Embryos either maternally (M-) or zygotically (Z-) compromised for wun display enhanced levels of ectodermal En and Ptc.

(A-B) Schematics depict the canonical Hh signalling pathway (A), where in the ‘signaling Off/Hh-unbound’ state, Ptc inhibits Smo, and in the absence of signal transduction downstream of Smo, Ci retains its repressor activity (CiR). In the ‘On’ state, Hh is bound to its receptor Ptc, which in turn relieves Smo inhibition. Smo dependent signaling results in generation of transcriptional activator (CiA) form. Panel (B) represents Hh’s role in the formation of segmental boundaries in the embryonic epidermis. En, expressed in the posterior of each para-segment, activates hh. Hh is secreted from the En-positive cells, and binds to Ptc receptor in the neighboring cells, wherein activation of Hh signaling leads to wg expression. Wg, in turn, is secreted from these neighboring cells, and signals reciprocally to the En-positive cells, setting up a tri-partite positive feedback loop between En/Wg/Hh. Together the activities of these three gene products determine the para-segmental boundary and thus defining the segment polarity. (C-E) nos-gal4 virgin females were mated with males of specified genotypes, including negative control (UAS-lex RNAi) and two different UAS-wun RNAi lines, denoted as wun Line1(L1) and wun Line2 (L2). Stage 10 embryos derived from mated females carrying both transgenes (nos-gal4/UAS-RNAi) were stained with anti-Engrailed (En) antibody. Compared to the Control lexM- embryos (A), a significantly higher percentage of wunM-embryos (B and C) display an increased level of En protein as well as broader En stripes. The two UAS-wun RNAi lines behaved similarly. Panel A1’-C1’ shows magnified insets that demonstrate both the elevation in the En levels and broadening of the stripes, indicating additional number of nuclei that stain for En. The ‘+’ mark denotes En positive cells. (F-G) Quantification of percentage of embryos showing increased En intensity and broader stripes (n=15, N=3, ****p<0.0001). Significance was estimated using Ordinary one-way ANOVA (H-J) Recombinant ptc-gal4 UAS-β-gal females were mated with the UAS-RNAi males of the indicated genotype. Stage 10/11 embryos (F1) derived from the respective crosses were stained with anti-β-gal antibody. Compared to the control, ptc-gal4 UAS-β-gal (H), all embryos ptc-gal4 UAS-β-gal/UAS-RNAi (n∼30) show considerably enhanced β-gal expression, which indicates Ptc activation (I and J).

Aberrant PGC migration, induced by ectopic expression of hmgcr can be effectively suppressed by simultaneously overexpressing wun.

(A-C) Stage 13 embryos. Compared to control CS (A) PGCs in elav-gal4 UAS-hmgcr/+ are greatly scattered away from the gonad (B). In elav-gal4 UAS-hmgcr/UAS-wun-GFP embryos, simultaneous overexpression of wun-GFP suppresses the migration defects induced by the ectopic expression of hmgcr in the nervous system (C) A2, B2, C2 are greyscale representations of the Vasa channel. (D-F) Stage 14 embryos of the indicated genotypes. Migration defects caused by ectopic expression of hmgcr in the nervous system are suppressed by simultaneous expression of wun-GFP. (A1-D1) Merged image with anti-Vas and counterstained with Hoechst. (A2-D2) PGCs stained with anti-Vas. D2, E2, F2 are greyscale representations of the vasa channel (G) Quantification of elav-gal4 UAS-hmgcr/+ and elav-gal4 UAS-hmgcr/UAS-wun-GFP embryos belonging to the mentioned classes of scattered PGCs (n=25, 3 replicates). Significance calculated using Two-way ANOVA and Tukey’s multiple comparison test ****p<0.0001, **p<0.01, *p<0.05.

wunM- embryos exhibit an increase in endogenous Hh-GFP puncta in the mesoderm and the adjacent PGCs.

(A-B) Either UAS-lexA RNAi or UAS-wun RNAi males were mated with nos-gal4. virgin females carrying both the transgenes were mated with endogenous Hh-GFP males. Compared to control lexAM- (A), wunM- embryos show a higher number of Hh-GFP puncta (Yellow) in the mesoderm. (C) Individual Hh-GFP puncta were counted in 15 ROIs dorsal to the gut, per genotype (gut outline is marked in all the panels). The increase in Hh-GFP is quantified using an unpaired T-test, ****p<0.0001. (A1-B1). Magnified insets shown in (A1’-B1’), with arrowheads marking Hh-GFP puncta. (D-F) Compared to control, lexAM- (D) wunM- embryos show a higher number of Hh-GFP puncta in their PGCs (E and F), marked with anti-Vasa antibody (red) staining. Magnified insets (D2’-F2’), with arrowheads marking Hh-GFP puncta. (G) The total number of puncta was counted in 15 PGCS per genotype across three replicates, and the percentages of PGCs with 1-2, 3-5, 6-8 and 9+ Hh-GFP puncta are plotted. Significance was calculated using Two-way ANOVA and Tukey’s multiple comparison test (****p<0.0001, ***p<0.001, **p<0.01).

Embryos compromised for wun display aberrant Smo localization.

(A-E) PGC migration through the gut in stage 10 embryos of the indicated genotype. In control lexAM-embryos (A) Smo is localized at the leading edge (A1, magnified inset A2-A4). In contrast, in wunM- PGCs, multiple foci of Smo localisation are observed (B1, magnified inset B2-B4) or are present all over the PGC (C1, magnified inset C2-C4). All panels were equally edited with minimum intensity value at 10 and maximum intensify value at 100 using Fiji Image J. (D) Comparison of percentages of embryos of indicated genotypes having Smo localization as per mentioned classes were quantified (n=40-70 across replicates) using 2-way ANOVA and Tukey’s multiple comparison test *** p<0.001** p<0.01. (E) Germ cell repulsion mediated by Wunen(s) is mediated by their ability to inhibit Hh signaling in the somatic tissues such as gut, ectoderm, nervous system. This influences the path of the migrating PGCs and guides them towards their destination i.e. SGPs, enriched in Hmgcr, which potentiates Hh activity. Additionally, germ cell-germ cell repulsion is also mediated by Wun and germ cell autonomous ability of Wun(s) to inhibit Hh signaling may be critical in this regard and upon compromising wun activity response to Hh signaling gets activated precociously.

Loss of wun leads to PGC migration defects that are qualitatively similar to loss of ptc.

All embryos shown are at Stage 10 (A-D) PGCs in control lexAM-embryo (A) exit the gut through its side and enter the mesoderm. In contrast, PGCs in wunM- embryos either clump precociously (B) or are delayed while migrating through the gut (C). PGCs of wunZ- (D) show a similar phenotype, although, as expected, the penetrance is lower. (E) PGC migration defects on depleting wun are quantified (n=15, 3 replicates) and significance determined using Ordinary one-way ANOVA and Tukey’s multiple comparison test (n=15, 3 replicates), ****p<0.0001, ***p<0.001. (F-G) wun2M- leads to a highly penetrant precocious clumping phenotype in stage 10 (E). wun2Z-embryos also show similar phenotype (F) although not as penetrant. Arrowheads mark mis-migrated PGCs. Additionally, dying PGCs, marked by a star, are observed (H). PGC migration defects on depleting wun2 are quantified (n=10, 2 replicates) and significance determined using Ordinary one-way ANOVA and Tukey’s multiple comparison test (n=10, 2 replicates) **p<0.01. Hatches represent the percentage of embryos that have PGCs that have not individualized by stage10. Embryos were stained with Hoechst to mark DNA (A1-D1 and E1-F1) and anti-Vasa (A2-D2 and E1-F1). (A3-D3 and E3-F3) display merged images.

Sac1 influences PGC migration.

(A-J) Embryos of indicated stages and specified genotypes stained with anti-Vas and Hoechst to mark DNA. (A-F) Embryos that are maternally deprived of indicated genes. Compared to control (A-C) lexAM-, the PGCs of sac1M- embryos have a variety of migration defects such as delay in exiting the gut (D), clumping (E) and inability to migrate towards the SGPs (F). (G-J) Embryos derived from nos-gal4 mothers crossed to UAS-RNAi of indicated genes. Compared to control (G-H), PGCs from sac1Z- embryos also show migration defects including delay in exiting the midgut (I) and have reduced number of PGCs in the coalesced embryonic gonads as compared to control (J). Arrowheads mark mis-migrated PGCs. In the relevant panels outline of the gut (stage 10) or the embryonic gonad (stage 14) are marked with dotted lines and circles respectively. (K-M) Stage 13 embryos derived from nos-gal4-VP16 females crossed to UAS-RNAi males of indicated genotypes resulting in embryos that are zygotically depleted for the individual gene expression in a PGC specific manner. sac1 and sst4 regulate Hh signaling in a qualitatively opposite manner. Compared to the control egfpZ- embryo (K), PGCs in sac1Z- embryos (L) are slow to reach the embryonic gonad. While PGC in stts4z-embryos scatter (M). (N-O) Stage 10 embryos which are maternally deprived of indicated genes. Compared to control (N), simultaneous maternal depletion of sac1 and wun result in worsening of PGC migration defects and leads to more precocious clumping of PGCs (O). (P) Quantification of comparison of PGC migration defects (n=15, 3 replicates) and significance calculated using Ordinary one-way ANOVA and Tukey’s multiple comparison test ****p<0.0001, ***p<0.01. Diagonal lines represent the percentage of embryos that have PGCs that have not individualised by stage10. The gut outline is marked. The gonads at stage 13-14 are marked within dashed circles.

Endogenous Hh-GFP specific puncta in the mesoderm and the adjacent PGCs are enhanced in sac1M- embryos.

(A-B) Either UAS-lexA RNAi (control) or UAS-sac1 RNAi males were mated with nos-Gal4 females. Virgin females carrying both the transgenes were subsequently mated with males expressing endogenous hh-GFP. Embryos resulting from this cross are maternally depleted of indicated genes, express endogenous Hh-GFP and are referred to as lexAM- or sac1M-. Compared to control (A) sac1M-embryos show higher number of Hh-GFP puncta (yellow) in the mesoderm(B). (C)The increase in Hh-GFP is quantified using an unpaired T-test. Individual Hh-GFP puncta were counted in 15 ROIs above gut, marked, per genotype. ****p<0.0001. (A1-B1) Embryos stained with anti-GFP (Yellow). Magnified insets are shown in (A1’-B1’). (D-E) Stage 10 embryos maternally depleted of the indicated gene, expressing endogenous Hh-GFP. Compared to control lexAM- (D), sac1M- embryos show higher number of Hh-GFP puncta in their PGCs. Hh-GFP stained with anti-GFP antibody (D1-E1, yellow) along with PGCS stained with anti-Vas (D2-E2, red). (F) Total number of puncta were counted in 12 PGCS per genotype across three replicates. The percentages of PGCs with 1-3, 4-6, 7-9 and 10+ Hh-GFP puncta are indicated in the graph and significance calculated using Two-way ANOVA and Dunnett’s multiple comparison test. ****p<0.0001, **p<0.01. (D2’-E2’) are magnified insets. Arrowheads mark Hh-GFP puncta.

Substrate specificity of Wun and Sac1.

A heat map plot showing relative lipid levels in 4-8 hour old whole embryos, post-fertilisation after maternal depletion of wun and sac1. lexA knockdown embryos were used as controls. Lipidomic analysis shows that loss of wun leads to a specific increase in DAGs, whereas reducing sac1 results in a considerable accumulation of PIs. Sac I is known to dephosphorylate Phospho-isoforms of Phosphatidylinositols to generate PIs with a reduced number of phosphate residues. The Y-axis represents the lipid classes identified in the embryonic lysates, with individual rows corresponding to different attached fatty acids, listed by chain length and degree of (un)saturation. The X-axis represents the multiple replicate experiments for the genotypes represented.

Embryos maternally compromised for wun2 display enhanced En expression in the ectoderm.

(A-B) Stage 10 embryos maternally depleted of control lexA and wun2. Compared to the negative control lexM- embryos (A), a higher percentage of wun2M-embryos display as broader En stripes and increased levels of En protein (B). (C-D) Quantification of En stripe broadening (C) and increased En expression(D) in embryos. Significance was estimated using Ordinary one-way ANOVA (n=10 N=2), *p<0.5.

Zygotic depletion or overexpression of wun affects En expression in the ectoderm.

(A-B) Stage 11 embryos derived from mat-gal4 mothers crossed to UAS-RNAi males of the indicated genotype. Compared to control egfpZ- (A), wunZ- embryos have increased En expression as well as broadening of En stripes (B). (C) On the contrary, zygotically overexpressing wun (wunOE) leads to decrease in En expression as well as thinning of the En stripe. All embryos were stained with anti-En antibodies and counter labeled with the DNA dye, Hoechst (A1-C1) Panels (A2-C2) display anti-En staining.

Maternal requirement of Hh receptors, ptc, and smo during PGC migration in early embryos.

(A-C) Stage 10 embryos maternally deprived of indicated genes were laid by the nos-gal4>UAS-RNAi of indicated genes. PGCs in control lexAM- embryo display clearly individualized PGCs that traverse across the gut through its dorsal side (A). In contrast, PGCs in ptcM embryos either clump and fail to individualize altogether (B) or show delayed migration through the gut possibly due to incomplete individualization. Conversely, PGCs of smoM- (C) are able to individualize like the control PGCs and exit the gut but start to scatter on their way to the mesoderm. Panels (A1-C1) display Hoechst staining, Panels (A3-C3) displayed merged images. Arrowheads mark mis-migrated embryos. Gut is outlined. (D) Aberrant migration of PGCs is quantified in graph using Ordinary one-way ANOVA and Tukey’s multiple comparison test (n=12,3 replicates), ****p<0.0001. Diagonal lines represent percentage of embryos that have PGCs that have not individualized by stage10.

Embryos derived from mothers compromised for sac1 (sac1M-) show elevated En expression in the ectoderm.

(A-B) Stage 10 Embryos derived from nos-gal4>UAS-RNAi were stained with anti-Engrailed (En) antibody (green) and DNA dye, Hoechst (not shown*). Compared to the negative control lexM- (A1 and A2) embryos a significantly higher percentage of sac1M- (B1 and B2) embryos display expansion in the width of the individual stripes and increased level of En protein and. ‘+’ mark is used for the cells positive for En in a stripe. Panels (A1’,-A2’ and B1’-B2’) show magnified insets. (C-D) Quantification of En stripe broadening (C) and increased En expression (D) in embryos. Significance calculated using Ordinary one-way ANOVA (n=10 N=3), ****p<0.0001.

Zygotic depletion of lipid modification enzymes required for regulating Hh signalling results in germ cell migration defects.

Bar graph showing the percentages of embryos of indicated genotypes with a given number of mis-migrating germ cells. Chi-square Goodness of Fit test was done for each genotype with the Control (n = 38), sac1Z-(n = 48) p = 2.01 x 10-54, stt4Z-(n = 32) p = 4.83 x 10-23.

Genetic interaction between wun and sac1 enhances aberrant Smo localization.

(A-C) Stage 10 embryos of the indicated genotype. In control lexAM-embryos (A) Smo is localized at the leading edge (A0, magnified inset A1-A3). In contrast, in sac1M-; wunM-PGCs, multiple foci of Smo localisation are observed (B0, magnified inset B1-B3) or are present all over the PGC (C0, magnified inset C1-C3). All panels were equally edited with minimum intensity value at 10 and maximum intensify value at 100 using Fiji Image J. (D) Comparison of percentages of embryos of indicated genotypes having Smo localization as per mentioned classes were quantified (n=40-70 across replicates) using 2-way ANOVA and Tukey’s multiple comparison test *** p<0.001** p<0.01.