Nucleoporin107 mediates female sexual differentiation via Dsx
- Department of Developmental Biology and Cancer Research, Institute of Medical Research Israel-Canada, The Hebrew University- Faculty of Medicine, Israel
- Medical Genetics Institute, Shaare Zedek Medical Center, the Hebrew University Hadassah Medical School, Israel
- Bioinformatics Unit of the I-CORE Computation Center, The Hadassah Hebrew University Medical Center, Israel
- Division of Pediatric Endocrinology, Hadassah Hebrew University Medical Center, Israel
- Department of Molecular Biology, Princeton University, United States
Figures
Figure 1 with 1 supplement
Phenotypic characterization of the ovaries compromised for Nup107.
(A) yw control Drosophila ovaries. In contrast, Nup107D364N ovarian samples display a variety of aberrations, including (B) small or (C) shriveled ovaries, and (D) bilateral dysgenesis. Knockdown of Nup107 using tj-Gal4 driver recapitulated the same phenotypes as Nup107D364N, with flies exhibiting (E) underdeveloped or (F) nondeveloped ovaries. (G) The percentages of under/nondeveloped ovaries in yw (n = 196), Nup107WT (n = 504), Nup107D364N (n = 890), tj > Nup107 (n = 316), and nos > Nup107 (n = 64) flies. Scale bar in A applies to all panels.
Figure 1—figure supplement 1
Gene knockdowns were validated through quantitative real-time PCR (qRT-PCR).
Analysis of relative gene expression was performed for (A) Nup107, (B) dsx, or (C) AdamTS-A in adult Drosophila ovaries of the indicated fly lines. Bars represent mean + standard error of the mean (SEM; 5, 2, and 2 biological replicates, 45 + animals each, respectively). p values are from a two-tailed t-test.
Figure 2 with 1 supplement
Nup107D364N larval gonads display aberrant cellular number and arrangement.
(A) Schematic representation of the LL3 larval gonad with its different cell types. (B) Confocal section of Nup107WT gonad containing on average 18 primordial germ cells (PGCs; VASA, green), compared to (C) 27 in Nup107D364N gonads. (D) Quantitation of the total PGCs per confocal section in each gonad, in yw, Nup107WT, Nup107D364N, and Nup107 KD (n = 42, 71, 88, 22). (E) Nup107 KD larval gonads contain excess PGCs (VASA, green) and abnormally dispersed intermingled cells (ICs; TJ, blue). Nup107D364N larval gonads exhibit a range of IC dispersion patterns from (F) normal to (G) moderate to (H) severe. (I) The percentage of each IC phenotype found in yw, Nup107WT, Nup107D364N, and Nup107 KD gonads (n = 22, 61, 58, 21). (J) Representation of the number of PGCs per gonad missing an immediately adjacent IC, due to abnormal dispersion.
Figure 2—figure supplement 1
Female larval gonads compromised for Nup107 show characteristic aberrations.
(A) Nup107D364N female larval gonads are present at LL3. (B) The difference between the average number of intermingled cells (ICs) in yw (n = 13), Nup107WT (n = 14), and Nup107D364N (n = 12) larval gonads is statistically insignificant. p value represents comparison of all three groups. (C) The difference between the total number of cells with spherical fusomes in yw (n = 11) Nup107WT (n = 13) and Nup107D364N (n = 17) larval gonads is significant. (D) Similar numbers of cells with round fusomes (anti-1B1) can be seen in yw and Nup107WT gonads, while being substantially elevated in (E) Nup107D364N gonads.
Figure 3
Nup107D364N adult ovaries demonstrate increased germline stem cell (GSC) number.
(A) A scheme of the Drosophila germarium. In region 1, GSCs divide four times to form a 16-cell cyst. Region 2a is identified by the presence of 16-cell cystocytes which are adjoining the follicle stem cell border. (B) Quantification of spherical fusomes (n = 57, 74, 100, 27), Bam (n = 127, 137, 139, 64), and pMad (n = 122, 121, 225, 48) phenotypes in yw, Nup107WT, Nup107D364N, and Nup107 KD, respectively. (C) Nup107WT germaria contain two to three cells with spherical fusomes, indicated by arrowhead, while (D) Nup107D364N and (E) tj > Nup107 germaria contain an average of six cells with spherical fusomes. (F) yw and (G) Nup107WT germaria show normal pMad (blue) and Bam (green) expression compared to (H) Nup107D364N and (I) tj > Nup107 germaria which show reduced Bam levels and expanded pMad expression.
Figure 4 with 3 supplements
Adult ovaries demonstrate missing escort cell (EC) extensions.
In germaria taken from (A) yw and (B) Nup107WT, the ECs extensions were easy to note. In contrast, ECs extensions from (C) Nup107D364N, (D) tj > Nup107 KD, (E) tj > dsx KD, and (F) tj > AdamTS-A KD germaria were dramatically reduced or lost. (G) Quantitation of Coracle extensions from yw (n = 88), Nup107WT (n = 32), Nup107D364N (n = 25), tj > Nup107 KD (n = 65), tj > dsx KD (n = 60), and tj > AdamTS-A KD (n = 54). Anti-Cora and anti-TJ staining are shown in green and magenta, respectively.
Figure 4—figure supplement 1
Knockdown of Nup107 in ECs results in loss of their cellular extensions acompanied with accumulation of undifferentiated GSCs.
(A) In c587-GFP control Drosophila germaria, the extensions (Cora, green) were clearly present, as well as two to three spherical fusomes (Cora, red) and normal pMad expression (blue). (B) c587-GFP > Nup107 RNAi germaria demonstrate absent escort cell (EC) extensions, high spherical fusome numbers, and expanded pMad expression. (C) Quantification of abnormal germaria with missing EC extensions, excess spherical fusomes and expanded pMad expression in c587-GFP (n = 54, 39, 67) and c587-GFP > Nup107 RNAi (n = 38) germaria. Bars represent mean + standard error of the mean (SEM). p values are from a two-tailed t-test.
Figure 4—figure supplement 2
Knockdown of cora in adult ovaries results in fewer escort cell (EC) extensions without affecting EC number.
(A) c587-GFP/+ germaria did not demonstrate a significant reduction in EC extensions. (B) c587-GFP > cora-RNAi1 germaria showed dramatically reduced or lost extensions. (C) Quantification of EC extensions in germaria taken from c587-GFP (n = 54), c587-GFP > cora-RNAi1 (n = 45), and c587-GFP > cora-RNAi2 (n = 41) flies. Bars represent mean + standard error of the mean (SEM). p values are from a two-tailed t-test.
Figure 4—figure supplement 3
cora KD in adult ovaries showed aberrant phenotypes similar to Nup-107 KD.
In c587-GFP/+ adult ovaries, the germaria contained (A) two to three spherical fusomes and (B) normal pMad expressing germ cells. Adult ovaries taken from c587-GFP > cora RNAi contained abnormal germaria with (C) excess numbers of spherical fusomes (1B1, green) and (D) expanded pMad expressing germ cells (pMad, green). (E) Quantification of the number of cells with round fusomes in c587-GFP/+, c587-GFP > cora-RNAi1, and c587-GFP > cora-RNAi2 (n = 39, 48, and 52, respectively), and pMad expression (n = 67, 50, and 67, respectively). Bars represent mean + standard error of the mean (SEM). p values are from a two-tailed t-test.
Figure 5
Knockdown of Nup107 specifically at the adult stage resulted in loss of escort cell (EC) extensions.
In germaria taken from (A) tjts > GFP flies, both the two to three germline stem cells (GSCs; pMad, red) and EC extensions (Cora, green) were easy to distinguish. (B) In contrast, pMad expression was expanded, and ECs extensions were absent in tjts > Nup107 RNAi germaria. (C) Quantification of abnormal germaria with missing EC extensions or expanded pMad expression in tjts > GFP (n = 69) and tjts > Nup107 RNAi (n = 74) flies. Bars represent mean + standard error of the mean (SEM). p values are from a two-tailed t-test.
Figure 6 with 1 supplement
Knockdown (KD) of dsx in the gonadal soma recapitulates Nup107D364N phenotypes.
(A) dsx KD in the germline (nos-Gal4) results in negligible effects, compared to KD in somatic cells (tj-Gal4), which results in both (B) underdeveloped and (C) nondeveloped ovaries. (D) The percentages of under/nondeveloped ovaries in tj-Gal4 dsx KD (n = 74) vs. nos-Gal4 dsx KD (n = 100). (E) Representation of the number of primordial germ cells (PGCs) per confocal section of each individual gonad in yw (n = 42) and dsx KD (n = 34) larvae. (F) Larval gonads where dsx is KD using tj-Gal4 contain excess PGCs and abnormally dispersed intermingled cells (ICs). (G) The percentage of IC severity phenotypes found in yw (n = 22) and dsx KD (n = 16) larval gonads. (H) Quantitation of the number of PGCs per gonad lacking an immediately adjacent IC, as a result of abnormal IC dispersion in yw (n = 22) and dsx KD (n = 34) larval gonads. (I) tj-driven dsx KD results in excess spherical fusomes, as well as (J) excess pMad (red) expression and missing Bam (green). (K) Quantitation of dsx KD germaria aberrant phenotypes (n = 53, 119, and 63, respectively).
Figure 6—figure supplement 1
Reduction in dsx and AdamTS-A transcription is observed upon compromising Nup107 activity in the Drosophila larval gonad.
Quantitative real-time PCR-based validation was performed by normalizing the relative expression levels to the abundance of housekeeping gene RpS17.
Figure 7
Overexpression of Dsx rescues Nup107 KD ovarian phenotypes.
(A) RNAi-KD of Nup107 using tj-Gal4 driver results in small, underdeveloped ovaries. (B) Coexpression of dsx with RNAi-KD of Nup107 rescues the underdeveloped phenotype, resulting in normal, robust ovaries. (C) Quantitation of under/nondeveloped ovaries in yw (196), Nup107-KD; UAS-GFP (n = 316) and Nup107 KD; dsx overexpression flies (n = 250). (D) tj-Gal4-driven Nup107 KD, dsx overexpression germarium contains normal pMad (red) expression and (D’) normal Bam expression. (E) Quantitation of Bam and pMad expression in yw (n = 127, 122), Nup107 KD; UAS-GFP (n = 64, 48), and Nup107 KD; dsx overexpression (n = 25, 33) germaria. (F) 55% of tj-Gal4-driven Nup107 KD; UAS-GFP ovarioles (n = 20) showed apoptosis, marked by anti-Caspase3 (red) compared to (G) zero Nup107 KD, dsx overexpression ovarioles (n = 15).
Figure 8
AdamTS-A KD larval and adult ovaries show aberrant phenotypes.
(A) Germline KD of AdamTS-A (nos-Gal4) results in negligible effects, compared to somatic KD (tj-Gal4), which results in (B) severely underdeveloped ovaries. (C) Quantitation of under/nondeveloped ovaries in tj- vs. nos-Gal4-driven AdamTS-A KD (n = 126, 108) flies. (D) Somatic KD (tj-Gal4) of AdamTS-A results in larval gonads containing excess numbers of primordial germ cells (PGCs; VASA, green) and abnormally dispersed intermingled cells (ICs; TJ, blue). (E) Quantitation of the total number of PGCs per confocal section in each individual gonad in yw (n = 42) and AdamTS-A KD (n = 17) larvae. (F) The percentage of IC severity phenotypes found in yw (n = 22) and AdamTS-A KD (n = 17) larval gonads. (G) Relative expression of AdamTS-A measured by RT-qPCR. (H) Quantification of cells with round fusomes (n = 57), Bam expression (n = 145, green), and pMad expression (n = 73, red) in yw and AdamTS-A KD ovaries. tj-Gal4-driven AdamTS-A KD results in (I) excess number of cells with spherical fusomes (anti-1B1), (J) expanded pMad (red), and (J’) reduced Bam expression.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72632/elife-72632-transrepform1-v4.docx
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Supplementary file 1
Processed data results of transcriptomic analysis performed on Nup107WT and Nup107D364N larval gonads.
- https://cdn.elifesciences.org/articles/72632/elife-72632-supp1-v4.xls
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Supplementary file 2
List of 82 candidate genes with differential expression following Nup107 loss identified in transcriptomic analysis, with emphasis on those found to be Dsx targets.
- https://cdn.elifesciences.org/articles/72632/elife-72632-supp2-v4.xlsx
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Nucleoporin107 mediates female sexual differentiation via Dsx
eLife 11:e72632.
https://doi.org/10.7554/eLife.72632
