk29e482 16-CC clones (outlined) 3
days after clonal induction. Testes were stained with pTyr (red) and Cindr (blue). Scale bars, 5 m. (I) Graph showing the
relative average RC pTyr intensities in GFP-positive control and Btk29e482 16-CCs from three independent experiments. Error
bars represent the standard error of the mean. The numbers of 16-CCs quantified were 25 for control and 24 for Btk29e482,
respectively. Differences between control and Btk29e482 are statistically non-significant. (J) Graph showing distribution of RC
diameters measured in 16-CCs from GFP-positive clones in MARCM FRT40A control and MARCM FRT40A Btk29e482 testes.
n=101 and 87 RCs, respectively. RC diameters are slightly increased in Btk29e482 versus control clones (p<0.05, Student’s t-test).
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Figure S4, Related to Figure 5. Localization of actin regulators and E-cadherin in wild type, Src64KO and abl4 germline cysts.
(A) Electron micrographs of a wild type testis tip. (A’) shows a zoomed view of the upper RC observed in the cyst (A). Note the
straightened membrane (arrow) at an almost perpendicular angle to the fusome plug within the RC. Scale bars, 5 and 1 m,
respectively. (B to D) Overview of testes tips in wild type and Src64KO males stained with antibodies against SCAR (B), Peanut (C)
or E-cadherin (D), and Cindr (green (B and C) or red (D)). Arrows indicate 2-CC RCs. Hoechst marks nuclei in blue. Scale bars, 10
m. See Table S2 for quantification of localization of SCAR, Peanut and E-cadherin in 2-CCs from wild type and Src64KO testes. (E
to H) 2-CCs in testes from Abl-GFP (E) or wild type (F to H) males stained with antibodies as indicated and phalloidin to visualize
actin (red). When comparing the localization of SCAR and Peanut to actin filaments around the RC, we observed only minor
colocalization with the actin filaments, but instead an enrichment of SCAR and Peanut at areas where actin filaments had not
accumulated at the membrane. E-cadherin marked a circular rim around the actin disk, perhaps outlining the borders of the cell-cell
contact surface in the 2-CCs. Scale bars, 5 m. (I to K) Confocal images of GFP-negative 2-CC clones (outlined) 3 days after clonal
induction in FRT2A control, FRT2A abl4 or FRT2A Src64KO testes stained with antibodies against E-cadherin (red) and Cindr (blue).
E-cadherin was normal in FRT2A control clones (n = 19 2-CCs), but irregular or absent in ~50% of FRT2A abl4 clones (n = 31 2CCs, 16 displayed irregular E-cadherin staining) and in ~95% of FRT2A Src64KO clones (n = 37 2-CCs, 35 displayed abnormal Ecadherin staining). Scoring is based on the results of three independent experiments. Scale bars, 5 m.
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Figure S5, Related to Figure 6. SCAR, but not Cortactin, is required for correct RC diameter in male
germline cysts.
(A and B) GFP-positive 16-CC clones (outlined) in MARCM FRT40A control and MARCM FRT40A SCAR∆37
testes stained with pTyr (red) and -Spectrin (green). Scale bars, 5 m. (C and D) Wild type and CortactinM7 16CCs stained with Cindr (green) and -Spectrin (red). Scale bars, 5 m. (E) Graph showing distribution of RC
diameters measured in 16-CCs from GFP-positive clones in MARCM FRT40A control and MARCM FRT40A
SCAR∆37 testes. n=68 and 72 RCs, respectively, p<0.05 using the Student’s t-test. (F) Graph showing distribution
of RC diameters measured in 16-CCs from wild type and CortactinM7 testes. n=126 and 142 RCs, respectively.
Development • Supplementary information
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Figure S6, Related to Figure 7. Rescue of Src64KO mutant phenotypes by expression of Src64 in the germline.
(A) Graph showing quantification of testes from the indicated genotypes, scored as normal, overproliferation with one or more tumors of
cells (tumor), or testes completely filled with mitotic cells (overgrown). n=51, 91 and 73 testes quantified for the three genotypes,
respectively. (B) Graph showing the frequency of spermatocyte cysts with 8, 16, 32 and 64 cells. n=108, 132 and 183 cysts quantified for
the three genotypes, respectively. (C to E) Phase contrast images of representative cysts of primary spermatocytes, 16 cells in the wild type
(C), 32 cells in the Src64KO mutant (D) and 16 cells in the testes of NosGAL4/UAS-Src64;Src64KO males (E). (F to H) Confocal images of
testes from Src64KO or NosGAL4/UAS-Src64;Src64KO males stained with antibodies as indicated. Note re-appearance of pTyr staining in
the rescue males (G), accompanied by abnormal RC morphology (H). Scale bars, 20 (F-G) and 5 m (H).
Development • Supplementary information
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Figure S7, Related to Figure 7. Src64KO tumors, but not mitotic cysts, are asynchronous.
(A and B) Mitotic 8- and 16-CCs from Src64KO testes stained with antibodies against Anillin (red) and the fusome (green), as
well as with Hoechst (blue). Scale bars, 5 m. (C) Large cyst from Src64KO testis labeled as in (A). Arrow points to
asynchronous cell in prometaphase, determined by cortical Anillin and DNA morphology. Scale bar, 5 m. (D and E) Testes
from from Src64KO males labeled with EdU (red), stained with a fusome antibody (green) and with Hoechst (blue). Asterisk
indicates anterior tip of the testis, with synchronous mitotic zone, arrows point to large cysts with asynchronous incorporation
of the labeled nucleotide. Scale bars, 50 m. (F) Graph showing the distribution of RC diameters measured in 16-CCs and
tumors from Src64KO testes, n = 171 and n = 278 RCs, respectively. Data from Figures 2J and 7J were combined in this graph
to compare RC diameters in Src64KO 16-CCs and tumors. Differences in RC diameters are statistically non-significant.
Development • Supplementary information
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Figure S8, Related to Figure 7. Overexpression of Src64 in the germline is not sufficient to promote premature germ cell
differentiation.
(A) Confocal images of a testis from a NosGAL4/+ male, stained with antibodies as indicated. Scale bar, 20 m. (B) Phase contrast
image of representative 16-cell primary spermatocyte cyst with same genotype as in (A). (C) Confocal images of a testis from a
NosGAL4/UAS-Src64 male, stained with antibodies as indicated. Scale bar, 20 m. (D) Phase contrast image of representative 16-cell
primary spermatocyte cyst with same genotype as in (C). (E) Graph showing the frequency of spermatocyte cysts with 8, 16 or 32
cells in testes from control NosGAL4/+ and NosGAL4/UAS-Src64 males. n=184 and 167 cysts quantified for the two genotypes,
respectively. (F) Confocal images of a testis from a BamGAL4/+ male, stained with antibodies as indicated. Scale bar, 20 m. (G)
Phase contrast image of representative 16-cell primary spermatocyte cyst with same genotype as in (F). (H) Confocal images of a
testis from a BamGAL4/UAS-Src64 male, stained with antibodies as indicated. Scale bar, 20 m. (I) Phase contrast image of
representative 16-cell primary spermatocyte cyst with same genotype as in (H). (J) Graph showing the frequency of spermatocyte
cysts with 8, 16 or 32 cells in testes from control BamGAL4/+ and BamGAL4/UAS-Src64 males. n=210 and 231 cysts quantified for
the two genotypes, respectively.
Development • Supplementary information
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Development • Supplementary information
Movie 1 (related to Fig. 3). pTyr localization at an RC determined by structured
illumination microscopy. The video shows a 360 degree rotation of a reconstructed
M1 RC from a wild type testis tip stained with antibodies against Cindr (green) and
pTyr (red), imaged using structured illumination imaging. Two distinct pools of pTyr
can be observed at the RC edges. See Fig. 3E.
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Table S1 (related to Fig. 4). Proteins identified by mass spectrometry analysis
from anti-phosphotyrosine immunoprecipitates from Drosophila testes.
Protein
Accession
Nr
Nr of
peptides
E3 ubiquitin-protein ligase UBR1
RNA polymerase II subunit RPB1
Abelson kinase***
Ribosomal protein RpS27A
CAD protein
Clathrin heavy chain
Myosin heavy chain
Coracle***
Heat shock protein 70 kDa
Beta-tubulin
Alpha-tubulin
Hem***
CYFIP / Sra-1***
PABP
EF1A1
Actin
Ribonucleoprotein A1
Dynein heavy chain
60S ribosomal protein
40S ribosomal protein
14-3-3 protein epsilon
Vav***
Q9VX91
P04052
P00522
P15357
P05990
P29742
P05661
Q9V8R9
P29845
P61857
P06603
P55162
Q9VF87
P21187
P08736
P02572
P07909
P37276
P09180
Q06559
P92177
Q9NHV9
9
10
8
2
5
5
4
2
142
82
45
35
22
27
20
12
10
6
8
6
3
3
Molecular
weight
(kDa)
208
209
171
18
247
191
224
184
70
50
50
129
149
70
50
42
39
530
45
27
30
91
Notes
*
*
*
*
*
*
*
*
**, Additional similar proteins
**, Additional similar proteins
**
**
**
**
**
**
**, Additional similar proteins
**
**, Additional similar proteins
**, Additional similar proteins
**
**
Development • Supplementary information
* Proteins identified from gel slice between 150 and 200 kDa.
**Proteins identified from gel slice between between 25 and 150 kDa.
***Proteins involved in actin regulation.
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Table S2 (related to Fig. 5). Quantification of components localizing adjacent to
the RC in wild type and Src64KO 2-CCs.
Component
SCAR
Peanut
E-cadherin
Actin
Lifeact
Abl-GFP
Total 2-CCs
42
41
31
35
10
21
wild type
Normal
41
40
31
35
10
21
Irregular
1
1
0
0
0
0
Src64KO
Total 2-CCs Normal
38
4
42
2
34
0
40
2
41
8
29
1
Irregular
34
40
34
38
33
28
The localization of the indicated components in 2-CCs from wild type or Src64KO
testes was quantified as normal or irregular. See Figures 4E-F (Abl-GFP), 5A-B
(Lifeact-GFP) and 5E-L (Actin, SCAR, Peanut and E-cadherin) for representative
Development • Supplementary information
images. Data represent quantifications from three independent experiments.
Development 142: doi:10.1242/dev.124370: Supplementary information
Table S3 (related to Fig. 7). Quantification of spermatocyte cysts 10 days after
clonal induction.
Genotype
FRT2A control
FRT2A Src64KO
FRT2A abl4
Rac1J11 Rac2∆ FRT2A mtl∆/+
Testes scored
100
130
92
72
Clones
90
86
45
18
16-CC
90
82
41
18
32-CC
0
4
4
0
FRT-mediated recombination was induced by one 2h heat shock at 37 degrees during
pupation, and males of the correct genotype were dissected for analysis 10 days later.
GFP-negative clones of primary spermatocytes were identified, and the number of
cells in each clone quantified with help from Vasa and -Spectrin antibody stainings.
Development • Supplementary information
Data represent quantifications from three independent experiments.
Development 142: doi:10.1242/dev.124370: Supplementary information
Table S4 (related to Fig. 7). Quantification of spermatocyte cysts 5 days after
clonal induction.
Genotype
FRT40A control
FRT40A SCAR∆37
FRT40A Arpc1Q25sd
Testes scored
62
49
44
Clones
70
32
15
16-CC
70
32
15
32-CC
0
0
0
FRT-mediated recombination was induced by three 1h heat shocks at 37 degrees over
three days during pupation, and males of the correct genotype were dissected for
analysis 5 days later. GFP-positive MARCM clones of primary spermatocytes were
identified, and the number of cells in each clone quantified with help from Vasa and
-Spectrin antibody stainings. Data represent quantifications from three independent
Development • Supplementary information
experiments.