Figures and data in A conserved function of Human DLC3 and Drosophila Cv-c in testis development

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Videos
Tables
Additional files

6 figures, 3 videos, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement

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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 the p.R887C and the p.S993N mutations. The protein is shown in cartoon representation, with the GAP domain represented in orange and the StART domain in blue. (D) Structure of the StART domain of human DLC3/StARD8. The distance between the Cα atoms of residues S/N993 and L1079 (shown in licorice representation) was used to determine the open and closed transitions arising from the motion of the Ω1 loop. (E) Distribution of the distance between the Cα atoms of S/N993 and L1079 in atomistic simulations of the WT and S993N systems.

Figure 1—figure supplement 1

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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 omitted for clarity. (B) Time-trace of minimum distance between the protein and the bilayer, for eight replicas. (C) Normalized interaction frequency for protein residues with bilayers. The shaded region in cyan represents the Ω1 loop of the domain. (D) Structure of the StART domain model from AlphaFold, with the Ω1 loop residues that exhibit the highest frequency of interaction highlighted in cyan.

Figure 2 with 1 supplement

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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) and Cv-c (magenta) StART domains. (C) Schematic representation of the cell types in a *Drosophila* testis at st17. Germ cells, green; pigment cells, magenta; somatic gonadal cells, grey; male-specific somatic gonadal cells, pink. RNA in situ hybridization of male (D) and female (E) st17 embryos shows general transcription of *cv-c* in the mesoderm. The right panels in D–I are close ups of the arrowed region in the central panels. (D) In the testis, comparable levels of mRNA puncta can be detected in the somatic mesoderm and in the gonadal mesoderm cells surrounding the male germ cells as are clearly observed in the male-specific somatic gonad precursors (msSGPs) marked by Eya (magenta, indicated by an arrowhead in grey panels and an arrow in the close up). (E) In the ovary, marginal levels of *cv-c* mRNA expression are observed in the gonadal mesodermal cells, creating a halo of decreased number of puncta surrounding the female germ cells contrasting with the *cv-c* expressing adjacent somatic mesodermal cells (arrow in close up). (**F–I**) Cv-c::GFP protein expression in male and female embryos. (**F, H**) In the testis Cv-c::GFP is detected in the gonadal mesoderm surrounding the germ cells including the male msSGPs (Eya, magenta F) and the pigment cell precursors (Ems, magenta H). (**G, I**) In females, no substantial GFP signal is detected in the gonadal mesoderm surrounding the germ cells. Note in (**F, H**) that Cv-c::GFP signal in the gonad mesoderm cells allows tracing the testis contour, while in ovaries (**G, I**) this is not possible. Higher levels of Cv-c::GFP are present in the ectodermally derived trachea and hindgut. In close ups white arrows point to membranes close to the germ cells, yellow arrows to the membrane of gonad mesodermal cells. In males, Cv-c::GFP can be detected in the membranes between gonadal and somatic mesodermal cells (**F, H**) whilst in females GFP can only be detected outside the ovary in the membrane of the somatic mesoderm (G, I asterisks). Scale bar: 10 and 5 µm in close ups.

Figure 2—figure supplement 1

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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 of activation (right panels show Cv-c::GFP in grey). (**C–F**) JAK/STAT testis activity in *cv-c^C524* mutants in heterozygosis (**C, E**) or homozygous (D, F) testis. Neither STAT localization (magenta), (**C, D**) nor 10XSTAT-GFP reporter expression (green), (**E, F**) are modified in *cv-c^C524* mutants. Panels (**C, D**) are stained with anti-STAT (magenta or grey in right panels), (**C–F**) with anti-Ems (grey), (**E, F**) with anti-Vasa (magenta), and (**C, F**) with anti-GFP (green or grey in right panels). Scale bar: 10 µm.

Figure 3 with 1 supplement

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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 the AJs with anti-β-catenin (grey membranes). Right panels show Eya and β-catenin channel. (A) In the control testis, germ cells are ensheathed by thin mesoderm extensions produced by the interstitial cells detectable by β-catenin staining. Similar germ cell ensheathment is observed in *cv-c^C524* ovaries (B), while in *cv-c^C524* testis (C) some germ cells become extruded from the gonad and are not enveloped by β-catenin (arrowhead). (**D–G**) Testes labelled with mesodermal specific markers to detect the pigment cells (Ems, magenta D–G) or the msSGPs (Sox100B, green F, G) in heterozygous (**D, F**) or *cv-c^C524* homozygous mutant embryos (**E, G**). Grey channels in right panels correspond to β-catenin in (**D-G**), Ems in (**D, E**), or Sox100B in (**F, G**). All mesoderm cell types are specified in *cv-c* mutant testis despite morphological aberrations resulting in the pigment cell layer’s discontinuity (compare D and F with E and G). (**H–J**) Testes stained with anti-Vasa (green) to label the germ cells and phalloidin (grey and right panels) to show actin filaments in heterozygous (H) or homozygous *cv-c^C524* mutants (I). Germ cells in mutant testes present protrusions compatible with migratory movements (arrowheads). Z sections are shown below H, I panels. Scale bar: 10 µm. (J) Quantification of blebbing cells in wild-type or mutant *cv-c* background using Fisher test; ****p value <2.2e−16 (*nos::GFP N* = 575 and *nos::GFP;cv-c^C524 N* = 744) (Figure 3—source data 1).

Figure 3—source data 1 Raw data.: https://cdn.elifesciences.org/articles/82343/elife-82343-fig3-data1-v2.xlsx
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Figure 3—figure supplement 1

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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-c^C524* mutant females, germ cells form a continuous filamentous actin structure around the cortex. (**C, D**) In homozygous *cv-c^C524* mutant males, the actin filament is discontinuous at the position where blebs form (arrowheads). Scale bar: 10 µm.

Figure 4

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Rescue of *cv-c* mutant testes.

(A) Schematic representation of Cv-c and DLC3 protein variants studied. Rescue of the dysgenic testis of *cv-c^C524* homozygous mutant males after expressing the specified (B) DLC3 or (C) Cv-c protein variants under UAS control with the pan mesodermal *twi-Gal4* line. Phenotypic rescue is shown as percentage of testes where all germ cells are encapsulated inside the testis. Representative images of testes in (D) control homozygous *cv-c^C524* animals, or homozygous *cv-c^C524* animals expressing in the mesoderm either (E) *UAS-DLC3^WT*, (F) *UAS-DLC3^S993N*, (G) *UAS-Cv-c^WT,* (H) *UAS-Cv-c^GAPmut*, or (I) *UAS-Cv-c^ΔStART*. Arrows in D, F, I point to extruded germ cells that are not surrounded by β-catenin. Testes are stained with anti-Vasa to label the germ cells (green), anti-Ems to label the pigment cells (purple), and anti-Eya and anti-β-catenin to label the male-specific somatic gonad precursors (msSGPs) and the membranes ensheathing the germ cells, respectively (grey in lower panels). Scale bar: 10 µm. Fisher test, *Cv-c^WT* p = 0.0017 (N = 34)*, Cv-c^GAPmut* p < 0.0001 (N = 56), *DLC3^WT* p < 0.0001 (N = 28), *Cv-c^ΔStART* p = 0.3005 (N = 31), and *DLC3^S993N* p = 0.3180 (N = 38) (ns, p > 0.05; **p < 0.001; ****p < 0.0001) (Figure 4—source data 1).

Figure 4—source data 1 Raw data.: https://cdn.elifesciences.org/articles/82343/elife-82343-fig4-data1-v2.xlsx
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Figure 5 with 1 supplement

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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 are observed in hemizygous *cv-c⁷*/*Df(3R)Exel6267* embryos carrying a *cv-c⁷* allele which inactivates the RhoGAP domain’s function. (**C, D**) Hemizygous *cv-c^C524*/*Df(3R)Exel6267* or (**E, F**) *cv-c^M62*/*Df(3R)Exel6267* showing extruded germ cells. (**G, H**) *Rho1^72R* mutant embryos have smaller testes without extruded germ cells. (**I, J**) Homozygous *cv-c^M62* mutants present extruded germ cells. (**K, L**) Homozygous *Rho1^72R cv-c^M62* double mutant embryos present smaller testis with extruded germ cells. Arrows point to GCs outside the gonads. Note that β-catenin envelops all germ cells in (**A, B**) and (**G, H**) while in (**C–F**) and (**I–L**) some germ cells are not surrounded (arrows). Z sections are shown under all panels. Scale bar: 10 µm.

Figure 5—figure supplement 1

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*cv-c^C524* 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-c^C524* animals, (B) control *c587-Gal4 cv-c^C524* homozygous animals or (C) homozygous *cv-c^C524* animals expressing either *UAS-DLC3^WT* or (D) *UAS-DLC3^S993N*. Arrowheads in B and D point to extruded germ cells that are not surrounded by E-Cad. *DLC3^WT* expression rescues the *cv-c^C524* testis defects whilst *DLC3^S993N* does not. Germ cells are stained with anti-Vasa (green); E-Cad, to label the membranes ensheathing the germ cells (grey) and anti-Ems to label the pigment cells (magenta). Scale bar: 10 µm. (E) Percentage of abnormal testes (blue bars) versus phenotypically wild-type rescued testis (purple bars) in the different genotypes analysed. (A testis is considered abnormal when one or more germ cells are extruded from the gonad.) (F) Comparison of rescue capability of the same UAS lines when expressed with the *twi-Gal4* pan-mesodermal (orange bars) or the *c587-Gal4* somatic gonadal cell driver lines (blue bars) shown as percentage of abnormal testes (Figure 5—figure supplement 1). Fisher test, *Cv-c^WT* p = 0.0344 (N = 89)*, Cv-c^GAPmut* p < 0.0001 (N = 135), *Cv-c^ΔStART* p = 1 (N = 31), *DLC3^WT* p = 0.0053 (N = 45), and *DLC3^S993N* p = 0.5722 (N = 50) (ns; * p > 0.05; **p < 0.001; ****p < 0.0001).

Figure 5—figure supplement 1—source data 1 Raw data.: https://cdn.elifesciences.org/articles/82343/elife-82343-fig5-figsupp1-data1-v2.xlsx
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Figure 6 with 1 supplement

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Ensheathment defects in *cv-c* mutant testes.

(**A, C, E**) Wild-type and (**B, D, F**) *cv-c^C524* 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 membranes (grey in right panels). (**A, A’**) In the wild-type E-cad highlights contacts between the germ cells and the interstitial somatic gonadal cells surrounding each germ cell reflecting their correct ensheathment. (**B, B’**) In *cv-c* mutant testes, several germ cells inside the testis and all the extruded ones (arrowheads) are not surrounded by E-cad labelling membranes indicating incorrect ensheathment. (**C, C’**) Neurotactin in the wild-type testis reflects correct germ cell ensheathment. (**D, D’**) In *cv-c^C524* mutants little Neurotactin expression is detected inside the testis. Nuclei of Tj-labelled somatic gonadal cells (magenta and grey in right panels) are detected between the GCs inside wild-type testes (**E, E’**) but not in *cv-c^C524* mutant testes (**F, F’**). (**G, G’**) Heterozygous and (**H, H’**) homozygous *cv-c^C524* testes labelling the extracellular matrix with Perlecan::GFP (Pcl, grey in right panels) and stained with anti-Vasa (green) and anti-Ems (magenta) to label the GCs and the pigment cells, respectively. (**G, G’**) The wild-type testis is enclosed by a Perlecan containing extracellular matrix (white). In *cv-c^C524* mutant testis a discontinuous extracellular matrix is observed where the GCs are outside the gonad (H, H’, yellow arrowheads). Z sections are shown under all panels. Scale bar: 10 µm. (I) Quantification of broken and continuous Perlecan Extracellular matrix (ECM) layer in *cv-c^C525* heterozygous or homozygous mutants using Fisher test; ****p value less than 0.0001 (*cv-c^C524*/*TM6B N* = 54 and *cv-c^C524 N* = 24) (Figure 6—source data 1). (J) Interpretation of the testis degeneration in wild type and DLC3/Cv-c mutants. (1) The StART domain of DLC3/Cv-c has a Rho-independent function stabilizing cell interactions between germ cells and somatic support cells. Stable cell–cell interaction among the cells of the gonadal niche allows them to settle down in the testis. (2) In DLC3^S997N/Cv-c^ΔStART testis cell–cell interactions become compromised, cells lose their cohesion and separate from the gonadal niche. (3) After losing communication with somatic cells, germ cells become extruded from the gonadal niche initiating an erratic migrating behaviour. (4) Progressive loss of germ cells leads to gonad degeneration.

Figure 6—source data 1 Raw data.: https://cdn.elifesciences.org/articles/82343/elife-82343-fig6-data1-v2.xlsx
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Figure 6—figure supplement 1

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ECM rescue of *cv-c* mutant testes.

Representative images of testes in (A) control *twi-Gal4; cv-c^C524* heterozygous animals or homozygous *cv-c^C524* animals expressing in the somatic mesoderm cells either (B) *UAS-DLC3^WT*, (C) *UAS-DLC3^S993N*, (D) *UAS-Cv-c^WT,* (E) *UAS-Cv-c^GAPmut*, or (F) *UAS-Cv-c^ΔStART*. Germ cells are stained with anti-Vasa (magenta), pigment cells with anti-Ems (grey) and the ECM surrounding the gonad with anti-Perlecan (green and grey in lower panels). Scale bar: 10 µm. Comparison of rescue capability of the same UAS lines when expressed with the *twi-Gal4* pan-mesodermal (G) or the *C587-Gal4* somatic gonadal cell (H) driver lines shown as percentage of broken ECM detected by abnormal Perlecan distribution. Fisher test, (G) *UAS-DLC3^WT* p = 0.0001 (N = 39), *UAS-DLC3^S993N* p = 0.1442 (N = 28), *UAS-Cv-c^WT* p = 0.0001 (N = 74), *UAS-Cv-c^GAPmut* p < 0.0001 (N = 42), and *UAS-Cv-c^ΔStART* p = 1 (N = 18). (F) *UAS-DLC3^WT* p < 0.0001 (N = 32), *UAS-DLC3^S993N* p = 0.5435 (N = 38), *UAS-Cv-c^WT* p < 0.0001 (N = 35), *UAS-Cv-c^GAPmut* p < 0.0001 (N = 44), and *UAS-Cv-c^ΔStART* p = 0.2385 (N = 22) (ns, p > 0.05; ****p < 0.0001) (Figure 6—source data 1).

Figure 6—figure supplement 1—source data 1 Raw data.: https://cdn.elifesciences.org/articles/82343/elife-82343-fig6-figsupp1-data1-v2.xlsx
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Videos

Video 1

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posterframe for video — In vivo gonad coalescence of a control heterozygous *cv-c^C524/+* 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

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Video 3

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Tables

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Drosophila melanogaster)	Rho1^72R			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-c^M62			Denholm et al., 2005
Genetic reagent (Drosophila melanogaster)	cv-c⁷			Denholm et al., 2005
Genetic reagent (Drosophila melanogaster)	cv-c^C524			Denholm et al., 2005
Genetic reagent (Drosophila melanogaster)	UAS-cv-c^WT			Sotillos et al., 2018
Genetic reagent (Drosophila melanogaster)	UAS-cv-c^GAPmut			Sotillos et al., 2018
Genetic reagent (Drosophila melanogaster)	UAS-cv-c^ΔStART			Sotillos et al., 2018
Genetic reagent (Drosophila melanogaster)	UAS-Myc-DLC3^WT			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-DLC3^S993N	This paper		Methods
Recombinant DNA reagent	XhoI DLC3^WT::pBS	This paper		Methods
Recombinant DNA reagent	XhoI DLC3 ^S993N::pBS	This paper		Methods
Recombinant DNA reagent	pUASt::Myc-DLC3^S993N	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.