SxlPE activity does not precede SxlPM in the germline

A-B) RNA-FISH (+ Immunofluorescence) against the transcript from SxlPM only (PM RNA) versus the common transcript from SxlPE and SxlPM (PE+PM RNA) in embryonic stage 5 primordial germ cells (PGCs). A) Male PGCs with both PE+PM and PM-probe signals. B) Female PGCs with both PE+PM and PM-probe signals. Arrows mark fluorescent nuclear foci of RNA-FISH signals. Note that a single focus is observed in female PGCs suggesting that X chromosomes may be paired in the germ cells. VAS stains germ cells. C-D) Graphs showing percentage of PGCs with observable RNA-FISH probe signals between stages 3 and 11 of embryogenesis.

SxlPE is sex-specifically activated in the germline

A-B ’) Immunofluorescence of first instar (L1) gonads from flies bearing SxlPE-10.2kb reporter transgene. Note the presence of sex-specific nuclear GFP expression in female germ cells only. Arrows mark nuclei of germ cells. C-D’) Immunofluorescence of first instar (L1) gonads from flies bearing the HA:SxlE1 (Early Sxl) tag. Arrows mark cytoplasmic HA-background staining. E-F’) Immunofluorescence of second instar (L2) gonads from flies bearing the FLAG:SxlL2 (Late Sxl) tag. Arrows mark FLAG-positive germ cells. Note the presence of FLAG (Late Sxl) expression in female germ cells only. The anti-FLAG immunoreactivity in the fat body of both sexes is also present in wild-type stocks with no FLAG-tagged proteins, indicating it is antibody background. VAS stains germ cells.

sisA loss of function in the female germline results in ovarian tumors and germ cell loss

A-F’) Immunofluorescence of adult ovaries to characterize germ cell phenotypes. A-A’) Wildtype ovary from fly with germline-specific mCherry RNAi as control. B-B’) Ovary with germ cell tumors from fly with germline-specific Sxl RNAi. Knockdown of Sxl causes a germline tumor phenotype. C-C’) Ovary with germ cell tumors from fly with germline-specific sisA RNAi (sisA RNAi 1). D-D’) Ovary with severe germ cell loss from fly with strong germline-specific sisA RNAi (2x GAL4). E-G) Animals expressing Cas9 in the germline along with guide RNAs for control (gfp) or sisA to create de novo “G0” mutations in the germline. E-E’) Wildtype ovary from fly with control guide RNAs for gfp. F-F’) Ovary with germ cell tumors from fly with guide RNAs generating mutations in sisA. G-G’) Germ cell-less ovary from fly with guide RNAs generating mutations in sisA. VAS stains germ cells. DAPI stains DNA (nucleus).

sisA is zygotically expressed in the germline

A-C ’) RNA-FISH (+ Immunofluorescence) against sisA in embryonic PGCs (Stages 3-5 shown). Smaller white dashed boxes specify the region zoomed in on, and presented in larger white dashed boxes. Arrows mark fluorescent nuclear foci of RNA-FISH signals. Note that a single focus is observed in female PGCs suggesting that X chromosomes may be paired in the germ cells. VAS stains germ cells. D) Graph showing percentage of PGCs with observable RNA-FISH probe signals between stages 3 and 11 of embryogenesis. sisA is zygotically expressed in PGCs of both sexes. E-G’) Immunofluorescence of embryonic PGCs (Stages 3-5 shown) from flies bearing sfGFP:SisA (SisA tag). Note that this expression is nuclear, consistent with SisA’s characterization as a bZIP transcription factor. Arrows mark GFP-positive germ cell nuclei. VAS stains germ cells.

Sxl expression in the female germline depends on sisA

A-E’) Immunofluorescence of adult ovaries using anti-Sxl antibody (green). Anti-Vas stains the germ cells. A-A’) Wildtype ovary from fly with germline-specific mCherry RNAi. Sxl staining is highest in the early germ cells and decreases in differentiating germ cells. B-B’) Ovary with germ cell tumors from fly with germline-specific Sxl RNAi. Knockdown of Sxl causes a germline tumor phenotype and germ cells lack Sxl staining. C-C’) Ovary with germ cell tumors from fly with germline-specific sisA RNAi. Note that tumorous germ cells lack Sxl staining. Somatic Sxl remains unaffected. D-D’) bam mutant ovary with germline-specific mCherry RNAi. bam mutations cause a germline tumor phenotype and an expansion of germ cells that highly express Sxl. E-E’) bam mutant ovary with germline-specific sisA RNAi. Sxl staining is dramatically reduced in the germline. F-G’) Visualization of endogenous GFP expression (Green) from the SxlPE-10.2kb reporter in L1 stage males (F, F’), females (G, G’) and females expressing sisA RNAi in the germline (nos>sisARNAi). White arrowheads indicate examples of germ cells and yellow arrowheads indicate somatic gonadal cells. H) Quantification of GFP fluorescence intensity in germ cells from samples as in F-G.

sisA is necessary but not sufficient to feminize the germline

A-C ’) Immunofluorescence of adult testes to characterize anti-Sxl staining. A-A’) Wildtype testis from fly with germline-specific overexpression of gfp. B-B’) Testis from fly with germline-specific overexpression of Sxl. Sxl staining is observed in the early germ cells. The anterior tip of the testis looks mildly atrophied. C-C’) Testis from fly with germline-specific overexpression of sisA. No Sxl staining is observed in the germ cells. The testis resembles the wildtype control. D-E) Immunofluorescence of adult ovaries to characterize rescue of sisA loss-of-function by Sxl expression. All animals strongly express UAS-sisA RNAi using two copies of nos-Gal4. D-D’) Germ cell-less ovary from sisA-RNAi flies with ectopic expression of a control protein (GFP) using the otu promoter. Flies of this genotype are sterile. E-E’) Ovary from sisA-RNAi flies with ectopic expression of Sxl under the control of the otu promoter. Note the rescue of the germline and the wildtype appearance of the ovary, indicating normal oogenesis. VAS stains germ cells. SXL stains Sxl. DAPI stains DNA (nucleus).

RNA-FISH against nascent RNA can be used to study Sxl promoter initiation

A) Cartoon showing relevant portion of the Sxl gene locus and span of SxlPM and SxlPE+SxlPM transcripts probed by PM-probes (Cy5, false colored Green) and PE+PM-probes (Cy3, Red) respectively, using RNA-FISH. Locus is drawn to scale. Size of probes is not drawn to scale. CDS: Coding sequences. UTR: Untranslated Region. B-C’’) RNA-FISH (+ Immunofluorescence) against the transcript from SxlPM only (PM RNA) versus the common transcript from SxlPE and SxlPM (PE+PM RNA) in embryonic stage 3 somatic cells. B-B’’) Male somatic cells always show both PE+PM and PM-probe signals. Note that a single focus is observed indicating a single X chromosome. C-C’’) Female somatic cells first have PE+PM-probe signals, indicative of SxlPE activity alone. Note that two foci are observed per nucleus indicating the presence of two X chromosomes that are unpaired. Arrows mark fluorescent nuclear foci of RNA-FISH signals except in C’’ which has no RNA-FISH signals. PE+PM RNA is probed by PE+PM-probes. PM RNA is probed by PM-probes. DAPI stains DNA (nucleus).

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SxlPE requires different cis-regulatory elements in the soma and the germline

A) Cartoon showing relevant portion of the Sxl gene locus and design of transcriptional reporter constructs specific for SxlPE. EGFP reporters include a nuclear localization sequence (nls) to aid in visualization of expression. The 1.5kb somatic enhancer is illustrated. In SxlPE-5.2kb, EGFPnls replaces the CDS of Exon E1. In SxlPE-10.2kb, EGFPnls replaces the CDS of Exon 4. Locus is drawn to scale. EGFPnls is not drawn to scale. CDS: Coding sequences. UTR: Untranslated Region. B-I) Immunofluorescence of developing gonads to characterize SxlPE-5.2kb expression from stage 15 of embryogenesis to the third larval instar stage (L3). Note the presence of sex-specific nuclear GFP expression in female somatic cells. No GFP expression is observed in the germ cells at any stage. Arrows mark GFP-positive somatic cells. J-K) Immunofluorescence of embryonic stage 15 gonads to characterize SxlPE-10.2kb expression. Note the absence of GFP expression in female germ cells but presence of GFP expression in female somatic cells. Arrows mark GFP-positive somatic cells. VAS stains germ cells.

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Endogenous tagging of Early and Late Sxl isoforms

A) Cartoon showing relevant portion of the Sxl gene locus and location of endogenous tags for Early and Late Sxl isoforms. A 2X HA tag is inserted at the N-terminus of Exon E1’s CDS to generate the HA:SxlE1 ‘Early (E) Sxl’ tag. A 3X FLAG tag is inserted at the N-terminus of Exon L2’s CDS to generate the FLAG:SxlL2 ‘Late (L) Sxl’ tag. Locus is drawn to scale. Tags are not drawn to scale. CDS: Coding sequences. UTR: Untranslated Region. B-G) Immunofluorescence of developing gonads to characterize HA:SxlE1 expression from stage 11 of embryogenesis to the second larval instar stage (L2). Note the presence of sex-specific Early Sxl expression in the nuclei of female somatic cells. Arrows mark HA-positive somatic cells. H-I) Immunofluorescence of developing guts to character HA:SxlE1 expression at the first larval instar stage (L1). Note the presence of sex-specific Early Sxl expression in the nuclei of female gut cells. Arrows mark HA-positive gut cells. J-M’) Immunofluorescence of developing gonads to characterize FLAG:SxlL2 expression from the third larval instar stage (L3) to adult stage. Note the presence of sex-specific Late Sxl expression in the cytoplasm of female germ cells. Arrows mark FLAG-positive germ cells. VAS stains germ cells. DAPI stains DNA (nucleus).

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RNAi against most somatic XSEs does not affect the female germline

A-D ’) Immunofluorescence of adult ovaries to characterize germ cell phenotypes resulting from RNAi against somatic XSEs. A-A’) Wildtype ovary from fly with germline-specific mCherry RNAi. B-B’) Ovary from fly with germline-specific sisB RNAi. C-C’) Ovary from fly with germline-specific sisC RNAi. D-D’) Ovary from fly with germline-specific run RNAi. All ovaries resemble the wildtype control. VAS stains germ cells. DAPI stains DNA (nucleus).

sisA loss of function in the female germline

A-B’) Immunofluorescence of adult ovaries to characterize germ cell phenotypes. A-A’) Wildtype ovary from fly with germline-specific mCherry RNAi. B-B’) Ovary with germ cell tumors from fly with germline-specific sisA RNAi (sisA RNAi 2). Note that the severity of tumors is less than those observed with sisA RNAi 1. VAS stains germ cells. DAPI stains DNA (nucleus). C) Graph showing percentage of ovaries exhibiting either wildtype, germ cell tumor, or germ cell-less phenotypes resultant from different loss of function conditions. D) Cartoon showing extended sisA locus with target sites for RNAi and guide RNA targets for G0 CRISPR. Locus is drawn to scale. CDS: Coding sequences. UTR: Untranslated Region.

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Characterizing sisA expression

A-B’) RNA-FISH (+ Immunofluorescence) against sisA in embryonic somatic cells. A-A’) Female somatic cells at stage 3 with sisA RNA-FISH signals. Note that two foci are observed per nucleus indicating the presence of two X chromosomes that are unpaired. Arrows mark fluorescent nuclear foci of RNA-FISH signals. B-B’) Yolk cell nuclei with sisA RNA-FISH signals. Arrows mark fluorescent nuclear foci of RNA-FISH signals. DAPI stains DNA (nucleus). C) Cartoon showing the sisA gene locus and location of endogenous sfGFP tag. An sfGFP tag is inserted at the N-terminus of sisA’s CDS to generate the sfGFP:SisA tag. Locus is drawn to scale. Tag is not drawn to scale. CDS: Coding sequences. UTR: Untranslated Region. D-E’) Immunofluorescence of adult gonads to characterize germ cell phenotypes caused by the N-terminal sfGFP tag on SisA. The gonads resemble wildtype gonads, suggesting that the tag does not impair SisA function. F-F’) Immunofluorescence of female somatic cells at stage 3 expressing sfGFP:SisA. G-G’) Immunofluorescence of yolk cell nuclei expressing sfGFP:SisA. Note that this expression is nuclear, consistent with SisA’s characterization as a bZIP transcription factor. H-H’) Immunofluorescence of embryonic stage 10 PGCs from flies bearing sfGFP:SisA (SisA tag). LAM-B stains nuclear lamina. VAS stains germ cells. DAPI stains DNA (nucleus).

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sisA lies upstream of Sxl in the female germline

A-D ’) Immunofluorescence of adult ovaries to characterize Sxl expression. A-A’) Wildtype ovary from fly with germline-specific mCherry RNAi. Sxl expression is highest in the early germ cells and decreases in differentiating germ cells. B-B’) Ovary with germ cell tumors from fly with germline-specific sisA RNAi (sisA RNAi 2). Note that tumorous germ cells lack Sxl expression. Somatic Sxl remains unaffected. C-C’) Wildtype ovary from fly with mutations in the germline against gfp. Sxl expression is wildtype. D-D’) Ovary with germ cell tumors from fly with mutations in the germline against sisA. Note that tumorous germ cells lack Sxl expression. VAS stains germ cells. SXL stains Sxl. E) Graph showing percentage of ovaries exhibiting either wildtype, germ cell tumor, or germ cell-less phenotypes, with or without rescue of sisA loss of function by Sxl.