Nup107 depletion impairs metamorphosis.

(A) Growth profile of third instar larvae from Actin5C-Gal4 driven control and Nup107 knockdowns (Nup107GD and Nup107KK RNAi lines) at 96 h AEL (after egg laying) and 120 h AEL.

(B) Quantitation of Nup107 knockdown efficiency. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. **p = <0.001 and ****p = <0.0001.

(C) Immunodetection of Nup107 protein levels in third instar larval brain-complex lysates from control and Nup107 knockdown.

(D) Quantification of Nup107 protein levels seen in (C). Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ***p = <0.0002 and ****p = <0.0001.

(E) Comparison of pupariation profiles of control and Nup107 knockdown organisms.

Ubiquitous knockdown of Nup107 disrupts ecdysone signaling.

(A-B) Staining of third instar larval salivary glands from control (A) and ubiquitous Nup107 knockdown (B) with Ecdysone receptor (anti-EcR antibody, red) and Nup107 (anti-Nup107 antibody, green). DNA is stained with DAPI. Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(C) Quantification of the nucleocytoplasmic ratio of EcR under control and Nup107 knockdown conditions. At least 45 nuclei were analyzed from 7 to 8 pairs of salivary glands. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = < 0.0001.

(D-F) Analysis of ecdysone-inducible genes, EcR (D), Eip75A (E), and Eip74EF (F) expression, respectively, at the onset of metamorphosis (late third instar larvae stage).

Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. The error bars represent the SEM. ***p = <0.0004 and ****p = <0.0001.

Nup107 regulates Ecdysone receptor-dependent signaling.

(A-C) Detection of and quantitation of nucleocytoplasmic distribution of EcR (anti-EcR antibody, red) and Nup107 (anti-Nup107 antibody, green) in control (A), salivary gland-specific Nup107 depletion (B), and prothoracic gland-specific Nup107 depletion (C) from third instar larval salivary gland nuclei. DNA is stained with DAPI. Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(D) EcR nucleo-cytoplasmic quantification ratio from the salivary gland and prothoracic gland-specific Nup107 knockdown, respectively. At least 45 nuclei were analyzed from 7 to 8 pairs of salivary glands. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = <0.0001, and ns is non-significant.

(E-F) Quantitation of expression of Eip75A (E) and Eip74EF (F) ecdysone-inducible genes at the onset of metamorphosis (RNA isolated from late third instar larvae of control and prothoracic gland-specific Nup107 depletion). Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. **p = <0.008 and ****p = <0.0001.

Nup107 critically regulates the expression of ecdysone-biosynthetic genes.

(A) ELISA measurements of whole-body 20-hydroxyecdysone (20E) levels in control, ubiquitous (Actin5C-Gal4), and prothoracic gland-specific (Phm-Gal4) Nup107 depletion at 96 and 120 h AEL. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. **p = <0.0013, ***p = < 0.0009 and ns is non-significant.

(B) Schematic representation of a Prothoracic gland cell showing genes involved in ecdysone biosynthesis from cholesterol.

(C-G) Quantification of ecdysone-biosynthetic gene expression levels of Spookier (C), Phantom (D), Disembodied (E), Shadow (F), and Shade (G) in cDNA isolated from control, and Nup107 knockdown late third instar larvae. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. **p = <0.001, ***p = <0.0005 and, ****p = <0.0001.

20E supplementation rescues Nup107 depletion-specific EcR signaling defects.

(A-F) Visualization of the nucleocytoplasmic distribution of EcR (anti-EcR antibody, red) without 20E (A-C) and with 20E (D-F) treatment in larval salivary glands of control, ubiquitous (Actin5C-Gal4) Nup107 knockdown, and prothoracic gland-specific (Phm-Gal4) Nup107 knockdown. DNA is stained with DAPI. Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(G-H) Comparative quantification of expression ecdysone-inducible genes Eip75A (G) and Eip74EF (H) from 20E treated salivary glands. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. *p = <0.02, **p = <0.002, ***p = <0.0008 and ns is non-significant.

Torso and Nup107 act synergistically to activate the ecdysone signaling.

(A) A model of the Torso pathway and its components.

(B) Quantitation of torso transcript levels from control and Nup107 depleted larvae (ubiquitous depletion using Actin5C-Gal4). Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = <0.0001.

(C-D) Comparison of pupariation profiles of control, Nup107 knockdown, and torso and rasV12 overexpressing rescue organisms.

(E-G) Detection and quantitation of nucleocytoplasmic distribution of EcR (anti-EcR antibody, red) and Nup107 (anti-Nup107 antibody, green) in control, torso overexpressing ubiquitous Nup107 knockdown (Actin5C-Gal4>Nup107KK; UAS-torso) and torso overexpressing PG-specific Nup107 knockdown (Phm-Gal4>Nup107KK; UAS-torso) third instar larval salivary gland nuclei. DNA is stained with DAPI. Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(H-I) Quantification of expression of Eip75A (H) and Eip74EF (I) ecdysone-inducible genes, respectively. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. *p = <0.03, **p = <0.008, ****p = <0.0001 and ns is non-significant.

Theoretical Model of Nup107 functions in metamorphosis:

During metamorphosis, the prothoracic gland (PG) responds to prothoracicotropic hormone (PTTH) via Torso receptors. The MAP kinase pathway involving Raf, MEK, and ERK, initiated by Ras, leads to Halloween gene expression responsible for Ecdysone synthesis and release. Ecdysone is converted to active 20-hydroxyecdysone (20E) in peripheral tissues. The binding of 20E to the Ecdysone receptor (EcR) allows EcR nuclear translocation and EcR pathway activation, culminating in target gene expression facilitating the metamorphic transition. Nup107 depletion negatively impacts Torso levels and Torso pathway activation, inducing pupariation arrest, which can be rescued by autonomous activation of the Torso pathway. Image created with BioRender.com/x18z538.

Nup107 staining in salivary glands:

(A-B) Custom-generated polyclonal anti-Nup107 antibody colocalizes with pan-FG-Nup antibody, mAb414 (A), and mRFP-tagged Nup107 (B) at the nuclear rim of the third instar salivary gland. DNA stained with DAPI. Scale bars, 20 µm.

Nup107 CRISPR mutant generation:

(A) Schematic representation of Nup107KO generation. (Ai) The genomic locus of nup107 on chromosome-II (2L). The filled black box corresponds to the nup107 ORF (2779 bp) with gRNA(s) positions indicated by red arrows. The first and second gRNAs were designed near start and stop codons, respectively, of the nup107 locus. (Aii) Blue and green arrows indicate two sets of primers located in the 5’-UTR and 3’-UTR regions used for screening of Nup107 mutant. (Aiii) The black discontinuous line represents the nup107 (2752 bp) deletion allele.

(B) Confirmation of Nup107 deletion mutant (heterozygous) line assessed by the presence of a ∼630 bp band amplified from isolated genomic DNA.

Compromised organ size due to ubiquitous depletion of Nup107:

Actin5C-Gal4 was used as a ubiquitous driver.

(A-B) Third instar larval salivary gland (A), and brain complex (B) images of Control, Nup107KK RNAi and Nup107GD RNAi. DNA stained with DAPI. Scale bars are 200 µm and 100 µm in (A) and (B), respectively.

Ubiquitous knockdown of Nup107 using Nup107GD RNAi disrupts ecdysone signaling.

(A-B) Staining of third instar larval salivary glands from control (A) and ubiquitous Nup107GD knockdown (B) with Ecdysone receptor (anti-EcR antibody, red) and Nup107 (anti-Nup107 antibody, green). DNA is stained with DAPI, and Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(C) Quantification of nucleo-cytoplasmic ratio of EcR under control and Nup107 knockdown conditions. At least 45 nuclei were analyzed from 7 to 8 pairs of salivary glands. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = < 0.0001.

(D-F) Analysis of ecdysone-inducible genes, EcR (D), Eip75A (E) and Eip74EF (F) expression. Data are represented from at least three independent experiments.

Statistical significance was derived from the Student’s t-test. The error bars represent the SEM. ***p = < 0.0007 and ****p = < 0.0001.

Nup107GD RNAi mediated Nup107 depletion regulates Ecdysone receptor-dependent signaling.

(A-C) Detection of and quantitation of nucleocytoplasmic distribution of EcR (anti-EcR antibody, red) and Nup107 (anti-Nup107 antibody, green) in control (A), salivary gland-specific Nup107GD depletion (B), and prothoracic gland-specific Nup107GD depletion (C) from third instar larval salivary gland nuclei. DNA is stained with DAPI. Scale bars, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

(D) EcR nucleo-cytoplasmic quantification ratio from salivary gland, and prothoracic gland-specific Nup107 knockdown. At least 45 nuclei were analyzed from 7 to 8 pairs of salivary glands. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = < 0.0001 and ns is non-significant.

(E-F) Quantitation of expression of Eip75A (E), and Eip74EF (F) ecdysone-inducible genes at the onset of metamorphosis (RNA isolated from late third instar larval stage). Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = < 0.0001.

Nup107 regulates metamorphosis via Ecdysone synthesis.

(A) Growth profile of third instar larvae from AB1-Gal4 driven control and Nup107 knockdowns (Nup107KK and Nup107GD RNAi lines) at 96 h AEL (hours after egg laying) and 120 h AEL.

(B) Growth profile of third instar larvae from Phm-Gal4 driven control and Nup107 knockdowns (Nup107KK and Nup107GD RNAi lines) at 96 h AEL and 120 h AEL.

(C) Comparison of pupariation profiles of control, and Nup107 knockdown organisms.

Nup107 depletion compromises the PG size.

(A-C) Prothoracic gland specific driver Phm-Gal4 driven expression of GFP in the prothoracic glands of the third instar larva image of Control (A), Nup107KK RNAi (B) and Nup107GD RNAi (C). DNA stained with DAPI. Scale bars, 20 µm.

Nup107GD RNAi mediated depletion of Nup107 critically regulates expression of ecdysone-biosynthetic genes:

(A) ELISA measurements of whole-body 20-hydroxyecdysone (20E) levels in control and prothoracic gland-specific (Phm-Gal4) Nup107GD depletion at 96 and 120 h AEL. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. **p = < 0.0025 and ‘ns’ is non-significant.

(B-F) Quantification of ecdysone-biosynthetic gene expression levels of Spookier (B), Phantom (C), Disembodied (D), Shadow (E), and Shade (F) in cDNA isolated from control, and Nup107 knockdown late third instar larvae. Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. ****p = < 0.0001, ***p = <0.0005, **p = <0.001 and *p = <0.03.

Ecdysone (20E) supplementation rescues Nup107GD dependent Nup107 depletion specific EcR signaling defects.

(A-B) Visualization of the nucleocytoplasmic distribution of EcR (anti-EcR antibody, red) without 20E (A) and with 20E (B) treatment in larval salivary glands of control, ubiquitous (Actin5C-Gal4) Nup107GD knockdown, and prothoracic gland-specific (Phm-Gal4) Nup107GD knockdown. DNA is stained with DAPI. Scale bar, 20 μm. Charts represent the line scan intensity profile of EcR (Red) and DAPI (Cyan) in the salivary gland nucleus region.

Torso rescues metamorphic defects of Nup107 knockdown:

(A) Growth profile of third instar larvae from different genotypes (Control, Nup107KK, Nup107KK;UAS-torso, and Nup107KK;UAS-rasV12) at 96 hours AEL (after egg laying).

(B-E) Quantification of ecdysone-biosynthetic gene expression levels of Spookier (B), Phantom (C), Disembodied (D) and Shadow (E) in cDNA isolated from control and Nup107 depleted torso overexpressing larvae (by using Actin5C-Gal4 and Phm-Gal4). Data are represented from at least three independent experiments. Statistical significance was derived from the Student’s t-test. Error bars represent SEM. (*) represents p <0.03 and ‘ns’ is non-significant.

Exogenous 20E supplementation analysis.