JNK signaling triggers spermatogonial dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis
- New York University School of Medicine, United States
Figures
Figure 1 with 2 supplements
Blocking bam-lineage dedifferentiation does not protect the GSC pool under normal laboratory conditions.
(A) Schematic of the Drosophila testis. Germline stem cells (GSCs) undergo differentiation through several rounds of mitotic divisions with incomplete cytokinesis (the cells remain connected through …
Figure 1—figure supplement 1
Methodology used for blocking and labeling bam-lineage dedifferentiation.
GSCs undergo four rounds of mitotic divisions, and the 4- to 8-cell spermatogonia activate the bam-Gal4 driver. This Gal4 drives expression of UAS-bam, resulting in the production of more Bam …
Figure 1—figure supplement 2
Lineage and real-time expression analysis of bam-Gal4 in aging and starvation.
(A–A”) bam-Gal4 driving the expression ubiP63E-FRT-stop-FRT-GFP lineage tracing cassette marker (A’) and of UAS-RFP for real-time expression analysis (A”) in 0 day-old flies. Note the absence of bam-…
Figure 2 with 1 supplement
bam-lineage dedifferentiation accelerates the recovery of the GSC pool after starvation.
(A) 9-day time course of the proportion of bam-lineage dedifferentiated GSCs during refeeding following 15 days of starvation in control bam > LacZ testes. (B) The total number of GSCs in bam > LacZ …
Figure 2—figure supplement 1
Centrosome mis-orientation is not increased in bam-lineage GSCs compared to lineage-negative GSCs in aging and after challenging conditions.
Centrosome orientation in GSCs in testes from bam > LacZ control males was assessed at day 0, at 45 days fed and unmated, after 15 days of starvation and 5 days of refeeding, and after 4 cycles of …
Figure 3 with 1 supplement
bam-lineage dedifferentiation is required for maintaining a robust GSC pool under chronic challenging conditions.
(A–D) Representative images of the testis stem cell niche in control bam > LacZ (A,B) and bam > bam (C,D) testes at 0 days (A,C) and after 4 cycles of starvation, refeeding and mating (B,D). The bam …
Figure 3—figure supplement 1
bam-lineage dedifferentiation is required for maintaining a robust GSC pool under conditions of continuous mating.
(A) Percentage of phospho-Histone3 (pH3)-positive GSCs in testes from unmated or mated males after 15 days of adulthood. There are significantly more pH3-positive GSCs in testes from mated males …
Figure 4
Spermatogenesis is compromised under chronic challenging conditions when bam-lineage dedifferentiation is inhibited.
(A–H) Representative images of control bam > LacZ (A,C,E,G) and bam > bam (B,D,F,H) testes at 0 days (A,B), after 2 (C,D) or 4 (E,F) cycles of challenging conditions or in 45-day-old fed unmated …
Figure 5
bam-lineage GSCs are more proliferative than their lineage-negative siblings.
(A) Relative contribution to 1-, 2- or 4-cell spermatogonial cysts of bam-lineage GSCs (green line) and lineage-negative sibling GSCs (black line) in the same bam > LacZ testis (see Materials and …
Figure 6 with 2 supplements
JNK pathway activity is required for bam-lineage spermatogonial dedifferentiation.
(A–E) Representative images of testes where puckeredE69 (puc)-Gal4 was lineage-traced at 0 days fed (A), 15 days fed (B), 20 days fed (C), after 15 days starved (D) and after 15 days starved and 5 …
Figure 6—figure supplement 1
Example of fragmentation of 4-cell spermatogonia that experienced JNK activation.
Different planes of a 20 μm z-stack of a testis from a puc > GFP male after 15 days of starvation and 3 days of refeeding. Each plane is 1.0 μm thick. The cells of the fragmenting germ cyst are …
Figure 6—figure supplement 2
puc or bskDN mis-expression does not perturb germ cell differentiation.
(A–B) Confocal slice of a bam > puc (A) or a bam > bskDN (labelled K53R) (B) testis after 15 days of starvation and 5 days of refeeding. Note the presence of pre-meiotic spermatocytes (lower …
Figure 7
Model of spermatogonial dedifferentiation under chronic challenging conditions.
Top left, under normal conditions, GSCs divide and their offspring undergo normal differentiation into spermatogonia. Top right, under challenging conditions like starvation, there is a significant …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | Oregon-R (Ore)R | Bloomington Drosophila Stock Center (BDSC) | stock number: 5 | |
| Strain, strain background (Drosophila melanogaster) | Ubi-p63E(FRT.STOP)Stinger | Bloomington Drosophila Stock Center (BDSC) | stock number: 28282 | |
| Strain, strain background (Drosophila melanogaster) | UAS-RedStinger | Bloomington Drosophila Stock Center (BDSC) | stock number: 28281 | |
| Strain, strain background (Drosophila melanogaster) | UAS-LacZ | Bloomington Drosophila Stock Center (BDSC) | stock number: 3955 | |
| Strain, strain background (Drosophila melanogaster) | UAS-bskK53R | Bloomington Drosophila Stock Center (BDSC) | stock number: 9311 | |
| Strain, strain background (Drosophila melanogaster) | pucE69-Gal4 | Bloomington Drosophila Stock Center (BDSC) | stock number: 6762 | |
| Strain, strain background (Drosophila melanogaster) | puc-IT.Gal4 | Bloomington Drosophila Stock Center (BDSC) | stock number: 63509 | |
| Strain, strain background (Drosophila melanogaster) | UAS-puc | pmid: 9472024 | ||
| Strain, strain background (Drosophila melanogaster) | bam-Gal4:VP16 | pmid: 12571107 | ||
| Strain, strain background (Drosophila melanogaster) | UAS-bam:GFP | pmid: 12571107 | ||
| Strain, strain background (Drosophila melanogaster) | FRT40A | pmid: 26807580 | ||
| Strain, strain background (Drosophila melanogaster) | ywhsFlp112; tubGal80,FRT40A | pmid: 26807580 | ||
| Antibody | goat anti‐Vasa | Santa Cruz | catalog number: dC-13 | ‘1:200’ |
| Antibody | mouse anti-Fas3 | DHSB | catalog number: 3A9 | ‘1:50’ |
| Antibody | mouse anti-α-Spectrin | DSHB | catalog number: 7G10 | ‘1:50’ |
| Antibody | mouse anti-γTubulin | Sigma | catalog number: T6557 | ‘1:100’ |
| Antibody | 5‐ethynyl‐2′ ‐deoxyuridine (EdU) | Invitrogen | catalog number: C10340 | ‘1:50’ |
| Antibody | rabbit anti-phospho Histone H3-Ser10 | Millipore | catalog number: #06–570 | ‘1:200’ |
| Software, algorithm | Fiji-ImageJ | pmid: 22743772 | ||
| Software, algorithm | Photoshop | Adobe | ||
| Software, algorithm | Excel | Microsoft | ||
| Software, algorithm | GraphPad Prism | GraphPad Software | ||
| Software, algorithm | Paint3D | Microsoft |
Additional files
-
Supplementary file 1
Raw data collected for this work.
Data are presented as the mean ± S.E.
- https://doi.org/10.7554/eLife.36095.016
-
Supplementary file 2
Raw data of proliferation markers analyzed for this work.
- https://doi.org/10.7554/eLife.36095.017
-
Supplementary file 3
Raw data collected for this work.
Data are presented as mean ± S.E.n.a is not applicable
- https://doi.org/10.7554/eLife.36095.018
-
Transparent reporting form
- https://doi.org/10.7554/eLife.36095.019
