Introduction

The multi-protein assembly of nucleoporins (Nups) constitutes nuclear pore complexes (NPCs). In eukaryotic cells, NPCs serve as molecular conduits for the trafficking of proteins and RNAs between the nucleus and the cytoplasm. The NPC composition was revealed from proteomic analyses conducted in yeasts and vertebrates, have contributed to our understanding, revealing that Approximately 30 distinct Nups are arranged in sub-structures and are distributed on different faces of NPCs (Cronshaw et al., 2002; Rout et al., 2000). The substructures are the outer cytoplasmic and inner nuclear rings, which surround a central inner ring called the scaffold ring, primarily aiding in the process of nucleocytoplasmic transport (Lin & Hoelz, 2019).

The largest Nup107 sub-complex, also called the Y-complex, is symmetrically located on both sides of the nuclear membrane. In metazoans, the Y-complex is composed of ten distinct Nups (ELYS, Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1). ELYS is a sub-stoichiometric Y-complex member and is present only on the nucleoplasmic side (D’Angelo & Hetzer, 2008; Morchoisne-Bolhy et al., 2015). Nup107, along with Nup133, forms the stalk of the Y-complex and is critically required for the Y-complex stability. The Nup107 complex plays a pivotal role in facilitating ELYS-coordinated post-mitotic NPC assembly (Boehmer et al., 2003; Walther et al., 2003) and messenger RNA (mRNA) export (Bai et al., 2004). Additionally, The Nup107 complex members actively participate in mitosis, contributing to the regulation of kinetochore-microtubule polymerization (Mishra et al., 2010; Zuccolo et al., 2007). The multitude of cellular processes where the Y-complex performs vital functions suggests it to be a central component in maintaining cellular homeostasis.

As a stable constituent of NPC, many Nups, including the members of the Y-complex, associate with chromatin and exert transcriptional regulation. Notably, interactions between active genes and Nups occurring predominantly within the nucleoplasm have been reported for dynamic Nups such as Nup98, ELYS, and Sec13 (Capelson, Liang, et al., 2010; Kalverda et al., 2010; Kuhn et al., 2019). In Drosophila, the dual nucleoporin, ELYS, governs the development, and ELYS RNAi-induced developmental defects are due to the reactivation of the Dorsal (NF-κB) pathway even during the late larval stages (Mehta et al., 2020).

Nup107 is associated with actively transcribing genes at the nuclear periphery (Gozalo et al., 2020). The disruption of Nup107 in zebrafish embryos leads to significant developmental anomalies, including the absence of the pharyngeal skeleton (Zheng et al., 2012). Moreover, the biallelic Nup107 mutations (D157Y and D831A) correlate well with clinical conditions such as microcephaly and steroid-resistant nephrotic Syndrome (Miyake et al., 2015). In Drosophila, Nup107 co-localizes with Lamin during meiotic division, and Nup107 depletion perturbs lamin localization, leading to a higher frequency of cytokinesis failure during male meiosis (Hayashi et al., 2016). Nup107 influences the regulation of cell fate in aged and transformed cells by modulating EGFR signaling and the nuclear trafficking of ERK protein (Kim et al., 2010).

Drosophila undergoes elaborate metamorphosis initiated by the neuropeptide prothoracicotropic hormone (PTTH) (Rewitz et al., 2013). Bilateral neurons projecting into the prothoracic gland (PG), when stimulated by the PTTH, induce Ecdysone production, which is subsequently released into the circulatory system for conversion by peripheral tissues into its active form, 20-hydroxyecdysone (20E) (Johnson et al., 2013; McBrayer et al., 2007; Shimell et al., 2018). The 20E binds to the ecdysone receptor, and the whole complex translocates into the nucleus and binds to chromatin to activate ecdysone inducible genes (Johnston et al., 2011; Kozlova & Thummel, 2002; Tennessen & Thummel, 2011). In the prothoracic gland, the primary neuroendocrine organ, PTTH signals through the receptor tyrosine kinase (RTK), Torso. The Torso-dependent activation of the MAP-kinase pathway is responsible for the production and release of ecdysone hormone. However, ecdysone synthesis can also be regulated by the EGFR pathway (Cruz et al., 2020; Yamanaka et al., 2013). While Nup107 modulates EGFR pathway activation, the involvement of EGFR and torso pathways in ecdysone-dependent metamorphosis is undeniable. We dissect the involvement of Nup107 in Torso-mediated signaling and underlying mechanisms during Drosophila metamorphosis.

In a reverse genetic RNAi screening for Nup107 complex members, we noted that Nup107 RNAi induces a significant developmental arrest at the third instar larval stage. Further analysis revealed that the Ecdysone Receptor (EcR) signaling pathway is perturbed, and EcR fails to translocate into the nucleus in Nup107 knockdown. The failure of the EcR nuclear localization upon Nup107 depletion is due to significantly reduced levels of the ecdysone hormone during the late third instar larval stage. Interestingly, overexpression of the torso and the ras^V12 in Nup107 depleted larvae rescued the developmental arrest and subsequently initiated pupation. We propose that Nup107, an epistatic regulator of torso-pathway activation in the PG, enables ecdysone surge for efficient metamorphic transition.

Results

Nup107 is essential for larval to pupal metamorphic transition

Cell biological analyses in the mammalian cell culture system has shed light on critical regulatory roles of Nup107 in vertebrates. Yet its importance in development is poorly understood. In this context, we started the characterization of Drosophila Y-complex member Nup107 in greater detail. Utilizing RNAi lines (Nup107^KK and Nup107^GD), we performed ubiquitous depletion through Actin5C-GAL4. Interestingly, Nup107 depletion, leading to larvae arrest at the third instar stage, causing complete cessation of pupariation (120 h AEL, right panel, Figure 1A), was accompanied by an extension of larval feeding and growth periods. Quantitative assessment of Nup107 transcript levels in the Nup107^GD and Nup107^KK RNAi lines suggested efficient Nup107 knockdown (approximately 60-70%, Figure 1B). For further analyses, we generated polyclonal antibodies against the Nup107 amino-terminal antigenic fragment (amino acids 1–210; see methods for details). Purified anti-Nup107 polyclonal antibodies detected a band of approximately ∼100 kDa in lysates prepared from the control larval brain complex. The intensity of this ∼100 kDa band was significantly reduced in lysates prepared from organisms where Nup107 was knocked down ubiquitously (Figure 1C) using Actin5C-GAL4 driving Nup107 RNAi (denoted as ubiquitous hereon). Further, the immunostaining with Nup107 antibodies identified a conserved and robust nuclear rim staining pattern overlapping with mAb414 antibodies recognizing FG-nucleoporins (Figure S1A) and mRFP-tagged Nup107 expressed through its endogenous promoter (Figure S1B) in salivary gland tissues. These observations confirm the efficacy of Nup107 knockdown and provide a handle to assess levels and localization of Nup107 in affected tissues. To further investigate the role of Nup107 in development, we generated a Nup107 null mutant using CRISPR-Cas9-mediated gene editing. This comprehensive approach involving RNAi-mediated knockdown and CRISPR-Cas9 gene editing is expected to provide valuable insights into the significance of Nup107 in Drosophila development. The gRNAs targeting regions close to the start and stop codon of the nup107 gene generated knockout (Nup107^KO) mutants, which were confirmed by sequencing and PCR (Figures S2A and S2B). However, the Nup107^KO mutants could not be used as the Nup107^KO homozygous shows lethality at the embryonic stage. So, we carried out most of the analyses hereon with Nup107 RNAi lines.

Figure 1.

Nup107 contributes significantly to ecdysone signaling

The depletion of Nup107 in Drosophila resulted in a distinct halt in growth and a developmental arrest at the third instar stage (Figure 1A). To discern the pathways affecting juvenile to adult developmental transition in Drosophila, we focused on levels of the sole insect steroid hormone, ecdysone.

We examined the localization of EcR in the late third instar salivary glands of both the control and Nup107-depleted larvae (using the Nup107^KK line). Wild-type larvae exhibited normal EcR localization within the nucleus, but the nuclear translocation of EcR is perturbed in the Nup107-depleted larvae, with the bulk of the signal retained in the cytoplasm (Figures 2A and 2B). Quantitative analysis of EcR intensities in the cytoplasm and nucleus further established a significant decrease in EcR signals inside the nucleus when Nup107 is depleted (Figure 2C). This observation suggests that the Nup107 is required for EcR nuclear localization to mediate critical larval to pupal developmental transition. Furthermore, we noticed that significantly smaller size salivary glands and the brain complex in Nup107 depleted larvae (Figure S3).

Figure 2.

Prompted by the cytoplasmic accumulation of EcR, we investigated whether the mRNA levels of ecdysone-inducible genes were affected. When 20E binds with EcR, the activated EcR with enhanced affinity occupies the ecdysone response element (ERE) region in gene promoters. The ecdysone receptor is thought to function in a positive auto-regulatory loop, which may elevate EcR levels and sustain ecdysone signaling (Varghese & Cohen, 2007). Firstly, we examined the EcR transcript levels, revealing a reduction under Nup107-depletion conditions (Figure 2D). Subsequently, we measured the mRNA levels of known EcR target genes, Eip75A and Eip74EF, to find that the expression of each of these two target genes is reduced and correlates with Nup107 knockdown levels (Figures 2E and 2F). Similar results were observed in the depletion of Nup107 using the GD-based RNAi line (Figure S4). The defects observed during metamorphic transitions can be attributed to impaired ecdysone signaling upon Nup107-depletion, which prompted a closer examination of their regulatory relationship.

Nup107 is dispensable for the nuclear import of EcR in the target tissue

We established that the nuclear localization of EcR is impaired upon Nup107 knockdown (see Figure 2B). Typically, EcR nuclear localization follows an intricate mechanism where 20E binding to EcR allows its nuclear translocation and subsequent activation of its target genes (Cronauer et al., 2007; Johnston et al., 2011; Lenaerts et al., 2019). Biosynthesis of ecdysone hormone from dietary cholesterol requires a group of P450 enzymes coded by the Halloween genes, is a prerequisite for EcR activation (Kannangara et al., 2021; Niwa & Niwa, 2014). Moreover, the involvement of nucleoporin, Nup358, in facilitating nuclear transport of Met juvenile hormone receptors is well documented (He et al., 2017). Consequently, we posited the hypothesis that Nup107 either disallows active 20E-EcR complex formation by affecting 20E biosynthesis in ubiquitous Nup107 knockdown scenarios or directly regulates EcR nuclear translocation in the target tissue.

We chose salivary glands to address these hypotheses and depleted Nup107 using salivary glands-specific AB1-GAL4 and PG-specific Phm-GAL4. Surprisingly, in contrast to the ubiquitous knockdown of Nup107, nuclear localization of EcR remained unaffected in salivary gland-specific Nup107 knockdown (Figures 3A, 3B, S5A and S5B). Interestingly, the PG-specific Nup107 knockdown phenocopied ubiquitous Nup107 knockdown induced EcR nuclear localization defects (Figures 3C and S5C).

Figure 3.

Quantification of EcR signals from Nup107 depleted late third instar salivary gland cells and comparison with control salivary glands indicates that the nuclear/cytoplasmic ratios are drastically reduced in case of PG-specific depletion but unaltered in salivary gland-specific Nup107 depletion (Figures 3D and S5D). Consistent with these observations, expression levels of ecdysone-inducible genes Eip75A and Eip74EF were significantly reduced in PG-specific Nup107 knockdown (Figures 3E, 3F, S5E and S5F). In accordance with unperturbed EcR nuclear translocation, salivary glands-specific depletion of Nup107 yielded no discernible differences in larval growth and pupariation compared to the control (Figures S6A and S6C). However, the PG-specific knockdown induced an extended third instar stage lifespan (10-12 days after egg laying, Figures S6B and S6C). The observed decrease in ecdysone-inducible gene expression during late third-instar developmental stages can explain the potential impairment of metamorphosis induction seen upon ubiquitous or PG-specific Nup107 knockdown. In addition to the reduced size of the salivary gland and brain complex, we also noticed a compromise in the size of the Prothoracic gland upon Nup107 knockdown (Figure S7).

These observations suggest that Nup107 exerts a regulation on ecdysone biosynthesis and active 20E-EcR complex formation rather than playing a direct role in EcR nuclear translocation.

Nup107 exerts control on the EcR pathway through Ecdysone level regulation

We reasoned that Nup107 may regulate the ecdysone biosynthesis in PG to induce the larval stage growth arrest. We delved into analyzing the effect of Nup107 knockdown on ecdysone production. The considerable decrease in PG size due to Nup107 knockdown (Figure S7) can potentially reduce 20E production. This prompted us to explore the potential role of Nup107 in influencing ecdysone production, and we assessed the impact of Nup107 knockdown on ecdysone biosynthesis.

Utilizing an ELISA-based detection method, we assessed 20E levels in larvae at 96 hours and 120 hours after egg laying (AEL). Larvae from different experimental conditions, including control, ubiquitous Nup107-depletion, and PG-specific Nup107-depletion, were used in this analysis. Strikingly, the results indicated a substantial decrease (∼ 3-fold with ubiquitous and ∼10-fold for PG-specific knockdown) in total 20E levels upon Nup107 knockdown, particularly at 120 AEL which coincides with the 20E surge seen during metamorphosis (Figures 4A and S8A). This observation is crucial and suggests a potential defect in ecdysone biosynthesis in Nup107-depleted organisms.

Figure 4.

As depicted in Figure 4B, the PTTH hormone signaling in the PG upregulates the expression of Halloween genes (spookier, phantom, disembodied, and shadow) responsible for ecdysone biosynthesis, and the shade gene product is required for active 20E generation in peripheral tissues (Christensen et al., 2020; McBrayer et al., 2007; Shimell et al., 2018). We analyzed the Halloween genes transcript level in Nup107-depleted larvae and observed a significant downregulation for each of the Halloween quartet genes mentioned earlier. (Figures 4C-4F and S8B-S8E). Further, the level of the shade gene required for converting precursor ecdysone to active 20E was also reduced twofold in Nup107-depleted larvae (Figures 4G and S8F).

20E rescues Nup107-dependent EcR localization defects

The observed correlation between the expression levels of ecdysone biosynthetic genes and reduced levels of 20E upon Nup107 knockdown strongly suggests a role for Nup107 in 20E biosynthesis, active 20E-EcR complex formation, and its nuclear translocation. It is not surprising in this context that exogenous ecdysone supplementation through larval food rescues pupariation blocks arising from ecdysone deficiency (Garen et al., 1977; Ou et al., 2016; Shimell et al., 2018). We experimented the same under Nup107-depletion conditions and supplemented exogenous ecdysone to PG-specific Nup107-depleted larvae by feeding them with a diet enriched with 20-hydroxyecdysone (0.2 mg/ml). Supplementation of 20E to Nup107-depleted larvae significantly (70-80%) alleviated the developmental arrest, and the onset of pupariation was comparable to the control (Table S1). However, none of the pupae could eventually eclose successfully, probably due to other effects of Nup107 depletion. We asked if nuclear translocation of EcR can also be rescued by exogenous supplementation of 20E. While incubation of salivary glands of late third instar larva in S2 media control alone did not rescue EcR localization in any of the Nup107-knockdown genotypes (Figures 5A-5C and S9A), we noticed a complete EcR nuclear translocation rescue in ubiquitous as well as PG-specific Nup107-depleted salivary glands when incubated with 20E (Figures 5D-5F and S9B).

Figure 5.

The exogenous supplementation of 20E leads to active 20E-EcR complex formation as mRNA levels of the Eip75A and Eip74EF target genes were rescued significantly back to normal levels (Figures 5G and 5H). Overall, these findings highlight the important regulatory function of Nup107 in the ecdysone signaling pathway.

Torso is an effector of Nup107 mediated functions in metamorphosis

Next, we explored the mechanism of Nup107-driven regulation of 20E levels and metamorphosis. Receptor tyrosine kinase family of cell surface receptors signal by binding to ligands (growth factors and hormones) and participate in metabolism, cell growth, and development. Among these RTKs, the Torso, belonging to the platelet-derived growth factor receptor (PDGFR) class, plays a significant role during metamorphosis by serving as a receptor for the neuropeptide PTTH in the Drosophila brain (Sopko & Perrimon, 2013).

Functional engagement of PTTH-Torso activates the MAP kinase pathway, involving components of Ras, Raf, MEK, and extracellular signal-regulated kinase (ERK), thereby initiating metamorphosis (Figure 6A). The torso knockdown in the prothoracic glands resulted in the developmental arrest phenotype synonymous with what is observed with Nup107 knockdown. Feeding 20E to torso-depleted larvae completely rescued developmental delay and normal growth phenotypes (Rewitz et al., 2009). We observed a significant decrease in the torso levels (∼ four-fold) when Nup107 was depleted ubiquitously using Actin5C-GAL4 (Figure 6B), suggesting an epistatic regulation by Nup107 on the torso. Further, the phenotypic similarity between Nup107 and torso-depletion scenarios and diminished torso level upon Nup107-depletion prompted us to investigate whether the torso is an effector of Nup107.

Figure 6.

Overexpression of the torso and or ras^V12 have been utilized to rescue torso pathway-mediated defects (Cruz et al., 2020; Rewitz et al., 2009). We probed the possibility of Nup107 phenotype rescue by ubiquitous or PG-specific overexpression of the torso and ras^V12. Overexpression of either torso or ras^V12 in Nup107-depletion background completely rescued the pupariation defects (Figures 6C and 6D). The torso overexpression-mediated rescue of Nup107-depletion phenotypes is specific as overexpression of Egfr (another RTK implicated in metamorphosis) and Usp (co-receptor with EcR) in Nup107 depletion background could not rescue the pupariation defects (data not shown). The remarkable rescue demonstrated by torso overexpression prompted us to analyze the status of EcR nuclear translocation, ecdysone biosynthesis, and ecdysone-inducible gene expression. The restoration of EcR nuclear translocation (Figures 6E-6G), the ecdysone biosynthesis genes, spookier, phantom, disembodied, and shadow (Figures S10B-S10E), and ecdysone target genes Eip75A and Eip74EF (Figures 6H and 6I) to control levels indicated that torso could efficiently rescue Nup107 phenotypes.

These observations suggest that the torso is a downstream effector of Nup107 functions, and torso-dependent signaling, responsible for 20E synthesis in PG and metamorphosis, is regulated by Nup107 levels.

Discussion

Apart from its importance in mRNA export and cell division at the cellular level, the NPCs Y-complex member Nup107, when mutated, correlates with developmental abnormalities, microcephaly, and nephrotic syndrome (Miyake et al., 2015; Zheng et al., 2012). In several human cancer types, Nup107 exhibits high expression and serves as a biomarker for hepatocellular carcinoma. A strong structural and functional conservation between human and Drosophila Nup107 proteins allows Drosophila to be used to model to gain mechanistic insights in Nup107 functions in human diseases (Shore et al., 2022; Weinberg-Shukron et al., 2015). We observe that Nup107 is involved in critical developmental transitions from larva to pupa during Drosophila development as pupariation is completely arrested (Figure 1). The foraging third instar larva must acquire a critical weight and sufficient energy stores before it can metamorphose into a non-feeding pupa, molting into a healthy adult. The activation of the Torso receptor and sequent surge in ecdysone synthesis is essential for larval to pupal transition (Christensen et al., 2020; Hao et al., 2021; Luo et al., 2024). The elevated ecdysone levels trigger the translocation of the heterodimeric ecdysone receptor [in complex with Ultraspiracle (Usp)] into the nucleus to induce pupariation-specific transcriptional programming (Johnston et al., 2011). Similarly, when Nup107 is depleted, nuclear translocation of EcR is abolished, arresting metamorphosis at the level of the third instar larval stage (Figure 2).

Translocation of nuclear receptor proteins like the ecdysone receptor to the nucleus is a crucial step for transcriptional activation. Nup358, present at the cytoplasmic filament face of NPC, plays a vital role in the nuclear import of Met (juvenile hormone receptor) along with Hsp83 (He et al., 2017) to help maintain the larval stages of development. The marked absence of EcR from the nucleus was intriguing as Nup107 generally participates in the nuclear export process. Perhaps Nup107 regulates pupariation by modulating ecdysone synthesis, ecdysone signaling, and nuclear translocation of EcR. Surprisingly, Nup107 is dispensable for the nuclear translocation of EcR in the target tissue, the salivary gland cells (Figure 3). Nup107 exerts strong control over the expression of Halloween genes involved in ecdysone biosynthesis, resulting in diminished 20E titer, poor EcR activation, and delayed larva-to-pupa transition (Figures 4 and S6). The effect of Nup107 on Halloween genes adheres well with pupariation defects. These observations are in concurrence with reports of low ecdysone levels disrupting the pupariation process, leading to a halt in insect development (Christensen et al., 2020; Cruz et al., 2020).

In addition to ecdysone signaling, insect metamorphosis also has a strong contribution from receptor tyrosine kinase RTK pathway signaling. Notably, Torso signaling is essential for embryonic development and metamorphosis in Drosophila. The receptor expression level impacts the signaling output: high receptor levels trigger a robust and transient signal, while lower levels result in a weaker, sustained signal (Jenni et al., 2015; Konogami et al., 2016). Accordingly, the reduced ecdysone levels in the torso knockdown prothoracic glands induced pupariation delays (Rewitz et al., 2009). We observe a similar defect with Nup107 knockdown, indicating a possible reduction in torso levels. Interestingly, we observed significantly reduced torso levels in Nup107-depleted organisms, suggesting an essential upstream function for Nup107 in regulating RTK pathway activation and the associated Drosophila metamorphosis process. In the contrasting observation, Nup107 depleted larval tissues (salivary glands, brain complex, and prothoracic gland) are significantly smaller in size (Figures S3 and S7). It is important to know how Nup107 contributes to organ size maintenance and if a crosstalk with RTK signaling is required in this context.

The developmental delays observed in Nup107 knockdown larvae can be restored by exogenous supplementation of 20E, suggesting that Nup107 can modulate directly or indirectly the Torso receptor activity for ecdysone production and developmental regulation. Accordingly, we successfully rescued developmental delays by ubiquitous and PG-specific torso overexpression. Perhaps overexpression of the torso pathway mediator molecules can rescue the Nup107 developmental delay phenotypes. The oncogenic variant of the Ras-GTPase (ras^V12) has been explored in a similar analysis with torso pathway mutants (Cruz et al., 2020; Rewitz et al., 2009). The alleviation of the developmental delays in the Nup107 knockdown background upon ras^V12 overexpression is an indication of Nup107 serving as an epistatic regulator of the torso pathway in metamorphic transitions (Figure 6).

In essence, our findings indicate that Nup107 influences pupariation timing by regulating the torso levels, it’s signaling, and ecdysone biosynthesis (Figure 7). Previous research has shown that nuclear pore complexes (NPCs) are essential for maintaining global genome organization and regulating gene expression (Capelson, Doucet, et al., 2010; Capelson, Liang, et al., 2010; Iglesias et al., 2020). Particularly, Nup107 interacts with chromatin and targets active gene domains to regulate gene expression (Gozalo et al., 2020). Thus, Nup107 exerting its effects on torso transcription is the primary regulatory event in the Drosophila metamorphosis. It is important to note that the synthesis and availability of Torso ligand, PTTH, may not be affected by the Nup107 since torso or downstream effector ras^V12 overexpression is sufficient to rescue the developmental arrest phenotype. It is thus crucial to further delineate the mechanism of Nup107-dependent regulation on torso pathway activation. The whole genome transcriptomics from PG can help shed more light on the regulatory roles of Nup107. This informationwill offer valuable insights into how nucleoporins regulate gene expression and serve as a model for elucidating how they govern the temporal specificity of developmental processes in organisms. These analyses will help establish a link between Nup107, the PTTH-PG axis, and the regulation of maturation timing. Our observations indicate critical roles for Nup107 in both torso-ecdysone interplay in metamorphosis and torso-independent mechanism for organ size maintenance. Together they add valuable information to hitherto unknown functions of the Nup107 in organismal development. Further investigations are required to identify the interactors of Nup107 involved in these coordination mechanisms in developmental transition.

Figure 7.

Materials and methods

Key resources table

Fly stocks and genetics

Experimental Drosophila melanogaster stocks were reared on a standard cornmeal diet (Nutri-Fly Bloomington formulation) under controlled conditions of 25°C and 60% relative humidity unless otherwise specified. Fly lines used in this study were sourced from the Bloomington Drosophila Stock Centre (BDSC) at Indiana University or from the Vienna Drosophila Resource Center (VDRC), which are listed in the Key resources table. The UAS-GFP lines were received as a gift from Dr. Varun Chaudhary (IISER Bhopal, India). Control groups were generated through crosses between the driver line and w¹¹¹⁸ flies. For RNA interference (RNAi) experiments, crosses were maintained at 29°C to optimize GAL4 expression. The Nup107^GD line, in conjunction with Actin5C-GAL4, was specifically cultivated at 23°C to generate third-instar larvae for experimental purposes.

Transgenic fly generation

In the generation of transgenic flies containing gRNA, we employed a systematic approach. The online tool available at http://targetfinder.flycrispr.neuro.brown.edu was used for the initial sgRNA design, ensuring zero predicted off-targets. Subsequently, we utilized an additional tool available at https://www.flyrnai.org/evaluateCrispr/ to evaluate and score the predicted efficiency of sgRNAs in the targeted region. The most efficient sgRNAs, demonstrating high specificity with no predicted off-targets, were selected and cloned into the pCFD4 vector. The Fly Facility services at the Centre for Cellular and Molecular Platforms, National Center for Biological Sciences (C-CAMP-NCBS), Bangalore, India, were utilized to clone and generate transgenic flies. Primer sequences employed in this study can be found in the Key resources table.

CRISPR –Cas9 mediated mutant Generation

Virgin nanos.Cas9 (BL-54591) flies were crossed with males of gRNA transgenic lines, and approximately 10 F1 progeny males were crossed with balancer flies specific for the gene of interest. A single-line cross was established using the F2 progeny to assess the deletion of nup107. Subsequently, F3 progeny were subjected to nested PCR to confirm deletions. Positive individuals were further tested for the presence of gRNA and Cas9 transgenes, and flies lacking both were propagated into stocks.

Genomic DNA Isolation

Approximately ten flies were homogenized in 250 µL of solution A, comprising 0.1 M Tris-HCl, pH 9.0, 0.1 M EDTA, and 1% SDS, supplemented with Proteinase-K. The homogenate was incubated at 70°C for 30 min. Subsequently, 35 µL of 8 M potassium acetate was added, mixed gently without vortexing, and incubated on ice for 30 min. The homogenate was then centrifuged at 13,000 rpm at 4°C for 15 min, and the supernatant was carefully collected without disturbing precipitates or the interphase. 150 µL of Isopropanol was added to the supernatant and incubated on ice for 15 min. After centrifugation at 13,000 rpm for 5 min, the supernatant was discarded, and the pellet was washed with 1 mL of 70% ethanol by centrifuging at 13,000 rpm for 5 min. The supernatant was again discarded, and the pellet was dried at 55°C until the ethanol evaporated. Finally, the pellet was resuspended in 50 µL of Tris-EDTA buffer and incubated at 37°C.

Nested PCR

Nested PCR was employed to assess the presence of deletions in flies, utilizing genomic DNA as the template. 1 µL of DNA was used for the initial PCR with an outer set of primers (Key resources table). Following this, 1 µL of the initial PCR product was employed for a subsequent PCR with an inner set of primers. The resulting PCR products were then resolved on 0.8% agarose gel to visualize the desired bands indicative of deletions.

Measurement of developmental timing and pupariation

Flies were permitted to lay eggs for 3 hours on agar plates supplemented with yeast, synchronizing the larvae. Newly hatched L1 larvae were then collected and transferred to vials. Pupariation times and dates were recorded daily during the light cycle. Data from 8-10 vials were aggregated, organized by pupariation time and cumulative percentage pupariation, and analyzed using Microsoft Excel.

Antibody generation and western blotting

To generate antibodies against Drosophila Nup107 (CG6743), the N-terminal 210 amino acids, recognized as the most unique and antigenic region, were sub-cloned into the modified tag-less pET28a(+) vector, pET28a(+)-JK vector. The protein was expressed in Escherichia coli BL21 (DE3) cells, induced with 200 μM IPTG (Sigma), and incubated at 30°C for 4 hours. Following cell pelleting, the pellet was resuspended in lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA, and 25% Sucrose) with 1× protease inhibitor mixture (Roche Applied Science), and lysozyme (from 50 mg/mL stock) was added to facilitate bacterial lysis. After sonication and centrifugation, the pellet was successively resuspended in inclusion body buffer I (20 mM Tris pH 8.0, 0.2 M NaCl, 1% Sodium deoxycholate, and 2 mM EGTA) and buffer II (10 mM Tris pH 8.0, 0.25% Sodium deoxycholate and 1 mM EGTA) and centrifuged. This process was repeated three times within inclusion body buffer II, and the final pellet was dissolved in 8 M Urea buffer (10 mM Tris-HCl pH 8.0, 8 M Urea, 0.1 mM NaN_3, and 1 mM EGTA), diluted to 6 M Urea, and centrifuged. The supernatant was loaded onto SDS-PAGE, and the desired band was cut for protein elution. Rabbit polyclonal antibodies against Nup107 were generated at Bio Bharati Life Sciences Pvt. Ltd., Kolkata, West Bengal, India. The antibodies obtained were subjected to affinity purification. The purification involved the chemical cross-linking of purified antigens to N-hydroxy succinimidyl-Sepharose beads (Sigma). The elution process was carried out under low pH conditions, followed by neutralization. Subsequently, the eluted antibodies were dialyzed against phosphate-buffered saline (PBS) overnight at 4°C to remove any remaining impurities and optimize their stability and functionality for subsequent experimental use.

Larval brain complexes from third-instar larvae were dissected, lysed in Laemmli buffer, and resolved on 8% SDS-PAGE. Two equivalent head complexes were loaded per well. After transfer to a polyvinylidene difluoride membrane, blocking was performed with 5% fat-free milk. The membrane was then incubated with polyclonal anti-Nup107 antibody (1:500) and anti-α-tubulin (DSHB, 12G10) (1:5000) at 4°C overnight. Following three washes with TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20), the membrane was incubated with secondary antibodies, anti-rabbit-Alexa-Fluor Plus 680 (Thermo Fisher Scientific #A32734) and anti-mouse-Alexa-Fluor Plus 800 (Thermo Fisher Scientific #A32730). Following incubation with secondary antibodies, the membrane was washed three times for 10 minutes each with Tris-buffered saline containing Tween-20 (TBS-T) to remove unbound antibodies. The washed membrane was then subjected to imaging using the Li-Cor IR system (Model: 9120), allowing for the visualization and analysis of the protein bands on the membrane.

Immunostaining

For immunostaining Drosophila salivary glands, the previously reported protocol was followed (Mehta et al., 2021). The larvae were dissected in cold phosphate-buffered saline (PBS) to isolate salivary glands. Glands underwent pre-extraction with 1% PBST (PBS + 1% Triton X-100), then fixation in freshly prepared 4% paraformaldehyde for 30 minutes at room temperature. Subsequently, the glands were thoroughly washed with 0.3% PBST (0.3% Triton X-100 containing PBS). Blocking was performed for 1 h with 5% normal goat serum (#005-000-001, The Jackson Laboratory, USA). The glands were stained with the following primary antibodies: anti-Nup107 (1:100), mAb414 (1:500, Bio-legend) and anti-EcR (1:20, DDA2.7, DSHB) overnight at 4°C. Tissues were washed three times with 0.3% PBST (PBS + 0.3% Triton X-100), followed by incubation with secondary antibodies: anti-rabbit Alexa Fluor 488 (1:800, #A11034, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 568 (1:800, #A11036, Thermo Fisher Scientific), anti-mouse Alexa Fluor 488 (1:800, #A11029, Thermo Fisher Scientific), anti-mouse Alexa Fluor 568 (1:800, #A11004, Thermo Fisher Scientific), and anti-rabbit Alexa Fluor 647 (1:800, Jackson ImmunoResearch). Following secondary antibody incubation, tissues were washed three times with 0.3% PBST (PBS + 0.2% Triton X-100) and mounted with DAPI-containing Fluoroshield (#F6057, Sigma). The same protocol was followed for staining of the Brain complex and prothoracic gland.

Fluorescence Intensity quantification

Images were acquired using an Olympus Confocal Laser Scanning Microscope FV3000. Subsequent image processing was conducted using the Fiji software, and signal intensities were averaged using GraphPad software (Prism). To quantify the EcR nuclear to cytoplasmic ratio, three distinct regions of interest (ROI) were designated per nucleus and its surrounding cytoplasm. Fiji software measured the average intensity in each ROI, and the mean of these intensities was determined per nucleus and its adjacent cytoplasm. The final graph depicts the ratio of the mean intensities observed in the nucleus to that in the cytoplasm. All experiments were conducted with a minimum of three independent replicates.

Quantitative RT-PCR

Total RNA was extracted from various genotypes (control, ubiquitously depleted Nup107, and prothoracic gland-specific depletion of Nup107) of whole late third instar larvae utilizing the total tissue RNA isolation kit (Favorgen Biotech). One microgram of total RNA was employed for cDNA synthesis with the iScript cDNA synthesis kit (Bio-Rad). The resulting cDNA was diluted fivefold, and 1 μL from each genotype was utilized as a template. RT-PCR was conducted using SYBR Green PCR master mix on an Applied Biosystems QuantStudio 3 Real-Time PCR System. The Rpl69 gene served as the control, and relative transcript levels were determined using the CT value (2^-ΔΔCT). Differences in gene expression were analyzed using Student’s t-test, with a p-value < 0.05 considered significant. The primers used are detailed in the Key resources table. The resultant graph illustrated the fold change in gene expression, plotted using GraphPad software (Prism).

20-hydroxyecdysone (20E) level measurements

Three biological replicates of 25 mg larvae from each genotype were collected at the specified times after egg laying (AEL). The larvae were washed, dried, and weighed before being flash-frozen on dry ice and stored at −80°C. Ecdysone extraction was done by thoroughly homogenizing the frozen samples in 300 µL ice-cold methanol with a plastic pestle. After centrifugation at 17,000 x g for 10 minutes, the supernatant was divided into two Eppendorf tubes containing approximately 150 µL supernatant. Methanol from both tubes was evaporated in a vacuum centrifuge for 60 minutes, and the resulting pellets were redissolved by adding 200 µL Enzyme Immunoassay (EIA) buffer to one of the tubes. After vortexing, the same 200 µL EIA buffer was transferred to the second tube, followed by another round of vortexing. The Enzyme-Linked Immunosorbent Assay (ELISA) was performed using a 20-hydroxyecdysone ELISA kit (#EIAHYD) from Thermo Fisher Scientific.

20E Rescue Experiment

20-hydroxyecdysone (#H5142, Sigma) was dissolved in ethanol to achieve a 5 mg/mL concentration. Salivary glands from third instar larvae of different genotypes were extracted and then incubated for 6 hours in Schneider’s S2 media with 50 μM 20E. After the incubation, the glands underwent procedures for immunostaining and RNA extraction.

To facilitate the rescue of the larvae through feeding, 30-third instar larvae, Nup107 depleted larvae cultured at 29°C incubator, were first washed with water. They were then transferred to vials containing either 20E (at a final concentration of 0.2 mg/mL) or 95% ethanol (in the same amount as the 20E). Once the larvae were added to the vials, these were returned to the 29°C incubator and observed for pupariation, recording the time of this developmental stage.

Data and Resource Availability

All relevant data and resource can be found within the article and its supplementary information.

Supplementary Information

Figure S1.

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Table S1.

Acknowledgements

We thank the Bloomington Drosophila Stock Centre (BDSC) and Vienna Drosophila Resource Centre (VDRC) for generously providing the fly lines crucial for this study. Special recognition is given to Bio Bharati Life Sciences Pvt. Ltd., Kolkata, India, for their significant contribution to antibody generation and the Developmental Studies Hybridoma Bank (DSHB) for supplying antibodies. Our sincere thanks go to C-CAMP Bengaluru for their instrumental role in generating the transgenic fly. The Indian Institute of Science Education and Research Bhopal Central Instrumentation Facility is acknowledged for its valuable support in DNA sequencing and facilitating access to confocal microscopes. This work is supported by the Science and Engineering Research Board grant no. CRG/2020/000496 provided to RKM.

Additional information

Author Contribution

JK and RKM conceptualized the project and the experimental plan and wrote the manuscript. JK performed all experiments and analyzed data. PAJ made initial observations with Nup107 RNAi and analyzed and discussed data. RKM provided intellectual support in data analysis and secured funding for the project.