sisterless A is required for activation of Sex lethal in the Drosophila germline

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, expression of Sex-lethal (Sxl) protein determines sexual identity and drives female development. Functional Sxl protein is absent from males where splicing includes a termination codon-containing "poison" exon. Early during development, in the soma of female individuals, Sxl expression is initiated by an X chromosome counting mechanism that activates the Sxl establishment promoter (SxlPE) to produce an initial amount of Sxl protein. This then suppresses the inclusion of the "poison" exon, directing the constructive splicing of Sxl transcripts emerging from the Sxl maintenance promotor (SxlPM) which is activated at a later stage during development irrespective of sex. This autoregulatory loop maintains Sxl expression and commits to female development.

Sxl also determines the sexual identity of the germline. Here Sxl expression generally follows the same principles as in somatic tissues, but the way expression is initiated differs from the soma. This regulation has so far remained elusive.

In the presented manuscript, Goyal et al. show that activation of Sxl expression in the germline depends on additional regulatory DNA sequences, or sequences different from the ones driving initial Sxl expression in the soma. They further demonstrate that sisterless A (sisA), a transcription factor that is required for activation of Sxl expression in the soma, is also necessary, but not sufficient, to initiate the expression of functional Sxl protein in female germ cells. sisA expression precedes Sxl induction in the germline and its ablation by RNAi results in impaired expression of Sxl, formation of ovarian tumors, and germline loss, phenocopying the loss of Sxl. Intriguingly, this phenotype can be rescued by the forced expression of Sxl, demonstrating that the primary function of sisA in the germline is the induction of Sxl expression.

Strengths:

The clever design of probes (for RNA FISH) and reporters allowed the authors to dissect Sxl expression from different promoters to get novel insight into sex-specific gene regulation in the germline. All experiments are carefully controlled. Since Sxl regulation differs between the soma and the germline, somatic tissues provide elegant internal controls in many experiments, ensuring e.g. functionality of the reporters. Similarly, animals carrying newly generated alleles (e.g. genomic tagging of the Sxl locus) are fertile and viable, demonstrating that the genetic manipulation does not interfere with protein function. The conclusions drawn from the experimental data are sound and advance our understanding of how Sxl expression is induced in the female germline.

Weaknesses:

The assays employed by the authors provide valuable information on when Sxl promoters become active. However, since no information on the stability of the gene products (i.e. RNA and protein) is available, it remains unclear when the SxlPE promoter is switched off in the germline (conceptually it only needs to be active for a short time period to initiate production of functional Sxl protein). As correctly stated by the authors, the persisting signals observed in the germline might therefore not reflect the continuous activity of the SxlPE promoter.

Mapping of regulatory elements and their function: SxlPE with 1.5 kb of flanking upstream sequence is sufficient to recapitulate early Sxl expression in the soma. The authors now provide evidence that beyond that, additional DNA sequences flanking the SxlPE promoter are required for germline expression. However, a more precise mapping was not performed. Also, due to technical limitations, the authors could not precisely map the sisA binding sites. Since this protein is also involved in the somatic induction of Sxl, its binding sites likely reside in the region 1.5kb upstream of the SxlPE promoter, which has been reported to be sufficient for somatic regulation. The regulatory role of the sequences beyond SxlPE-1.5kb therefore remains unaddressed and it remains to be investigated which trans-acting factor(s) exert(s) its/their function(s) via this region.

The central question of how Sxl expression is initiated and controlled in the germline still remains unanswered. Since sisA is zygotically expressed in both the male and the female germline (Figure 4D), it is unlikely the factor that restricts Sxl expression to the female germline.

How does weak expression of Sxl in male tissues or expression above background after knockdown of sisA reconcile with the model that an autoregulatory feedback loop enforces constant and clonally inheritable Sxl expression once Sxl is induced? Is the current model for Sxl expression too simple or are we missing additional factors that modulate Sxl expression (such as e.g. Sister of Sex-lethal)? While I do not expect the authors to answer these questions, I would expect them to appropriately address these intriguing aspects in the discussion.

Reviewer #2 (Public Review):

Summary:

The authors wanted to determine whether cis-acting factors of Sxl - two different Sxl promoters in somatic cells - regulate Sxl in a similar way in germ cells. They also wanted to determine whether trans-acting factors known to regulate Sxl in the soma also regulate Sxl in the germline.

Regarding the cis-acting factors, they examine the Sxl "establishment promoter" (SxlPE) that is activated in female somatic cells by the presence of two X chromosomes. Slightly later in development, dosage compensation equalizes X chromosome expression in males and females and so X chromosomes can no longer be counted. The second Sxl promoter is the "maintenance promoter," (SxlPM), which is activated in both sexes. The mRNA produced from the maintenance promoter has to be alternatively splicing from early Sxl protein generated earlier in development by the PE. This leads to an autoregulatory loop that maintains Sxl expression in female somatic cells. The authors used fluorescent in situ hybridization (FISH) with oligopaints to determine the temporal activation of the PE or PM promoters. They find that - unlike the soma - the PE does not precede the PM and instead is activated contemporaneously or later than the PM - this is confusing with the later results (see below). Next, they generated transcriptional reporter constructs containing large segments of the Sxl locus, the 1.5 kb used in somatic studies, a 5.2 kb reporter, and a 10.2 kb. Interestingly the 1.5 kb reporter that was reported to recapitulate Sxl expression in soma and germline was not observed by the authors. The 5.2 kb reporter was observed in female somatic cells but not in germ cells. Only when they include an additional 5 kb downstream of the 5.2 kb reporter (here the 10.2 kb reporter) they did see expression in germ cells but this occurred at the L1 stages. Their data indicate that Sxl activity in the germ requires different cis-regulation than the soma and that the PE is activated later in germ cells than in somatic cells. The authors next use gene editing to insert epitope tags in two distinct strains in the hopes of creating an early Sxl and a later Sxl protein derived from the PE and PM, respectively. The HA-tagged protein from the PE was seen in somatic cells but never in the germline, possibly due to very low expression. The FLAG-tagged late Sxl protein is observed in L2 germ cells. Because the early HA-Sxl protein is not perceptible in germ cells, it is not possible to conclude its role in the germline. However, because late FLAG-Sxl was only observed in L2 germ cells and the PE was detected in L1, this leaves open the possibility that PE produces early HA-Sxl (which currently cannot be detected), which then alternatively splices the transcript from the PM. In other words, the soma and germline could have a similar temporal relationship between the two Sxl promoters. While I agree with the authors about this conclusion, the earlier work with the oligopaints leads to the conclusion that SE is active after PM. This is confusing.

Next, the authors wanted to turn their attention to the trans-acting factors that regulate Sxl in the soma, including Sisterless A (SisA), SisB, Runt, and the JAK/STAT ligand Unpaired. Using germline RNAi, the authors found that only knockdown of SisA causes ovarian tumors, similar to the loss of Sxl, suggesting that SisA regulates Sxl (ie the PE) in both the soma and the germline. They generated a SisA null allele using CRISPR/Cas9 and these animals had ovarian tumors and germ cell-less ovaries. FISH revealed that sisA is activated in primordial germ cells in stages 3-6 before the activation of Sxl. They used CRISPR-Cas9 to generate an endogenously-tagged SisA and found that tagged SisA was expressed in stage 3-6 PCGs, which is consistent with activating PE in the germline. They showed that sisA is upstream of Sxl as germline depletion of sisA led to a significant decrease in expression from the 10.2 kb PE reporter and in SXL protein. The authors could rescue the ovarian tumors and loss of Sxl protein upon germline depletion of sisA by supplying Sxl from another protein (the otu promoter). These data indicate that sisA is necessary for Sxl activation in the germline. However, ectopic sisA in germ cells in the testis did not lead to ectopic Sxl, suggesting that sisA is not sufficient to activate Sxl in the germline.

Strengths:

(1) The genetic and genomic approaches in this study are top-notch and they have generated reagents that will be very useful for the field.

(2) Excellent use of powerful approaches (oligo paint, reporter constructs, CRISPR-Cas9 alleles).

(3) The combination of state of art approaches and quantification of phenotypes allows the authors to make important conclusions.

Weaknesses:

(1) Confusion in line 127 (this indicates that SxlPE is not activated before SxlPM in the germline) about PE not being activated before the PM in the germline when later figures show that PE is activated in L1 and late Sxl protein is seen in L2. It would be helpful to the readers if the authors edited the text to avoid this confusion. Perhaps more explanation of the results at specific points would be helpful.

Reviewer #3 (Public Review):

Summary:

The mechanisms governing the initial female-specific activation of Sex-lethal (Sxl) in the soma, the subsequent maintenance of female-specific expression and the various functions of Sxl in somatic sex determination and dosage compensation are well documented. While Sxl is also expressed in the female germline where it plays a critical role during oogenesis, the pathway that is responsible for turning Sxl on in germ cells has been a long-standing mystery. This manuscript from Goyal et al describes studies aimed at elucidating the mechanism(s) for the sex-specific activation of the Sex-lethal (Sxl) gene in the female germline of Drosophila.

In the soma, the Sxl establishment promoter, Sxl-Pe, is regulated in pre-cellular blastoderm embryos in somatic cells by several X-linked transcription factors (sis-a, sis-b, sis-c and runt). At this stage of development, the expression of these transcription factors is proportional to gene dose, 2x females and 1x in males. The cumulative two-fold difference in the expression of these transcription factors is sufficient to turn Sxl-Pe on in female embryos. Transcripts from the Sxl-Pe promoter encode an "early" version of the female Sxl protein, and they function to activate a splicing positive autoregulatory loop by promoting the female-specific splicing of the initial pre-mRNAs derived from the Sxl maintenance promoter, Sxl-Pm (which is located upstream of Sxl-Pm). These female Sxl-Pm mRNAs encode a Sxl protein with a different N-terminus from the Sxl-Pe mRNAs, and they function to maintain female-specific splicing in the soma during the remainder of development.

In this manuscript, the authors are trying to understand how the Sxl-Pm positive autoregulatory loop is established in germ cells. If Sxl-Pe is used and its activation precedes Sxl-Pm as is true in the soma, they should be able to detect Sxl-Pe transcripts in germ cells before Sxl-Pm transcripts appear. To test this possibility, they generated RNA FISH probes complementary to the Sxl-Pe first exon (which is part of an intron sequence in the Sxl-Pm transcript) and to a "common sequence" that labels both Sxl-Pe and Sxl-Pm transcripts. Transcripts labeled by both probes were detected in germ cells beginning at stage 5 (and reaching a peak at stage 10), so either the Sxl-Pm and Sxl-Pe promoters turn on simultaneously, or Sxl-Pe is not active.

They next switched to Sxl-Pe reporters. The first Sxl-Pe:gfp reporter they used has a 1.5 kb upstream region which in other studies was found to be sufficient to drive sex-specific expression in the soma of blastoderm embryos. Also like the endogenous Sxl gene it is not expressed in germ cells at this early stage. In 2011, Hashiyama et al reported that this 1.5 kb promoter fragment was able to drive gfp expression in Vasa-positive germ cells later in development in stage 9/10 embryos. However, because of the high background of gfp in the nearby soma, their result wasn't especially convincing. Though they don't show the data, Goyal et al indicated that unlike Hashiyama et al they were unable to detect gfp expressed from this reporter in germ cells. Goyal et al extended the upstream sequences in the reporter to 5 kb, but they were still unable to detect germline expression of gfp.

Goyal et al then generated a more complicated reporter which extends 5 kb upstream of the Sxl-Pe start site and 5 kb downstream-ending at or near 4th exon of the Sxl-Pm transcript (the Sxl-Pe10 kb reporter). (The authors were not explicit as to whether the 5 kb downstream sequence extended beyond the 4th exon splice junction-in which case splicing could potentially occur with an upstream exon(s)-or terminated prior to the splice junction as seems to be indicated in their diagram.) With this reporter, they were able to detect sex-specific gfp expression in the germline beginning in L1 (first instar larva). With the caveat that gfp detection might be delayed compared to the onset of reporter activation, these findings indicated that the sequences in the reporter are able to drive sex-specific transcription in the germline at least as early as L1.

The authors next tagged the N-terminal end of the Sxl-Pe protein with HA (using Crispr/Cas9) and the N-terminal end of Sxl-Pm protein with Flag. They report that the HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis. Somatic HA-Sxl-Pe protein persists into L1, but is no longer detected in L2. However, while somatic HA-Sxl-Pe protein is detected, they were unable to detect HA-Sxl-Pe protein in germ cells. In the case of FLAG-Sxl-Pm, it could first be detected in L2 germ cells indicating that at this juncture the Sxl-positive autoregulatory loop has been activated. This contrasts with Sxl-Pm transcripts which are observed in a few germ cells at stage 5 of embryogenesis, and in most germ cells by stage 10. The authors propose (based on the expression pattern of the Sxl-Pe10kb reporter and the appearance of Flag-Sxl-Pm protein) that Sxl-Pe comes on in germ cells in L1, and that the Sxl-Pe protein activates the female splicing of Sxl-Pm transcripts, giving detectable Flag-Sxl-Pm proteins beginning in L2.

To investigate the signals that activate Sxl-Pe in germ cells, the authors tested four of the X-linked genes (sis-a, sis-b, sis-c, and runt) that function to activate Sxl-Pe in the soma in early embryos. RNAi knockdown of sis-b, sis-c, and runt had no apparent effect on oogenesis. In contrast, knockdown of sis-a resulted in tumorous ovaries, a phenotype associated with Sxl mutations. (Three different RNAi transgenes were tested-two gave this phenotype, the third did not.) Sxl-Pe10kb reporter activity in L1 female germ cells is also dependent on sis-A.

Several approaches were used to confirm a role for sis-a in a) oogenesis and b) the activation of the Sxl-Pm autoregulatory loop. They showed that sis-a germline clones (using tissue-specific Crispr/Cas9 editing) resulted in the tumorous ovary phenotype and reduced the expression of Sxl protein in these ovaries. They found that sis-a transcripts and GFP-tagged Sis-A protein are present in germ cells. Finally, they showed tumorous ovary phenotype induced by germline RNAi knockdown of sis-a can be partially rescued by expressing Sxl in the germ cells.

Critique:

While this manuscript addresses a longstanding puzzle - the mechanism activating the Sxl autoregulatory loop in female germ cells-and likely identified an important germline transcriptional activator of Sxl, sis-a, the data that they've generated doesn't make a compelling story. At every step, there are puzzle pieces that don't fit the narrative. In addition, some of their findings are inconsistent with many previous studies.

(1) The authors used RNA FISH to time the expression of Sxl-Pe and Sxl-Pm transcripts in germ cells. Transcripts complementary to Sxl-Pe and Sxl-Pm were detected at the same time in embryos beginning at stage 5. This is not a definitive experiment as it could mean a) that Sxl-Pe and Sxl-Pm turn on at the same time, b) that Sxl-Pe comes on after Sxl-Pm (as suggested by the Sxl-Pe10kb reporter) or c) Sxl-Pe never comes on.

(2) Hashiyama et al reported that they detected gfp expression in stage 9/10 germ cells from a 1.5 kb Sxl-Pe-gfp. As noted above, this result wasn't entirely convincing and thus it isn't surprising that Goyal et al were unable to reproduce it. Extending the upstream sequences to just before the 1st exon of Sxl-Pm transcripts also didn't give gfp expression in germ cells. Only when they added 5 kb downstream did they detect gfp expression. However, from this result, it isn't possible to conclude that the Sxl-Pe promoter is actually driving gfp expression in L1 germ cells. Instead, the Sxl promoter active in the germ line could be anywhere in their 10 kb reporter.

(3) At least one experiment suggests that Sxl-Pe never comes on in germ cells. The authors tagged the N-terminus of the Sxl-Pe protein with HA and the N-terminus of the Sxl-Pm protein with Flag. Though they could detect HA-Sxl-Pe protein in the soma, they didn't detect it in germ cells. On the other hand, the Flag-Sxl-Pm protein was detected in L2 germ cells (but not earlier). These results would more or less fit with those obtained for the 10 kb reporter and would support the following model: Prior to L1, Sxl-Pm transcripts are expressed and spliced in the male pattern in both male and female germ cells. During L1, Sxl protein expressed via a mechanism that depends upon a 10 kb region spanning Sxl-Pe (but not on Sxl-Pe) is produced and by L2 there are sufficient amounts of this protein to switch the splicing of Sxl-Pm transcripts from a male to a female pattern-generating Flag-tagged Sxl-Pm protein.

(4) The 10kb reporter is sex-specific, but not germline-specific. The levels of gfp in female L1 somatic cells are equal to if not greater than those in L1 female germ cells. That the Sxl-Pe10kb reporter is active in the soma complicates the conclusion that it represents a germ line-specific promoter. Germline activity is, however, sensitive to sis-A knockdowns which is plus. Presumably, somatic expression of the reporter wouldn't be sensitive to a (late) sis-A knockdown- but this wasn't shown.

(5) Their results with the HA-Sxl-Pe protein don't fit with many previous studies-assuming that the authors have explained their results properly. They report that HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis and that it then persists till L2. However, previous studies have shown that Sxl-Pe transcripts and then Sxl-Pe proteins are first detected in ~NC11-NC12 embryos. In RNase protection experiments, the Sxl-Pe exon is observed in 2-4 hr embryos, but not detected in 5-8 hr, 14-12 hr, L1, L2, L3, or pupae. Northerns give pretty much the same picture. Western blots also show that Sxl-Pe proteins are first detectable around the blastoderm stage. So it is not at all clear why HA-Sxl-Pe proteins are first observed at stage 9 which, of course, is well after the time that the Sxl-Pm autoregulatory loop is established.

Given the obvious problems with the initial timing of somatic expression described here, it is hard to know what to make of the fact that HA-tagged Sxl-Pe proteins aren't observed in germ cells.

As for the presence of HA-Sxl-Pe proteins later than expected: While RNase protection/Northern experiments showed that Sxl-Pe mRNAs are expressed in 2-4 hr embryos and disappear thereafter, one could argue from the published Western experiments that the Sxl-PE proteins expressed at the blastoderm stage persist at least until the end embryogenesis, though perhaps at somewhat lower levels than at earlier points in development. So the fact that Goyal et al were able to detect HA-Sxl-Pe proteins in stage 9 embryos and later on in L1 larva probably isn't completely unexpected. What is unexpected is that the HA-Sxl-Pe proteins weren't present earlier.

(6) The authors use RNAi and germline clones to demonstrate that sis-A is required for proper oogenesis: when sis-A activity is compromised in germ cells, i) tumorous ovary phenotypes are observed and ii) there is a reduction in the expression of Sxl-Pm protein. They are also able to rescue the phenotypic effects of sis-a knockdown by expressing a Sxl-Pm protein. While the experiments indicating sis-a is important for normal oogenesis and that at least one of its functions is to ensure that sufficient Sxl is present in the germline stem cells seem convincing, other findings would make the reader wonder whether Sis-A is actually functioning (directly) to activate Sxl transcription from promoter X.

The authors show that sis-a mRNAs and proteins are expressed in stage 3-5 germ cells (PGCs). This is not unexpected as the X-linked transcription factors that turn Sxl-Pe on are expressed prior to nuclear migration, so their protein products should be present in early PGCs. The available evidence suggests that their transcription is shut down in PGCs by the factors responsible for transcriptional quiescence (e.g., nos and pgc) in which case transcripts might be detected in only one or two PGC-which fits with their images. However, it is hard to believe that expression of Sis-A protein in pre-blastoderm embryos is relevant to the observed activation of the Sxl-Pm autoregulatory loop hours later in L2 larva.

It is also not clear how the very low level of gfp-Sis-A seen in only a small subset of migrating germ cells in stage 10 embryos (Figure S6) would be responsible for activating the Sxl-Pe10kb reporter in L1. It seems likely that the small amount of protein seen in stage 10 embryos is left over from the pre-cellular blastoderm stage. In this case, it would not be surprising to discover that the residual protein is present in both female and male stage 10 germ cells. This would raise further doubts about the relevance of the gfp-Sis-A at these early stages.

In fact, given the evidence presented implicating sis-a in activating Sxl, (the germline activation of the Sxl-Pe10kb reporter, the RNAi knockdowns, and the germ cell-specific sis-a clones) it is clear that the sis-A RNAs and proteins seen in pre-cellular blastoderm PGCs aren't relevant. The germline clone experiment (and also the RNAi knockdowns) indicates that sis-A must be transcribed in germ cells after Cas9 editing has taken place. Presumably, this would be after transcription is reactivated in the germline (~stage 10) and after the formation of the embryonic gonad (stage 14) so that the somatic gonadal cells can signal to the germ cells. With respect to the reporter, the relevant time frame for showing that sis-A is present in germ cells would be even later in L1.

(7) As noted above, the data in this manuscript do not support the idea that Sxl-Pe proteins activate the Sxl-Pm female splicing in the germline. Flybase indicates that there is at least one other Sxl promoter that could potentially generate a transcript that includes the male exon but still could encode a Sxl protein. This promoter "Sxl-Px" is located downstream of Sxl-Pm and from its position it could have been included in the authors' 10 kb reporter. The reported splicing pattern of the endogenous transcript skips exon2, and instead links an exon just downstream of Sxl-Px to the male exon. The male exon is then spliced to exon4. If the translation doesn't start and end at one of the small upstream orfs in the exons close to Sxl-Px and the male exon, a translation could begin with an AUG codon in exon4 that is in frame with the Sxl protein coding sequence. This would produce a Sxl protein that lacks aa sequences from N-terminus, but still retains some function.

Another possible explanation for how gfp is expressed from the 10 kb reporter is that the transcript includes the "z" exon described by Cline et al., 2010.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, expression of Sex-lethal (Sxl) protein determines sexual identity and drives female development. Functional Sxl protein is absent from males where splicing includes a termination codon-containing "poison" exon. Early during development, in the soma of female individuals, Sxl expression is initiated by an X chromosome counting mechanism that activates the Sxl establishment promoter (SxlPE) to produce an initial amount of Sxl protein. This then suppresses the inclusion of the "poison" exon, directing the constructive splicing of Sxl transcripts emerging from the Sxl maintenance promotor (SxlPM) which is activated at a later stage during development irrespective of sex. This autoregulatory loop maintains Sxl expression and commits to female development.

Sxl also determines the sexual identity of the germline. Here Sxl expression generally follows the same principles as in somatic tissues, but the way expression is initiated differs from the soma. This regulation has so far remained elusive.

In the presented manuscript, Goyal et al. show that activation of Sxl expression in the germline depends on additional regulatory DNA sequences, or sequences different from the ones driving initial Sxl expression in the soma. They further demonstrate that sisterless A (sisA), a transcription factor that is required for activation of Sxl expression in the soma, is also necessary, but not sufficient, to initiate the expression of functional Sxl protein in female germ cells. sisA expression precedes Sxl induction in the germline and its ablation by RNAi results in impaired expression of Sxl, formation of ovarian tumors, and germline loss, phenocopying the loss of Sxl. Intriguingly, this phenotype can be rescued by the forced expression of Sxl, demonstrating that the primary function of sisA in the germline is the induction of Sxl expression.

Strengths:

The clever design of probes (for RNA FISH) and reporters allowed the authors to dissect Sxl expression from different promoters to get novel insight into sex-specific gene regulation in the germline. All experiments are carefully controlled. Since Sxl regulation differs between the soma and the germline, somatic tissues provide elegant internal controls in many experiments, ensuring e.g. functionality of the reporters. Similarly, animals carrying newly generated alleles (e.g. genomic tagging of the Sxl locus) are fertile and viable, demonstrating that the genetic manipulation does not interfere with protein function. The conclusions drawn from the experimental data are sound and advance our understanding of how Sxl expression is induced in the female germline.

Weaknesses:

The assays employed by the authors provide valuable information on when Sxl promoters become active. However, since no information on the stability of the gene products (i.e. RNA and protein) is available, it remains unclear when the SxlPE promoter is switched off in the germline (conceptually it only needs to be active for a short time period to initiate production of functional Sxl protein). As correctly stated by the authors, the persisting signals observed in the germline might therefore not reflect the continuous activity of the SxlPE promoter.

Mapping of regulatory elements and their function: SxlPE with 1.5 kb of flanking upstream sequence is sufficient to recapitulate early Sxl expression in the soma. The authors now provide evidence that beyond that, additional DNA sequences flanking the SxlPE promoter are required for germline expression. However, a more precise mapping was not performed. Also, due to technical limitations, the authors could not precisely map the sisA binding sites. Since this protein is also involved in the somatic induction of Sxl, its binding sites likely reside in the region 1.5kb upstream of the SxlPE promoter, which has been reported to be sufficient for somatic regulation. The regulatory role of the sequences beyond SxlPE-1.5kb therefore remains unaddressed and it remains to be investigated which trans-acting factor(s) exert(s) its/their function(s) via this region.

We agree that a more precise mapping of the essential elements within the 10.2 kb reporter is an important direction in which to proceed. Unfortunately, this is out of the scope of the current manuscript given current lab personnel. In regard to the 1.5 kb promoter that activates SxlPE in the soma, we do not feel that the Sisa binding sites are necessarily in this region. It is important to note that, while the 1.5 kb promoter is sufficient for female-specific expression in the soma, it may not contain all of the regulatory elements that normally regulate PE from the endogenous locus. Activation of PE in the soma is thought to be regulated by a combination of positive-acting factors (SisA, SisB, etc.) and repressive factors (e.g. Dpn) that set a threshold for PE activation. Much more work would need to be done to determine whether all of these factors bind to the 1.5 kb promoter, or whether additional sequences are also involved to control the proper timing and robustness of normal Sxl PE activation in the soma.

The central question of how Sxl expression is initiated and controlled in the germline still remains unanswered. Since sisA is zygotically expressed in both the male and the female germline (Figure 4D), it is unlikely the factor that restricts Sxl expression to the female germline.

X chromosome “counting” elements like SisA are always expressed in both males and females, but it is thought that the 2X does of them in females activates PE, while the 1X does in males does not. Thus, we do expect SisA to be expressed in both males and females as we observed.

How does weak expression of Sxl in male tissues or expression above background after knockdown of sisA reconcile with the model that an autoregulatory feedback loop enforces constant and clonally inheritable Sxl expression once Sxl is induced? Is the current model for Sxl expression too simple or are we missing additional factors that modulate Sxl expression (such as e.g. Sister of Sex-lethal)? While I do not expect the authors to answer these questions, I would expect them to appropriately address these intriguing aspects in the discussion.

It is difficult to know what is “background” and what is actual weak Sxl expression in males. We agree that, if it is real, then why it doesn’t activate autoregulation of the Sxl PM transcript is mysterious. And yes, the current model for female-specific expression of Sxl in the soma may well be incomplete. Sxl PM transcript is present in the testis based on community RNA-seq data and our own analysis of male vs. female bam-mutant gonads (PMID 31329582), but it is at lower levels. Whether the lower level in the testis is due to tissue differences or sex-specific regulation of RNA levels is unknown. Our observations that the HA-tagged Sxl Early protein remains present in somatic cells in L1 larvae, and that GFP expression from the 10.2 kb Sxl PE-GFP can be detected in the soma until L2 could either be due to perdurance of the protein products, or continued sex-specific expression of PE long after the time that it was thought to shut off. This is also long after dosage compensation should have equalized the expression of X chromosome gene expression, meaning that X chromosomes can no longer be “counted” by factors like SisA and SisB. Thus, sex-specific expression of PE at this time would require another mechanism besides the current model (such as feedback regulation of Sxl PE transcription from downstream factors).

Reviewer #2 (Public Review):

Summary:

The authors wanted to determine whether cis-acting factors of Sxl - two different Sxl promoters in somatic cells - regulate Sxl in a similar way in germ cells. They also wanted to determine whether trans-acting factors known to regulate Sxl in the soma also regulate Sxl in the germline.

Regarding the cis-acting factors, they examine the Sxl "establishment promoter" (SxlPE) that is activated in female somatic cells by the presence of two X chromosomes. Slightly later in development, dosage compensation equalizes X chromosome expression in males and females and so X chromosomes can no longer be counted. The second Sxl promoter is the "maintenance promoter," (SxlPM), which is activated in both sexes. The mRNA produced from the maintenance promoter has to be alternatively splicing from early Sxl protein generated earlier in development by the PE. This leads to an autoregulatory loop that maintains Sxl expression in female somatic cells. The authors used fluorescent in situ hybridization (FISH) with oligopaints to determine the temporal activation of the PE or PM promoters. They find that - unlike the soma - the PE does not precede the PM and instead is activated contemporaneously or later than the PM - this is confusing with the later results (see below). Next, they generated transcriptional reporter constructs containing large segments of the Sxl locus, the 1.5 kb used in somatic studies, a 5.2 kb reporter, and a 10.2 kb. Interestingly the 1.5 kb reporter that was reported to recapitulate Sxl expression in soma and germline was not observed by the authors. The 5.2 kb reporter was observed in female somatic cells but not in germ cells. Only when they include an additional 5 kb downstream of the 5.2 kb reporter (here the 10.2 kb reporter) they did see expression in germ cells but this occurred at the L1 stages. Their data indicate that Sxl activity in the germ requires different cis-regulation than the soma and that the PE is activated later in germ cells than in somatic cells. The authors next use gene editing to insert epitope tags in two distinct strains in the hopes of creating an early Sxl and a later Sxl protein derived from the PE and PM, respectively. The HA-tagged protein from the PE was seen in somatic cells but never in the germline, possibly due to very low expression. The FLAG-tagged late Sxl protein is observed in L2 germ cells. Because the early HA-Sxl protein is not perceptible in germ cells, it is not possible to conclude its role in the germline. However, because late FLAG-Sxl was only observed in L2 germ cells and the PE was detected in L1, this leaves open the possibility that PE produces early HA-Sxl (which currently cannot be detected), which then alternatively splices the transcript from the PM. In other words, the soma and germline could have a similar temporal relationship between the two Sxl promoters. While I agree with the authors about this conclusion, the earlier work with the oligopaints leads to the conclusion that SE is active after PM. This is confusing.

The temporal relationship between Sxl PE and Sxl PM in the germline is indeed confusing. One source of confusion comes from whether one is discussing Sxl protein production or promoter activity. As the reviewer nicely summarizes, our transcription analysis with oligopaints indicates that, unlike in the soma, Sxl PE is NOT on in the germline prior to PM. Our other data indicate that PE is instead likely only active well after transcription from PM has begun. However, this still means that the temporal order of the EARLY and LATE Sxl proteins can be the same as the soma. Even if PM is active well before PE in the germline, the PE transcript cannot produce any functional protein in the absence of being alternatively spliced by the Sxl protein (Sxl autoregulation). Thus, even if PM is active before PE in the germline, we would not expect to observe any LATE Sxl protein until the PE promoter comes on, and produces a pulse of EARLY Sxl protein. The fact that we observe LATE Sxl protein at L2 is consistent with our observation that the 10.2 kb Sxl PE reporter is active at L1. We will attempt to explain all of this better in a revised manuscript.

Next, the authors wanted to turn their attention to the trans-acting factors that regulate Sxl in the soma, including Sisterless A (SisA), SisB, Runt, and the JAK/STAT ligand Unpaired. Using germline RNAi, the authors found that only knockdown of SisA causes ovarian tumors, similar to the loss of Sxl, suggesting that SisA regulates Sxl (ie the PE) in both the soma and the germline. They generated a SisA null allele using CRISPR/Cas9 and these animals had ovarian tumors and germ cell-less ovaries. FISH revealed that sisA is activated in primordial germ cells in stages 3-6 before the activation of Sxl. They used CRISPR-Cas9 to generate an endogenously-tagged SisA and found that tagged SisA was expressed in stage 3-6 PCGs, which is consistent with activating PE in the germline. They showed that sisA is upstream of Sxl as germline depletion of sisA led to a significant decrease in expression from the 10.2 kb PE reporter and in SXL protein. The authors could rescue the ovarian tumors and loss of Sxl protein upon germline depletion of sisA by supplying Sxl from another protein (the otu promoter). These data indicate that sisA is necessary for Sxl activation in the germline. However, ectopic sisA in germ cells in the testis did not lead to ectopic Sxl, suggesting that sisA is not sufficient to activate Sxl in the germline.

Strengths:

(1) The genetic and genomic approaches in this study are top-notch and they have generated reagents that will be very useful for the field.

(2) Excellent use of powerful approaches (oligo paint, reporter constructs, CRISPR-Cas9 alleles).

(3) The combination of state of art approaches and quantification of phenotypes allows the authors to make important conclusions.

Weaknesses:

(1) Confusion in line 127 (this indicates that SxlPE is not activated before SxlPM in the germline) about PE not being activated before the PM in the germline when later figures show that PE is activated in L1 and late Sxl protein is seen in L2. It would be helpful to the readers if the authors edited the text to avoid this confusion. Perhaps more explanation of the results at specific points would be helpful.

We agree--see response above.

Reviewer #3 (Public Review):

Summary:

The mechanisms governing the initial female-specific activation of Sex-lethal (Sxl) in the soma, the subsequent maintenance of female-specific expression and the various functions of Sxl in somatic sex determination and dosage compensation are well documented. While Sxl is also expressed in the female germline where it plays a critical role during oogenesis, the pathway that is responsible for turning Sxl on in germ cells has been a long-standing mystery. This manuscript from Goyal et al describes studies aimed at elucidating the mechanism(s) for the sex-specific activation of the Sex-lethal (Sxl) gene in the female germline of Drosophila.

In the soma, the Sxl establishment promoter, Sxl-Pe, is regulated in pre-cellular blastoderm embryos in somatic cells by several X-linked transcription factors (sis-a, sis-b, sis-c and runt). At this stage of development, the expression of these transcription factors is proportional to gene dose, 2x females and 1x in males. The cumulative two-fold difference in the expression of these transcription factors is sufficient to turn Sxl-Pe on in female embryos. Transcripts from the Sxl-Pe promoter encode an "early" version of the female Sxl protein, and they function to activate a splicing positive autoregulatory loop by promoting the female-specific splicing of the initial pre-mRNAs derived from the Sxl maintenance promoter, Sxl-Pm (which is located upstream of Sxl-Pm). These female Sxl-Pm mRNAs encode a Sxl protein with a different N-terminus from the Sxl-Pe mRNAs, and they function to maintain female-specific splicing in the soma during the remainder of development.

In this manuscript, the authors are trying to understand how the Sxl-Pm positive autoregulatory loop is established in germ cells. If Sxl-Pe is used and its activation precedes Sxl-Pm as is true in the soma, they should be able to detect Sxl-Pe transcripts in germ cells before Sxl-Pm transcripts appear. To test this possibility, they generated RNA FISH probes complementary to the Sxl-Pe first exon (which is part of an intron sequence in the Sxl-Pm transcript) and to a "common sequence" that labels both Sxl-Pe and Sxl-Pm transcripts. Transcripts labeled by both probes were detected in germ cells beginning at stage 5 (and reaching a peak at stage 10), so either the Sxl-Pm and Sxl-Pe promoters turn on simultaneously, or Sxl-Pe is not active.

They next switched to Sxl-Pe reporters. The first Sxl-Pe:gfp reporter they used has a 1.5 kb upstream region which in other studies was found to be sufficient to drive sex-specific expression in the soma of blastoderm embryos. Also like the endogenous Sxl gene it is not expressed in germ cells at this early stage. In 2011, Hashiyama et al reported that this 1.5 kb promoter fragment was able to drive gfp expression in Vasa-positive germ cells later in development in stage 9/10 embryos. However, because of the high background of gfp in the nearby soma, their result wasn't especially convincing. Though they don't show the data, Goyal et al indicated that unlike Hashiyama et al they were unable to detect gfp expressed from this reporter in germ cells. Goyal et al extended the upstream sequences in the reporter to 5 kb, but they were still unable to detect germline expression of gfp.

Goyal et al then generated a more complicated reporter which extends 5 kb upstream of the Sxl-Pe start site and 5 kb downstream-ending at or near 4th exon of the Sxl-Pm transcript (the Sxl-Pe10 kb reporter). (The authors were not explicit as to whether the 5 kb downstream sequence extended beyond the 4th exon splice junction-in which case splicing could potentially occur with an upstream exon(s)-or terminated prior to the splice junction as seems to be indicated in their diagram.) With this reporter, they were able to detect sex-specific gfp expression in the germline beginning in L1 (first instar larva). With the caveat that gfp detection might be delayed compared to the onset of reporter activation, these findings indicated that the sequences in the reporter are able to drive sex-specific transcription in the germline at least as early as L1.

The authors next tagged the N-terminal end of the Sxl-Pe protein with HA (using Crispr/Cas9) and the N-terminal end of Sxl-Pm protein with Flag. They report that the HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis. Somatic HA-Sxl-Pe protein persists into L1, but is no longer detected in L2. However, while somatic HA-Sxl-Pe protein is detected, they were unable to detect HA-Sxl-Pe protein in germ cells. In the case of FLAG-Sxl-Pm, it could first be detected in L2 germ cells indicating that at this juncture the Sxl-positive autoregulatory loop has been activated. This contrasts with Sxl-Pm transcripts which are observed in a few germ cells at stage 5 of embryogenesis, and in most germ cells by stage 10. The authors propose (based on the expression pattern of the Sxl-Pe10kb reporter and the appearance of Flag-Sxl-Pm protein) that Sxl-Pe comes on in germ cells in L1, and that the Sxl-Pe protein activates the female splicing of Sxl-Pm transcripts, giving detectable Flag-Sxl-Pm proteins beginning in L2.

To investigate the signals that activate Sxl-Pe in germ cells, the authors tested four of the X-linked genes (sis-a, sis-b, sis-c, and runt) that function to activate Sxl-Pe in the soma in early embryos. RNAi knockdown of sis-b, sis-c, and runt had no apparent effect on oogenesis. In contrast, knockdown of sis-a resulted in tumorous ovaries, a phenotype associated with Sxl mutations. (Three different RNAi transgenes were tested-two gave this phenotype, the third did not.) Sxl-Pe10kb reporter activity in L1 female germ cells is also dependent on sis-A.

Several approaches were used to confirm a role for sis-a in a) oogenesis and b) the activation of the Sxl-Pm autoregulatory loop. They showed that sis-a germline clones (using tissue-specific Crispr/Cas9 editing) resulted in the tumorous ovary phenotype and reduced the expression of Sxl protein in these ovaries. They found that sis-a transcripts and GFP-tagged Sis-A protein are present in germ cells. Finally, they showed tumorous ovary phenotype induced by germline RNAi knockdown of sis-a can be partially rescued by expressing Sxl in the germ cells.

Critique:

While this manuscript addresses a longstanding puzzle - the mechanism activating the Sxl autoregulatory loop in female germ cells-and likely identified an important germline transcriptional activator of Sxl, sis-a, the data that they've generated doesn't make a compelling story. At every step, there are puzzle pieces that don't fit the narrative. In addition, some of their findings are inconsistent with many previous studies.

We respect and appreciate this reviewer for the detailed comments. However, we feel that the claim that our work doesn’t “make a compelling story” and that many “pieces…don’t fit the narrative” is incorrect. The main issue that this reviewer raises is that we do not know if Sxl “early” transcription in the germline initiates from the Pe promoter. This is true, which we fully acknowledge, but the detail of whether “germline early” transcription of Sxl initiates from Pe or from other, as yet undefined, germline promoter does not affect the main conclusions of the paper. These conclusions are that a) regulation of Sxl in the germline is fundamentally different from in the soma and 2) despite point (1), sisA acts as an activator of Sxl in both the soma and the germline. Neither of these main points is disputed by this reviewer.

(1) The authors used RNA FISH to time the expression of Sxl-Pe and Sxl-Pm transcripts in germ cells. Transcripts complementary to Sxl-Pe and Sxl-Pm were detected at the same time in embryos beginning at stage 5. This is not a definitive experiment as it could mean a) that Sxl-Pe and Sxl-Pm turn on at the same time, b) that Sxl-Pe comes on after Sxl-Pm (as suggested by the Sxl-Pe10kb reporter) or c) Sxl-Pe never comes on.

When designing this experiment, we wanted to test whether the “soma model” of Pe activation before Pm was also true in the germ cells. Our data clearly demonstrate that transcripts beginning downstream of Pe are not expressed prior to transcripts beginning downstream of Pm. Thus, we can state that the “soma model” of Pe first and then Pm does not occur in the germline, which is very interesting. However, we cannot make any other conclusions about Pe in the germline from these data, as the reviewer indicates.

(2) Hashiyama et al reported that they detected gfp expression in stage 9/10 germ cells from a 1.5 kb Sxl-Pe-gfp. As noted above, this result wasn't entirely convincing and thus it isn't surprising that Goyal et al were unable to reproduce it. Extending the upstream sequences to just before the 1st exon of Sxl-Pm transcripts also didn't give gfp expression in germ cells. Only when they added 5 kb downstream did they detect gfp expression. However, from this result, it isn't possible to conclude that the Sxl-Pe promoter is actually driving gfp expression in L1 germ cells. Instead, the Sxl promoter active in the germ line could be anywhere in their 10 kb reporter.

We agree that we have not determined the transcriptional start sites for Sxl in the germline and it is possible that the 10.2 kb reporter uses a different promoter than Pe, as long as that transcript can also be spliced into exon 4 where the GFP tag has been placed. The three types of experiments conducted—FISH to regions of the nascent transcripts, tagged versions of the different predicted ORFs, and promoter-GFP constructs—are extensive, but all have different limitations. Indeed, it would be challenging to determine the transcription start sites in the germline, as it would require obtaining enough L1 larvae to be able to dissociate the animals, or isolated gonads, into single cells in order to FACS purify the germ cells for RACE or long-read sequencing (I’m not sure that L1 larval single-nucleus seq would be enough for calling start sites). Otherwise, there would be no way to determine if expected or unexpected transcripts came from the soma or the germline. We can consider these experiments in the future.

Fortunately, the main conclusions from this paper do not require knowing whether the germline uses Pe or some other “germline early” promoter that can produce Sxl protein in the absence of autoregulation by existing Sxl protein. The observations that a nascent transcript including the region downstream of Pm is observed in embryonic germ cells, but that the tagged LATE protein is not observed until L2, suggest that the transcript produced in early germ cells cannot produce a functional protein. This is consistent with the need for Sxl autoregulation of the Pm transcript in the germline as in the soma, as was previously thought. This is further supported by the observations that activity of the 10.2 kb reporter is only observed in L1 germ cells, and that the LATE Sxl protein is only observed in germ cells after this point. Thus, we can conclude that either Pe, or another “germline early” promoter, acts to produce female-specific Sxl protein to initiate autoregulation of Sxl splicing and protein production in the germline. We feel that this is a significant advance for the field, and we will make it more clear in the text that the initial expression of Sxl in the germline may not be from the Pe promoter.

Other conclusions of the manuscript are unaffected by the start site for “germline early” Sxl transcription, including that the germline activates Sxl protein expression much later than the soma, which calls into question previous work indicating an early role for Sxl in the germline. Also unaffected is our conclusion that different enhancer sequences are required for activation of Sxl expression in the germline than in the soma, consistent with previous work demonstrating that the genetics of Sxl activation in the germline are different than in the soma. Lastly, our conclusions that sisA acts upstream of Sxl, and is required for Sxl germline expression, either directly or indirectly, are also unaffected by the nature of the Sxl “germline early” start site.

(3) At least one experiment suggests that Sxl-Pe never comes on in germ cells. The authors tagged the N-terminus of the Sxl-Pe protein with HA and the N-terminus of the Sxl-Pm protein with Flag. Though they could detect HA-Sxl-Pe protein in the soma, they didn't detect it in germ cells. On the other hand, the Flag-Sxl-Pm protein was detected in L2 germ cells (but not earlier). These results would more or less fit with those obtained for the 10 kb reporter and would support the following model: Prior to L1, Sxl-Pm transcripts are expressed and spliced in the male pattern in both male and female germ cells. During L1, Sxl protein expressed via a mechanism that depends upon a 10 kb region spanning Sxl-Pe (but not on Sxl-Pe) is produced and by L2 there are sufficient amounts of this protein to switch the splicing of Sxl-Pm transcripts from a male to a female pattern-generating Flag-tagged Sxl-Pm protein.

As described above, it is indeed possible that another promoter besides Pe is active as the “germline early” promoter. We will make this more clear in a revised version, but the major conclusions of the manuscript are unaffected.

(4) The 10kb reporter is sex-specific, but not germline-specific. The levels of gfp in female L1 somatic cells are equal to if not greater than those in L1 female germ cells. That the Sxl-Pe10kb reporter is active in the soma complicates the conclusion that it represents a germ line-specific promoter. Germline activity is, however, sensitive to sis-A knockdowns which is plus. Presumably, somatic expression of the reporter wouldn't be sensitive to a (late) sis-A knockdown- but this wasn't shown.

We are confused by this comment because we do not conclude that the Pe is a germline-specific promoter. Pe is known to be expressed in the soma, from considerable previous work cited by this reviewer, and the simplest model is that Pe is used in both the soma and the germline, as reflected by our 10.2 kb reporter. It is actually quite interesting how late this promoter seems active in the soma, contrary to current dogma, but we did not study somatic activation of Sxl in this work.

(5) Their results with the HA-Sxl-Pe protein don't fit with many previous studies-assuming that the authors have explained their results properly. They report that HA-Sxl-Pe protein is first detected in the soma at stage 9 of embryogenesis and that it then persists till L2. However, previous studies have shown that Sxl-Pe transcripts and then Sxl-Pe proteins are first detected in ~NC11-NC12 embryos. In RNase protection experiments, the Sxl-Pe exon is observed in 2-4 hr embryos, but not detected in 5-8 hr, 14-12 hr, L1, L2, L3, or pupae. Northerns give pretty much the same picture. Western blots also show that Sxl-Pe proteins are first detectable around the blastoderm stage. So it is not at all clear why HA-Sxl-Pe proteins are first observed at stage 9 which, of course, is well after the time that the Sxl-Pm autoregulatory loop is established.

Given the obvious problems with the initial timing of somatic expression described here, it is hard to know what to make of the fact that HA-tagged Sxl-Pe proteins aren't observed in germ cells.

As for the presence of HA-Sxl-Pe proteins later than expected: While RNase protection/Northern experiments showed that Sxl-Pe mRNAs are expressed in 2-4 hr embryos and disappear thereafter, one could argue from the published Western experiments that the Sxl-PE proteins expressed at the blastoderm stage persist at least until the end embryogenesis, though perhaps at somewhat lower levels than at earlier points in development. So the fact that Goyal et al were able to detect HA-Sxl-Pe proteins in stage 9 embryos and later on in L1 larva probably isn't completely unexpected. What is unexpected is that the HA-Sxl-Pe proteins weren't present earlier.

We thank the reviewer for this detailed analysis. Since we were not focused on somatic expression of Sxl in this work, it is possible that stage 9 was the earliest stage we observed in our experiments, rather than the earliest stage in which it is ever observed. We will repeat these experiments to verify when the HA-tagged early Sxl protein is first observed. However, these comments have no bearing on our conclusions about Sxl expression in the germline, which is the focus of this manuscript.

(6) The authors use RNAi and germline clones to demonstrate that sis-A is required for proper oogenesis: when sis-A activity is compromised in germ cells, i) tumorous ovary phenotypes are observed and ii) there is a reduction in the expression of Sxl-Pm protein. They are also able to rescue the phenotypic effects of sis-a knockdown by expressing a Sxl-Pm protein. While the experiments indicating sis-a is important for normal oogenesis and that at least one of its functions is to ensure that sufficient Sxl is present in the germline stem cells seem convincing, other findings would make the reader wonder whether Sis-A is actually functioning (directly) to activate Sxl transcription from promoter X.

It is true that we do not know the binding specificity for SisA, which is why we have made no claims about the directness of SisA regulation of Sxl. This does not change our conclusions that sisA is upstream of Sxl activation, since loss of sisA function has a similar phenotype to loss of Sxl, loss of sisA blocks Sxl protein expression, and expression of Sxl rescues the sisA mutant phenotype.

The authors show that sis-a mRNAs and proteins are expressed in stage 3-5 germ cells (PGCs). This is not unexpected as the X-linked transcription factors that turn Sxl-Pe on are expressed prior to nuclear migration, so their protein products should be present in early PGCs. The available evidence suggests that their transcription is shut down in PGCs by the factors responsible for transcriptional quiescence (e.g., nos and pgc) in which case transcripts might be detected in only one or two PGC-which fits with their images. However, it is hard to believe that expression of Sis-A protein in pre-blastoderm embryos is relevant to the observed activation of the Sxl-Pm autoregulatory loop hours later in L2 larva.

It is also not clear how the very low level of gfp-Sis-A seen in only a small subset of migrating germ cells in stage 10 embryos (Figure S6) would be responsible for activating the Sxl-Pe10kb reporter in L1. It seems likely that the small amount of protein seen in stage 10 embryos is left over from the pre-cellular blastoderm stage. In this case, it would not be surprising to discover that the residual protein is present in both female and male stage 10 germ cells. This would raise further doubts about the relevance of the gfp-Sis-A at these early stages.

In fact, given the evidence presented implicating sis-a in activating Sxl, (the germline activation of the Sxl-Pe10kb reporter, the RNAi knockdowns, and the germ cell-specific sis-a clones) it is clear that the sis-A RNAs and proteins seen in pre-cellular blastoderm PGCs aren't relevant. The germline clone experiment (and also the RNAi knockdowns) indicates that sis-A must be transcribed in germ cells after Cas9 editing has taken place. Presumably, this would be after transcription is reactivated in the germline (~stage 10) and after the formation of the embryonic gonad (stage 14) so that the somatic gonadal cells can signal to the germ cells. With respect to the reporter, the relevant time frame for showing that sis-A is present in germ cells would be even later in L1.

The reviewer is correct in wondering how early sisA transcription can affect late Sxl activation, and we are clear about this conundrum in our manuscript. However, they are incorrect about the early sisA expression. Our experiments examining nascent sisA transcripts indicate that sisA is zygotically expressed in the formed germ cells rather than being leftover from expression in early nuclei. The fact that only a portion of germ cells express sisA at any time may well be due to a timing issue, where not all germ cells express sisA at the same time. They are also incorrect about the timing of Cas9 editing in the germline—the guide RNAs are expressed from a general promoter that is active both maternally and in the early embryo, and the Cas9 RNA from the nos promoter is deposited in the germ plasm where it is translated long before cellularization, meaning that sisA CRISPR knockout can begin at the earliest stages of germ cell formation or before.

(7) As noted above, the data in this manuscript do not support the idea that Sxl-Pe proteins activate the Sxl-Pm female splicing in the germline. Flybase indicates that there is at least one other Sxl promoter that could potentially generate a transcript that includes the male exon but still could encode a Sxl protein. This promoter "Sxl-Px" is located downstream of Sxl-Pm and from its position it could have been included in the authors' 10 kb reporter. The reported splicing pattern of the endogenous transcript skips exon2, and instead links an exon just downstream of Sxl-Px to the male exon. The male exon is then spliced to exon4. If the translation doesn't start and end at one of the small upstream orfs in the exons close to Sxl-Px and the male exon, a translation could begin with an AUG codon in exon4 that is in frame with the Sxl protein coding sequence. This would produce a Sxl protein that lacks aa sequences from N-terminus, but still retains some function.

Another possible explanation for how gfp is expressed from the 10 kb reporter is that the transcript includes the "z" exon described by Cline et al., 2010.

As discussed above, the exact location of the start site for the Sxl transcript in the germline remains to be determined, but does not affect the main conclusions of the paper.