Nucleoporin107 mediates female sexual differentiation via Dsx
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Curated by eLife
Evaluation Summary:
This manuscript focuses on a missense mutation of Nucleoporin107 (Nup107) gene, which encodes a nuclear pore complex protein. Phenotypic analyses demonstrate similar phenotypes compared to syndromes identified in human XX-ovarian-dysgenesis patients. Further RNA-seq analyses revealed potential targets of Nup107, through which the authors identified the doublesex (dsx) gene as a critical target with functional readouts. Both Nup107 and Dsx act in the somatic gonadal cells to regulate germ cell function, and have connections with the BMP signaling pathway. This study provides a great example to use Drosophila as a model organism to study the molecular and cellular mechanisms of a known human disease and should be of general interest.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx ) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma–germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.
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Author Response
Reviewer #2 (Public Review):
In this manuscript Shore et al determine that Nucleoporin 107 (Nup107) is required in developing female somatic cells [intermingled cells (ICs)] for proper ovarian development. The authors propose that Nup107 is required for proper orientation of ICs during development to ensure proper function of escort cells during adulthood. They show that loss of Nup107 results in ectopic germline stem cells (GSCs) away from the GSC niche (primarily cap cells and terminal filament cells) and that these ectopic GSCs display hypermorphic Bone Morphogenic Protein (BMP) signaling. The authors also find that Nup107 regulates expression of the transcription factor doublesex (dsx) and share a common transcriptional target of AdamTS-A. Through knockdown/rescue experiments, the authors show that expression of …
Author Response
Reviewer #2 (Public Review):
In this manuscript Shore et al determine that Nucleoporin 107 (Nup107) is required in developing female somatic cells [intermingled cells (ICs)] for proper ovarian development. The authors propose that Nup107 is required for proper orientation of ICs during development to ensure proper function of escort cells during adulthood. They show that loss of Nup107 results in ectopic germline stem cells (GSCs) away from the GSC niche (primarily cap cells and terminal filament cells) and that these ectopic GSCs display hypermorphic Bone Morphogenic Protein (BMP) signaling. The authors also find that Nup107 regulates expression of the transcription factor doublesex (dsx) and share a common transcriptional target of AdamTS-A. Through knockdown/rescue experiments, the authors show that expression of Dsx-F can rescue phenotypes observed with ovarian somatic knockdown of Nup107 and that ovarian somatic knockdown of AdamTS-A mimics loss of Nup107 and dsx (including loss of escort cell membrane protrusions and enhanced BMP signaling). These data provide an interesting non-cytoplasmic to nuclear transport mechanism for Nup107 in regulating oogenesis.
This is a well written manuscript with proper experimental analysis (sufficient n's, proper statistics). However, it is unclear how the authors came to some of their conclusions and how their results significantly enhance findings from prior studies exploring how disruption of organization of somatic cells of the developing female gonad influences adult escort cell protrusions and preventing expansion of BMP signaling to ensure proper germline stem cell cyst differentiation.
Points to consider:
- It was unclear reading the first part of the paper how the adult phenotypes were connecting to defects during larval development. It would further strengthen the rationale for describing the adult phenotypes first if the authors perhaps show the larval gonad defects (abnormal IC stacking/arrangement) prior to showing adult phenotypes. This would also help with citing previous studies that have also found the correlation with incorrect IC placement and ectopic GSC-like cells in adulthood (e.g., Tseng et al., 2018, Stem Cell Reports).
We appreciate this insight and have changed the manuscript as advised, beginning with the larval gonads (line number 116) and then progressing to the adult ovaries (line number 156).
- It would also help the reader to know that nuclear pore complex proteins have roles outside of nuclear-cytoplasmic transport early in the manuscript as opposed to only in the discussion.
As suggested, we have included this in the introduction (line number 63).
- It is unclear the model the authors are proposing for the Nup107-Dsx-AdamTs relationship. Are the authors proposing that Nup107 can regulate the import of Dsx or is directly regulating dsx transcription (based on the RNA-sequencing results)? A little more explanation would be helpful.
We have elaborated on this in the discussion (lines numbers 482-512).
Reviewer #3 (Public Review):
This is an interesting story, providing molecular explanation for XX-OD, caused by mutations in Nup107 gene. Overall, the experiments are thoroughly conducted, and the results are important in understanding XX-OD. However, there are some issues that need to be addressed, as the data presented in this study still leaves some gaps that need attention. Whereas I do not think that all the gaps need to be filled for a paper to be published, the gaps that remain in this paper leaves confusions and inconsistencies, they need to be addressed.
- Dsx is the major target of Nup107. Although it is clear that Dsx is downregulated in Nup107 mutant, how exactly Nup107 regulates Dsx expression remains entirely unclear. Does Nup107 functions as transcriptional regulator of Nup107?
As previously mentioned, this is an important question that we hope to answer in future. However, we think that the answer to this question is beyond the scope of this work as our data have clearly demonstrated that Dsx-F acts downstream of Nup107 to regulate activities of unique cell types in the larval as well as adult ovaries to modulate germline soma interactions underlying ovarian morphology and GSC development.
- Nup107 Dsx axis is required for escort cells to encapsulate germ cells to allow the downregulation of BMP signaling in germ cells, which in turn allows differentiation of germ cells. Whereas this axis appears to operate, the relationship between germ cell encapsulation and BMP signaling is quite unclear. Is encapsulation upstream or downstream of BMP modulation? Authors provide the evidence that adamTS-A, which modulates the amount of extracellular BMP ligands, is the downstream target of Dsx, and adamTS-A appears to regulate encapsulation. This makes it unclear whether encapsulation is required to down regulate BMP, or BMP regulates encapsulation (and if the latter, what is achieved by encapsulation that leads to germ cell differentiation?)
This is an excellent point and we followed reviewer’s suggestion to resolve this issue. To answer this question, we knocked down coracle activity specifically just in the ECs (new Figure 4-Figure supplement 2 & 3). This turned out to be highly informative and we thank the reviewer for encouraging us to do this experiment.
This manipulation revealed that encapsulation is likely upstream of BMP, as disruption of the cellular processes responsible for the encapsulation of germ cells, leads to dysregulation of BMP signaling. Our data thus argue that Nup107, Dsx and AdamTS-A likely function in ECs and are necessary for the formation and maintenance of the cellular protrusions which are required for restricting the BMP signal emanating from the GSC niche. These observations also suggest that Adam-TS-A, which typically resides in the extracellular matrix, is essential for the proper formation and/or maintenance of these cellular protrusions. Altogether these observations indicate that AdamTS-A modulates BMP signal distribution indirectly by regulating the density/length/ activity of the cellular protrusions that restrict the propagation of BMP signal. We have included a short discussion of this possibility at a relevant juncture in the revised discussion. See new addition to discussion, “AdamTS-A modulates BMP signaling via regulation of EC extensions (line number 556).
- Dsx regulates germ cell differentiation in a female specific manner. I am somewhat puzzled by distinct phenotypes of Dsx depletion described in this paper (germ cell differentiation defect in female only) compared to other previous reports on Dsx function in male and female germline. Is there any sex-transformation phenotype? The part of this manuscript that describes Dsx appears to be detached from the context (published literature on Dsx), and it's somewhat difficult for me to interpret the results.
As mentioned previously (and also in the manuscript), we have not addressed the effect of compromising dsx in males since the effect of Nup107 is largely female specific and males are not obviously affected by Nup107 mutation. Whether this is unique to the specific point mutation in nup107 remains to be determined, however.
We have addressed the question regarding previous reports in great detail above, in point number two of the editor and in the manuscript as well (See new addition to discussion, “Dsx is essential for proper ovarian development” line number 535).
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Evaluation Summary:
This manuscript focuses on a missense mutation of Nucleoporin107 (Nup107) gene, which encodes a nuclear pore complex protein. Phenotypic analyses demonstrate similar phenotypes compared to syndromes identified in human XX-ovarian-dysgenesis patients. Further RNA-seq analyses revealed potential targets of Nup107, through which the authors identified the doublesex (dsx) gene as a critical target with functional readouts. Both Nup107 and Dsx act in the somatic gonadal cells to regulate germ cell function, and have connections with the BMP signaling pathway. This study provides a great example to use Drosophila as a model organism to study the molecular and cellular mechanisms of a known human disease and should be of general interest.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
This manuscript focuses on a missense mutation of Nucleoporin107 (Nup107) gene, which encodes a nuclear pore complex protein. Phenotypic analyses demonstrate similar phenotypes compared to syndromes identified in human XX-ovarian-dysgenesis patients. Further RNA-seq analyses revealed potential targets of Nup107, through which the authors identified the doublesex (dsx) gene as a critical target with functional readouts. Both Nup107 and Dsx act in the somatic gonadal cells to regulate germ cell function, and have connections with the BMP signaling pathway. This study provides a great example to use Drosophila as a model organism to study the molecular and cellular mechanisms of a known human disease and should be of general interest.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study uses Drosophila as a model to study a specific mutant in a gene encoding a nuclear pore protein, whose counterpart in human leads to a rare disease called XX-ovarian-dysgenesis. Intriguingly, the fly mutants mimic the syndromes identified in human patients, such as failures in ovary development and function. The authors use fly as a model to study the molecular and cellular mechanisms underlying the phenotypes, which should provide insight to our understanding of this known human disease.
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Reviewer #2 (Public Review):
In this manuscript Shore et al determine that Nucleoporin 107 (Nup107) is required in developing female somatic cells [intermingled cells (ICs)] for proper ovarian development. The authors propose that Nup107 is required for proper orientation of ICs during development to ensure proper function of escort cells during adulthood. They show that loss of Nup107 results in ectopic germline stem cells (GSCs) away from the GSC niche (primarily cap cells and terminal filament cells) and that these ectopic GSCs display hypermorphic Bone Morphogenic Protein (BMP) signaling. The authors also find that Nup107 regulates expression of the transcription factor doublesex (dsx) and share a common transcriptional target of AdamTS-A. Through knockdown/rescue experiments, the authors show that expression of Dsx-F can rescue …
Reviewer #2 (Public Review):
In this manuscript Shore et al determine that Nucleoporin 107 (Nup107) is required in developing female somatic cells [intermingled cells (ICs)] for proper ovarian development. The authors propose that Nup107 is required for proper orientation of ICs during development to ensure proper function of escort cells during adulthood. They show that loss of Nup107 results in ectopic germline stem cells (GSCs) away from the GSC niche (primarily cap cells and terminal filament cells) and that these ectopic GSCs display hypermorphic Bone Morphogenic Protein (BMP) signaling. The authors also find that Nup107 regulates expression of the transcription factor doublesex (dsx) and share a common transcriptional target of AdamTS-A. Through knockdown/rescue experiments, the authors show that expression of Dsx-F can rescue phenotypes observed with ovarian somatic knockdown of Nup107 and that ovarian somatic knockdown of AdamTS-A mimics loss of Nup107 and dsx (including loss of escort cell membrane protrusions and enhanced BMP signaling). These data provide an interesting non-cytoplasmic to nuclear transport mechanism for Nup107 in regulating oogenesis.
This is a well written manuscript with proper experimental analysis (sufficient n's, proper statistics). However, it is unclear how the authors came to some of their conclusions and how their results significantly enhance findings from prior studies exploring how disruption of organization of somatic cells of the developing female gonad influences adult escort cell protrusions and preventing expansion of BMP signaling to ensure proper germline stem cell cyst differentiation.
Points to consider:
1. It was unclear reading the first part of the paper how the adult phenotypes were connecting to defects during larval development. It would further strengthen the rationale for describing the adult phenotypes first if the authors perhaps show the larval gonad defects (abnormal IC stacking/arrangement) prior to showing adult phenotypes. This would also help with citing previous studies that have also found the correlation with incorrect IC placement and ectopic GSC-like cells in adulthood (e.g., Tseng et al., 2018, Stem Cell Reports).
2. It would also help the reader to know that nuclear pore complex proteins have roles outside of nuclear-cytoplasmic transport early in the manuscript as opposed to only in the discussion.
3. It is unclear the model the authors are proposing for the Nup107-Dsx-AdamTs relationship. Are the authors proposing that Nup107 can regulate the import of Dsx or is directly regulating dsx transcription (based on the RNA-sequencing results)? A little more explanation would be helpful. -
Reviewer #3 (Public Review):
This is an interesting story, providing molecular explanation for XX-OD, caused by mutations in Nup107 gene. Overall, the experiments are thoroughly conducted, and the results are important in understanding XX-OD. However, there are some issues that need to be addressed, as the data presented in this study still leaves some gaps that need attention.
1. Dsx is the major target of Nup107. Although it is clear that Dsx is downregulated in Nup107 mutant, how exactly Nup107 regulates Dsx expression remains entirely unclear. Does Nup107 functions as transcriptional regulator of Nup107?
2. Nup107 Dsx axis is required for escort cells to encapsulate germ cells to allow the downregulation of BMP signaling in germ cells, which in turn allows differentiation of germ cells. Whereas this axis appears to operate, the …
Reviewer #3 (Public Review):
This is an interesting story, providing molecular explanation for XX-OD, caused by mutations in Nup107 gene. Overall, the experiments are thoroughly conducted, and the results are important in understanding XX-OD. However, there are some issues that need to be addressed, as the data presented in this study still leaves some gaps that need attention.
1. Dsx is the major target of Nup107. Although it is clear that Dsx is downregulated in Nup107 mutant, how exactly Nup107 regulates Dsx expression remains entirely unclear. Does Nup107 functions as transcriptional regulator of Nup107?
2. Nup107 Dsx axis is required for escort cells to encapsulate germ cells to allow the downregulation of BMP signaling in germ cells, which in turn allows differentiation of germ cells. Whereas this axis appears to operate, the relationship between germ cell encapsulation and BMP signaling is quite unclear. Is encapsulation upstream or downstream of BMP modulation? Authors provide the evidence that adamTS-A, which modulates the amount of extracellular BMP ligands, is the downstream target of Dsx, and adamTS-A appears to regulate encapsulation. This makes it unclear whether encapsulation is required to down regulate BMP, or BMP regulates encapsulation (and if the latter, what is achieved by encapsulation that leads to germ cell differentiation?)
3. Dsx regulates germ cell differentiation in a female specific manner. I am somewhat puzzled by distinct phenotypes of Dsx depletion described in this paper (germ cell differentiation defect in female only) compared to other previous reports on Dsx function in male and female germline. Is there any sex-transformation phenotype? The part of this manuscript that describes Dsx appears to be detached from the context (published literature on Dsx), and it's somewhat difficult for me to interpret the results.
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