Author response:
Public Reviews:
Reviewer #1 (Public review):
This study provides a thorough analysis of Nup107's role in Drosophila metamorphosis, demonstrating that its depletion leads to developmental arrest at the third larval instar stage due to disruptions in ecdysone biosynthesis and EcR signaling. Importantly, the authors establish a novel connection between Nup107 and Torso receptor expression, linking it to the hormonal cascade regulating pupariation.
However, some contradictory results weaken the conclusions of the study. The authors claim that Nup107 is involved in the translocation of EcR from the cytoplasm to the nucleus. However, the evidence provided in the paper suggests it more likely regulates EcR expression positively, as EcR is undetectable in Nup107-depleted animals, even below background levels.
We appreciate the concern raised in this public review. However, we must clarify that we do not claim that Nup107 regulates the translocation of EcR from the cytoplasm. It is important to note that we posited this hypothesis if Nup107 will regulate EcR nuclear translocation (9th line of 2nd paragraph on page 6). We have spelled this out more clearly as the 3rd sub-section title of the Results section, and in the discussion (8th line of 2nd paragraph on page 11). Overall, we have expressed surprise that Nup107 is not directly involved in the nuclear translocation of EcR.
Ecdysone hormone acts through the EcR to induce the transcription of EcR also and creates a positive autoregulatory loop that enhances the EcR level through ecdysone signaling (1). Since Nup107 depletion leads to a reduction in ecdysone levels, it disrupts the transcription autoregulatory EcR expression loop. This can contribute to the reduced EcR levels seen in Nup107-depleted animals.
Additionally, the link between Nup107 and Torso is not fully substantiated. While overexpression of Torso appears to rescue the lack of 20E production in the prothoracic gland, the distinct phenotypes of Torso and Nup107 depletion-developmental delay in the former versus complete larval arrest in the latter complicate understanding of Nup107's precise role.
We understand that there are differences in the developmental delay when Tosro and Nup107 depletion is analyzed. However, the two molecules being compared here are very different, and the extent of Torso depletion is not evident in other studies (2). Even if the extent of depletion of Torso and Nup107 is similar, we believe that Nup107, being a more widely expressed protein, induces stronger defects owing to its importance in cellular physiology. We think that RNAi-mediated depletion of Nup107 causes a defect in 20E biosynthesis through the Halloween genes, inducing a developmental arrest.
To clarify these discrepancies, further investigation into whether Nup107 interacts with other critical signaling pathways related to the regulation of ecdysone biosynthesis, such as EGFR or TGF-β, would be beneficial and could strengthen the findings.
In summary, although the study presents some intriguing observations, several conclusions are not well-supported by the experimental data.
We agree with the reviewer’s suggestion. As noted in the literature, five RTKs-torso, InR, EGFR, Alk, and Pvr-stimulate the PI3K/Akt pathway, which plays a crucial role in the PG functioning and controlling pupariation and body size (3). We have checked the torso and EGFR signaling. We rescued Nup107 defects with the torso overexpression, however, constitutively active EGFR (BL-59843) did not rescue the phenotype (data was not shown). Nonetheless, we plan to examine the EGFR pathway activation by measuring the pERK levels in Nup107-depleted PGs.
Reviewer #2 (Public review):
Summary:
The manuscript by Kawadkar et al investigates the role of Nup107 in developmental progression via the regulation of ecdysone signaling. The authors identify an interesting phenotype of Nup107 whole-body RNAi depletion in Drosophila development - developmental arrest at the late larval stage. Nup107-depleted larvae exhibit mis-localization of the Ecdysone receptor (EcR) from the nucleus to the cytoplasm and reduced expression of EcR target genes in salivary glands, indicative of compromised ecdysone signaling. This mis-localization of EcR in salivary glands was phenocopied when Nup107 was depleted only in the prothoracic gland (PG), suggesting that it is not nuclear transport of EcR but the presence of ecdysone (normally secreted from PG) that is affected. Consistently, whole-body levels of ecdysone were shown to be reduced in Nup107 KD, particularly at the late third instar stage when a spike in ecdysone normally occurs. Importantly, the authors could rescue the developmental arrest and EcR mislocalization phenotypes of Nup107 KD by adding exogenous ecdysone, supporting the notion that Nup107 depletion disrupts biosynthesis of ecdysone, which arrests normal development. Additionally, they found that rescue of the Nup107 KD phenotype can also be achieved by over-expression of the receptor tyrosine kinase torso, which is thought to be the upstream regulator of ecdysone synthesis in the PG. Transcript levels of the torso are also shown to be downregulated in the Nup107KD, as are transcript levels of multiple ecdysone biosynthesis genes. Together, these experiments reveal a new role of Nup107 or nuclear pore levels in hormone-driven developmental progression, likely via regulation of levels of torso and torso-stimulated ecdysone biosynthesis.
Strengths:
The developmental phenotypes of an NPC component presented in the manuscript are striking and novel, and the data appears to be of high quality. The rescue experiments are particularly significant, providing strong evidence that Nup107 functions upstream of torso and ecdysone levels in the regulation of developmental timing and progression.
Weaknesses:
The underlying mechanism is however not clear, and any insight into how Nup107 may regulate these pathways would greatly strengthen the manuscript. Some suggestions to address this are detailed below.
Major questions:
(1) Determining how specific this phenotype is to Nup107 vs. to reduced NPC levels overall would give some mechanistic insight. Does knocking down other components of the Nup107 subcomplex (the Y-complex) lead to similar phenotypes? Given the published gene regulatory function of Nup107, do other gene regulatory Nups such as Nup98 or Nup153 produce these phenotypes?
We thank this public review to raise this concern. Working with a Nup-complex like the Nup107 complex, this concern is anticipated but difficult to address as many Nups function beyond their complex identity. Our observations with all other members of the Nup107-complex, including dELYS, suggest that except Nup107, none of the other Nup107-complex members could induce larval developmental arrest.
In this study, we primarily focused on the Nup107 complex (outer ring complex) of the NPC. We have not examined other nucleoporins outside of this complex, such as Nup98 and Nup153. However, previous studies have reported that Nup98 and Nup153 interact with chromatin, with these investigations conducted in Drosophila S2 cells (4, 5, 6). In the future, we may check whether Nup98 and Nup153 depletion can produce the arrest phenotype.
(2) In a related issue, does this level of Nup107 KD produce lower NPC levels? It is expected to, but actual quantification of nuclear pores in Nup107-depleted tissues should be added. These and the above experiments would help address a key mechanistic question - is this phenotype the result of lower numbers of nuclear pores or specifically of Nup107?
We agree with the concern raised here, and we plan to assess nucleoporin intensity using mAb414 antibody (exclusively FG-repeat Nup recognizing antibody) in the Nup107 depletion background. Our past observations suggest that Nup107-depletion does not affect the overall nuclear pore complex assembly in Drosophila salivary glands (Data is not shown).
(3) Additional experiments on how Nup107 regulates the torso would provide further insight. Does Nup107 regulate transcription of the torso or perhaps its mRNA export? Looking at nascent levels of the torso transcript and the localization of its mRNA can help answer this question. Or alternatively, does Nup107 physically bind the torso?
While the concern regarding torso transcript level is genuine, we have already reported in the manuscript that Nup107 levels directly regulate torso expression. When Nup107 is depleted torso levels go down, which in turn controls ecdysone production and subsequent EcR signaling (Figure 6B of the manuscript). However, the exact nature of Nup107 regulation on torso expression is still unclear. Since the Nup107 is known to interact with chromatin (7), it may affect torso transcription. The possibility of a physiologically relevant interaction between Nup107 and the torso in a cellular context is unlikely due to their distinct sub-cellular localizations. If we investigate this further, it will require a significant amount of time for having reagents and experimentation, and currently stands beyond the scope of this manuscript.
(4) The depletion level of Nup107 RNAi specifically in the salivary gland vs. the prothoracic gland should be compared by RT-qPCR or western blotting.
Although we know that the Nup107 protein signal is reduced in SG upon knockdown (Figure 3B), we have not compared the Nup107 transcript level in these two tissues (SG and PG). As suggested here, we will knock down Nup107 using SG and PG-specific drivers and quantify the Nup107 depletion level by RT-qPCR.
(5) The UAS-torso rescue experiment should also include the control of an additional UAS construct - so Nup107; UAS-control vs Nup107; UAS-torso should be compared in the context of rescue to make sure the Gal4 driver is functioning at similar levels in the rescue experiment.
This is a very valid point, and we took this into account while planning the experiment. To maintain the GAL4 function, we used the Nup107KK;UAS-GFP as control alongside the Nup107KK;UAS-torso. This approach ensures that GAL4 dilution does not affect observations made in the experiments. It can be noticed in Figure S7 that the presence of GFP signal in prothoracic glands and their reduced size indicates genes downstream to both UAS sequences are transcribed, and GAL4 dilution does not play a role here.
Minor:
(6) Figures and figure legends can stand to be more explicit and detailed, respectively.
We will revisit all figures and their corresponding legends to ensure appropriate and explicit details are provided.
Reviewer #3 (Public review):
Summary:
In this study by Kawadkar et al, the authors investigate the developmental role of Nup107, a nucleoporin, in regulating the larval-to-pupal transition in Drosophila through RNAi knockdown and CRISPR-Cas9-mediated gene editing. They demonstrate that Nup107, an essential component of the nuclear pore complex (NPC), is crucial for regulating ecdysone signaling during developmental transitions. The authors show that the depletion of Nup107 disrupts these processes, offering valuable insights into its role in development.
Specifically, they find that:
(1) Nup107 depletion impairs pupariation during the larval-to-pupal transition.
(2) RNAi knockdown of Nup107 results in defects in EcR nuclear translocation, a key regulator of ecdysone signaling.
(3) Exogenous 20-hydroxyecdysone (20E) rescues pupariation blocks, but rescued pupae fail to close.
(4) Nup107 RNAi-induced defects can be rescued by activation of the MAP kinase pathway.
Strengths:
The manuscript provides strong evidence that Nup107, a component of the nuclear pore complex (NPC), plays a crucial role in regulating the larval-to-pupal transition in Drosophila, particularly in ecdysone signaling.
The authors employ a combination of RNAi knockdown, CRISPR-Cas9 gene editing, and rescue experiments, offering a comprehensive approach to studying Nup107's developmental function.
The study effectively connects Nup107 to ecdysone signaling, a key regulator of developmental transitions, offering novel insights into the molecular mechanisms controlling metamorphosis.
The use of exogenous 20-hydroxyecdysone (20E) and activation of the MAP kinase pathway provides a strong mechanistic perspective, suggesting that Nup107 may influence EcR signaling and ecdysone biosynthesis.
Weaknesses:
The authors do not sufficiently address the potential off-target effects of RNAi, which could impact the validity of their findings. Alternative approaches, such as heterozygous or clonal studies, could help confirm the specificity of the observed phenotypes.
This is a very valid point raised, and we are aware of the consequences of the off-target effects of RNAi. To assert the effects of authentic RNAi and reduce the off-target effects, we have used two RNAi lines (Nup107GD and Nup107KK) against Nup107. Both RNAi induced comparable levels of Nup107 reduction, and using these lines, ubiquitous and PG specific knockdown produced similar phenotypes. Although the Nup107GD line exhibited a relatively stronger knockdown compared to the Nup107KK line, we preferentially used the Nup107KK line because the Nup107GD line is based on the P-element insertion, and the exact landing site is unknown. Furthermore, there is an off-target predicted for the Nup107GD line, where a 19bp sequence aligns with the bifocal (bif) sequence. The bif-encoded protein is involved in axon guidance and regulation of axon extension. However, the Nup107KK line does not have a predicted off-target molecule, and we know its precise landing site on the second chromosome. Thus, the Nup107KK line was ultimately used in experimentation for its clearer and more reliable genetic background.
We are also investigating Nup107 knockdown in the prothoracic gland, which exhibits polyteny. Additionally, the number of cells in the prothoracic gland is quite limited, approximately 50-60 cells (8). Given this, there is a possibility that a clonal study may not yield the phenotype. However, we will consider moving forward with this approach also.
NPC Complex Specificity: While the authors focus on Nup107, it remains unclear whether the observed defects are specific to this nucleoporin or if other NPC components also contribute to similar defects. Demonstrating similar results with other NPC components would strengthen their claims.
We thank this public review to raise this concern. Working with a Nup-complex like the Nup107 complex, this concern is anticipated but difficult to address as many Nups function beyond their complex identity. Our observations with all other members of the Nup107-complex, including dELYS, suggest that except Nup107, none of the other Nup107-complex members could induce larval developmental arrest. Since the study is primarily focused on the Nup107 complex (outer ring complex) of the NPC, we have not examined other nucleoporins outside of this complex.
Although the authors show that Nup107 depletion disrupts EcR signaling, the precise molecular mechanism by which Nup107 influences this process is not fully explored. Further investigation into how Nup107 regulates EcR nuclear translocation or ecdysone biosynthesis would improve the clarity of the findings.
We appreciate the concern raised. Through our observation, we have proposed the upstream effect of Nup107 on the PTTH-torso-20E-EcR axis regulating developmental transitions. We know that Nup107 regulates torso levels, but we do not know if Nup107 directly interacts with torso. We would like to address whether Nup107 exerts control on PTTH levels also.
We must emphasize that Nup107 does not directly regulate the translocation of EcR. On the contrary, we have demonstrated that EcR translocation is 20E dependent and Nup107 independent. Through our observations, we have argued that Nup107 regulates the expression of Halloween genes required for ecdysone biosynthesis. We are interested in identifying if Nup107 associates directly or through some protein to chromatin to bring about the changes in gene expression required for normal development.
There are some typographical errors and overly strong phrases, such as "unequivocally demonstrate," which could be softened. Additionally, the presentation of redundant data in different tissues could be streamlined to enhance clarity and flow.
We thank the reviewer for this observation. We will remove all typographical errors and make reasonable statements based on our conclusions.
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