A conserved function of Human DLC3 and Drosophila Cv-c in testis development
- Centro Andaluz de Biología del Desarrollo, Spain
- Department of Endocrinology, Metabolism and Cardiovascular research, University of Fribourg, Switzerland
- Department of Biology, University of Fribourg, Switzerland
- Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Ukraine
- Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Switzerland
Figures
Figure 1 with 1 supplement
DLC3 variants associated to Differences of Sex Development (DSD) patients.
(A, B) Family diagrams showing the segregation of two different DLC3 alleles (panel A is modified from Ilaslan et al., 2018). (C) Structure of DLC3 GAP and StART domains showing the localization of …
Figure 1—figure supplement 1
Binding of DLC3-StART domain to lipid bilayers.
(A) System setup containing the StART domain and a lipid bilayer. The protein is shown in cartoon representation, the lipid head-groups in grey and the tails in yellow. Water and ions beads are …
Figure 2 with 1 supplement
Cv-c and DLC3 structure and cv-c expression in the Drosophila gonad mesoderm.
(A) Linear representation of the DLC3-α, DLC3-β, and Cv-c proteins. The SAM domain is represented in yellow, the GAP domain in orange, and the StART domain in blue. (B) Alignment of the DLC3 (blue) …
Figure 2—figure supplement 1
JAK/STAT pathway and cv-c-independent functions.
(A, B) cv-c::GFP stage 17 testis stained against GFP (green), Vasa (magenta), and Ems (grey) in a wild-type (A) or Df(1)os1A mutants. Cv-c expression and localization are unaffected by JAK/STAT lack …
Figure 3 with 1 supplement
Gonad morphology in cv-c mutant embryos.
(A–C) Gonads with germ cells labelled with nos-nod::GFP (green), pigment cells with anti-Ems (magenta), and male-specific somatic gonad precursor (msSGP) with anti-Eya (nuclear grey staining) and …
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Figure 3—source data 1
Raw data.
- https://cdn.elifesciences.org/articles/82343/elife-82343-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Actin cystoskeleton in cv-c mutants testes.
(A–D) Embryos labelled with phalloidin (grey) to reveal the filamentous actin cytoskeleton and with nos-nod::GFP (green) to label the germ cells. (A) In heterozygous males and (B) homozygous cv-cC524…
Figure 4
Rescue of cv-c mutant testes.
(A) Schematic representation of Cv-c and DLC3 protein variants studied. Rescue of the dysgenic testis of cv-cC524 homozygous mutant males after expressing the specified (B) DLC3 or (C) Cv-c protein …
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Figure 4—source data 1
Raw data.
- https://cdn.elifesciences.org/articles/82343/elife-82343-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Testes in embryos with altered small GTPase regulation.
(A–L) Testes of various genotypes stained with anti-Vasa (green), β-catenin (grey), and Ems (magenta). β-Catenin and Vasa channels are shown separately in right panels. (A, B) No extruded gem cells …
Figure 5—figure supplement 1
cv-cC524 homozygous mutant testes expressing different Cv-c and DLC3 protein variants exclusively in the somatic gonadal mesoderm with the c587-Gal4 driver line.
Representative images of testes in (A) heterozygous cv-cC524 animals, (B) control c587-Gal4 cv-cC524 homozygous animals or (C) homozygous cv-cC524 animals expressing either UAS-DLC3WT or (D) UAS-DLC3…
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Figure 5—figure supplement 1—source data 1
Raw data.
- https://cdn.elifesciences.org/articles/82343/elife-82343-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
Ensheathment defects in cv-c mutant testes.
(A, C, E) Wild-type and (B, D, F) cv-cC524 testes. Germ cells are labelled with nos-nod::GFP (green A–F) and labelled in grey with anti-E-cad (A–B) or anti-Nrt (C–D) to highlight the ensheathing …
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Figure 6—source data 1
Raw data.
- https://cdn.elifesciences.org/articles/82343/elife-82343-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
ECM rescue of cv-c mutant testes.
Representative images of testes in (A) control twi-Gal4; cv-cC524 heterozygous animals or homozygous cv-cC524 animals expressing in the somatic mesoderm cells either (B) UAS-DLC3WT, (C) UAS-DLC3S993N…
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Figure 6—figure supplement 1—source data 1
Raw data.
- https://cdn.elifesciences.org/articles/82343/elife-82343-fig6-figsupp1-data1-v2.xlsx
Videos
Video 1
In vivo gonad coalescence of a control heterozygous cv-cC524/+ testis.
The germ cells are labelled with nos-nod::GFP and the male-specific somatic gonadal precursors with six-moe::GFP. Movie taken from st14 (prior to gonad coalescence) up to st16 (after coalescence).
Video 2
In vivo gonad coalescence of a homozygous cv-cC524 testis.
The germ cells are labelled with nos-nod::GFP and the male-specific somatic gonadal precursors with six-moe::GFP. Movie runs from st14 (prior to coalescence) to st16 (after coalescence) when germ …
Video 3
Selected planes from the testis presented in Video 2 to show more clearly the extrusion of an internal germ cell (asterisk).
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | Rho172R | Strutt et al., 1997 | ||
| Genetic reagent (Drosophila melanogaster) | y[1]w[*];Mi{y[+mDint2]=MIC}cv-c[MI00245] | BDSC | RRID:BDSC_30677 | |
| Genetic reagent (Drosophila melanogaster) | P{Sxl-Pe-EGFP.G}G5b, w* | BDSC | RRID:BDSC_32565 | |
| Genetic reagent (Drosophila melanogaster) | w1118; P{10XStat92E-GFP}1 | BDSC | RRID:BDSC_26197 | Bach et al., 2007 |
| Genetic reagent (Drosophila melanogaster) | cv-cM62 | Denholm et al., 2005 | ||
| Genetic reagent (Drosophila melanogaster) | cv-c7 | Denholm et al., 2005 | ||
| Genetic reagent (Drosophila melanogaster) | cv-cC524 | Denholm et al., 2005 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-cv-cWT | Sotillos et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-cv-cGAPmut | Sotillos et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-cv-cΔStART | Sotillos et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-Myc-DLC3WT | Sotillos et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | nos-nod::GFP | A. González Reyes | ||
| Genetic reagent (Drosophila melanogaster) | c587-Gal4 | E. Matunis | RRID:BDSC_67747 | |
| Genetic reagent (Drosophila melanogaster) | six4-moe::GFP | S. DiNardo | Sano et al., 2012 | |
| Genetic reagent (Drosophila melanogaster) | Vasa::GFP | P. Lasko | ||
| Genetic reagent (Drosophila melanogaster) | Perlecan::GFP | Morin et al., 2001 | ||
| Genetic reagent (Drosophila melanogaster) | twist-G4 | Greig and Akam, 1993 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-Myc-DLC3S993N | This paper | Methods | |
| Recombinant DNA reagent | XhoI DLC3WT::pBS | This paper | Methods | |
| Recombinant DNA reagent | XhoI DLC3 S993N::pBS | This paper | Methods | |
| Recombinant DNA reagent | pUASt::Myc-DLC3S993N | This paper | Methods | |
| Sequence-based reagent | DLC3S993N-For | This paper (Sigma-Aldrich) | PCR primers | 5’-TGTACCACTATGTCACCGACA-A-CATGGCACC-3’ |
| Sequence-based reagent | DLC3S993N-Rev | This paper (Sigma-Aldrich) Methods | PCR primers | 5’-TGGGGTGCCATG-T-TGTCGGTGACATAGTG-3’ |
| Antibody | Rat monoclonal anti-VASA | DSHB | AB_760351 | (1:20) |
| Antibody | Mouse monoclonal anti-Nrt | DSHB | AB_528404 | (1:100) |
| Antibody | rat monoclonal anti-DE-cad | DSHB | AB_528120 | (1:50) |
| Antibody | Rabbit polyclonal anti-STAT | E. Bach | Flaherty et al., 2010 | (1:500) |
| Antibody | Mouse monoclonal anti-β-catenin | DSHB | AB_528089 | (1:100) |
| Antibody | mouse monoclonal anti-Eya | DSHB | AB_528232 | (1:100) |
| Antibody | guinea pig polyclonal anti-Ems | U. Walldorf | Walldorf and Gehring, 1992 | (1:5.000) |
| Antibody | Rabbit polyclonal anti-Sox100B | S. Russell | Nanda et al., 2009 | (1:1000) |
| Antibody | guinea pig polyclonal anti-Tj | A. González-Reyes | Díaz-Torres et al., 2021 | (1:1000) |
| Antibody | rabbit polyclonal anti-Perlecan | A. González-Reyes | Díaz-Torres et al., 2021 | (1:1000) |
| Antibody | Rabbit polyclonal anti-GFP | Invitrogen | A11122 | (1:300) |
| Antibody | Chicken polyclonal anti-GFP | Abcam | ab13970 | (1:500) |
| Antibody | mouse anti-βgal | Promega | Z378A | (1:1000) |
| Antibody | Goat polyclonal anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A-11029 | (1:400) |
| Antibody | Goat polyclonal anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-21424 | (1:400) |
| Antibody | Goat polyclonal anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21236 | (1:400) |
| Antibody | Goat polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A-11034 | (1:400) |
| Antibody | Goat polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-21429 | (1:400) |
| Antibody | Goat polyclonal anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21245 | (1:400) |
| Antibody | Goat polyclonal anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-21435 | (1:400) |
| Antibody | Goat polyclonal anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21450 | (1:400) |
| Antibody | Goat polyclonal anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A-48262 | (1:400) |
| Antibody | Goat polyclonal anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 | Invitrogen | A-48263 | (1:400) |
| Antibody | Goat polyclonal anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A32931 | (1:400) |
| Antibody | Donkey polyclonal anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-21432 | (1:400) |
| Software | ImageJ/Fiji | Fiji | http://fiji.sc/ | |
| Software | AdobePhotoshop/Illustrator | Adobe | https://www.adobe.com/ | |
| Software | Prism | GraphPad | https://www.graphpad.com/data-analysis-resource-center/ | |
| Software | Imaris | Oxford Instruments | https://imaris.oxinst.com/ | |
| Software | SeqBuilder/SeqMan | DNASTAR | https://www.dnastar.com/ | |
| Commercial assay, kit | DIG RNA Labelling Kit | Roche | 11 175 025 910 | |
| Commercial assay, kit | Qiagen Plasmid Midi Purification Kit | Qiagen | 12143 | |
| Other | Rhodamine Phalloidin (1:100) | Invitrogen | R415 | High-affinity F-actin probe conjugated to red fluorescence dye TRITC |
| Other | VECTASHIELD Mounting Medium | Vector Laboratories | âH-1000 | Antifade Mounting Medium for preserving fluorescence |
| Other | DAPI (1:10.000) | Thermo Fisher Scientific | 62248 | Blue-fluorescent DNA stain |
| Other | Pfu polymerase | Promega | M774A | PCR polymerase |
| Other | DpnI restriction enzyme | Roche | 10742988001 | DNA restriction enzyme |
| Other | Halocarbon oil 27 | Sigma | H8773 | Inert oil to avoid Drosophila embryos/tissues desiccation. |
