Competitiveness for the niche and mutual dependence of the germline and somatic stem cells in the Drosophila testis are regulated by the JAK/STAT signaling

Abstract

In many tissues, two or more types of stem cells share a niche, and how the stem cells coordinate their self-renewal and differentiation is poorly understood. In the Drosophila testis, germ line stem cells (GSCs) and somatic cyst progenitor cells (CPCs) contact each other and share a niche (the hub). The hub expresses a growth factor unpaired (Upd) that activates the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in GSCs to regulate the stem cell self-renewal. Here, we demonstrate that the JAK/STAT signaling also regulates CPCs self-renewal. We also show that a negative regulator, the suppressor of cytokine signaling 36E (SOCS36E), suppresses JAK/STAT signaling in somatic cells, preventing them from out-competing the GSCs. Furthermore, through selectively manipulating the JAK/STAT signaling level in either CPCs or GSCs, we demonstrate that the somatic JAK/STAT signaling is essential for self-renewal and maintenance of both CPCs and GSCs. These data suggest that a single JAK/STAT signal from the niche orchestrate the competitive and dependent co-existence of GSCs and CPCs in the Drosophila testis niche.

Fig. 1

JAK/STAT signaling is required for CPCs self-renewal. (A) Testes are labeled for Tj (red); GFP (green); and Dapi (blue). (B–F) Testes are labeled for Tj (red); GFP (green); and Arm (blue). The hubs are marked by stars. Testes with wild-type (WT) GFP-marked clones are shown at 2 days (A), 4 days (C), and 6 days (E) ACI. Testes with stat92E^j6C8 homozygous mutant GFP-marked clones are shown at 2 days (B), 4 days (D), and 6 days (F) ACI. Testes of the following genotypes were shifted from RT to 29°C for 4 days: C587-Gal4/Y;stat92E^F/stat92E^j6C8 (G, H); C587-Gal4/Y;stat92E^F/UAS-stat92E.stat92E^j6C8 (C, D). (H, I) Testes were immunostained with anti-Tj (green) and anti-Arm (red). (G, J) Testes were immunostained with anti-Vasa (red), anti-Arm (green), mAb 1B1 (green), and DAPI (blue). The hub cells are marked by stars (G, I–J). Arrows point to GSCs (J). Arrowheads point to spermatogonia (G). GFP-marked CPCs are highlighted by arrows. (K, L) C587-Gal4/Y; stat92E^F/stat92E^j6C8 testes shifted to 29°C for 3 days from RT. Testis in (K) is immunostained with anti-Tj (green) and anti-Arm (red). Testis in (L) is immunostained with anti-Socs36E (red) and anti-Eya (green). Tj-positive cells move away from the hub (arrowhead), and late-stage somatic cells (arrows in L) move up to the tip. (M, N) C587-Gal4/Y; stat92E^F/stat92E^j6C8 testes shifted to 29°C for 3 days and then returned to 18°C for 4 days. Testis in (M) is immunostained with anti-Arm (red, arrowhead); anti-Tj (green); and anti-Eya (red). Testis in (N) is immunostained with anti-Socs36E (red) and anti-Eya (green). Tj-positive cells move back toward the hub (arrowhead), and late-stage somatic cells (arrows in N) move away from the hub. Scale bars represent 10 μm.

Fig. 2

Analysis of socs36E mutation. (A) socs36E exons are indicated by filled boxes; breaks between the boxes indicate introns. Triangle indicates insertion site of the P element. The black arrow at the bottom of the figure indicates the orientation of the transcription. (B) Molecular analysis of socs36E¹ mutant. Lanes 1 and 2 are representing results of using Northern blotting; the black arrow in lane 1 points to the band of the socs36E transcript, which is undetectable in socs36E¹ mutant flies (lane 2). Lanes 3 and 4 represent results using RT-PCR. The socs36E transcript is undetectable in socs36E¹ mutant flies (lane 4). socs16D serves as an internal control. (C) A sagittal section of the Drosophila testis apex is drawn schematically and leaves out most of the cells for clarity. Both the GSCs (pink) and the CPCs (yellow) are anchored around the hub (green) through adherens junctions. Asymmetric division of both stem cells results in spermatogenic cysts, in which each gonialblast (red) is encased by two somatic cyst cells (white). Four more consecutive divisions produce a cyst of 16 spermatogonia. (D) A wild-type testis was immunostained with anti-Fas III antibody to label the hub (green, arrowhead) and anti-Socs36E (red). Socs36E is expressed in somatic cyst cells. (E, F) Wild-type (E) and socs36E¹ (F) testes were stained with anti-Eya (green) and anti-Socs36E (red). The expression of Socs36E and Eya partially overlaps. Socs36E is totally absent in the socs36E¹ testis (F). The hubs are marked by stars. (G,H) Testes immunostained for germ cells (with anti-Vasa, red), the hub (with anti-Arm, green) fusomes (with mAb1B1, green). The hubs are marked by arrow. Scale bars in D, E, and F represent 10 μm and in G and H represent 30μm.

Fig. 3

Socs36E nonautonomously regulates GSCs self-renewal and maintenance. (A, B) Wild-type (A) and socs36E¹ (B) testes were immunostained with anti-Fas III (green), mAb 1B1 (green), and anti-Vasa (red). Arrows point to GSCs. (C to E) Wild-type (C) and socs36E¹ (D and E) testes were immunostained with anti-Fas III (red), mAb 1B1 (red), and anti-STAT92E (green). Arrow in C points to GSCs. (F) Confocal section through the apex of the testis containing socs36E¹ clones 11 days ACI. The testis is immunostained with anti-β-galactosidase (red); anti-Fas III (green); and mAb1B1 (green). One socs36E null GSCs clone (outlined) and a number of socs36E null spermatogonia clones that are β-galactosidase–negative are found in this testis. The hubs are marked by stars. Scale bars represent 10 μm.

Fig. 4

Socs36E regulates CPCs self-renewal through the JAK/STAT signal transduction pathway. (A to C) Testes immunostained with anti-Tj antibody to label somatic cells (green) and anti-Arm antibody to label the hub (red, stars). Their genotypes are (A) wild-type: CPCs (red arrow) contact the Arm-positive hub. (B) A socs36E¹/socs36E¹; Increased numbers of CPCs contact the hub. (C) C587-Gal4/Y; UAS-socs36E socs36E¹/socs36E¹. Expression of socs36E in somatic cells rescued the phenotype of socs36E¹ to wild-type. (D) A C587-Gal4/Y;UAS-socs36E/+ testis. Overexpression of socs36E (C587-Gal4/UAS-socs36E) resulted in a decrease of Tj-positive somatic cells. (E–F). Testes immunostained with anti-β-galactosidase (green) from an 842 marker and anti-Arm (red, white arrowheads). (E) A wild-type testis with the 842 marker. β-galactosidase (green) is expressed in the hub, CPCs (red arrow), and early somatic cyst cells. (F) A C587-Gal4Y;/UAS-socs36E/842 testis. β-galactosidase stainings are significantly reduced in CPCs and early somatic cyst cells. (G–J) Testes are labeled for Tj (red), GFP (green), and Arm (blue). The hubs are marked by stars. (G) Testis with GFP-marked homozygous socs36E¹ mutant clones 2 days ACI. (H, I, and J) Testes with GFP-marked homozygous socs36E¹ mutant clones 6 days ACI. GFP-marked CPCs are highlighted by arrows. (K–M) Testes are immunostained with anti-Arm (red) and anti-Tj (green). The hubs are marked by arrowheads. (K) A C587-Gal4/Y; UAS-socs36E/+; stat92E^F/stat92E^j6C8 testis shifted to 29°C for 2 days. (L) A C587-Gal4/Y; UAS-socs36E/+; stat92E^F/stat92E^j6C8 testis shifted to 29°C for 2 days and then returned to 18°C for 4 days. (M) A C587-Gal4/Y; UAS-socs36E/+; stat92E^F/UAS-stat92E.stat92E^j6C8 testis shifted to 29°C for 2 days. Scale bars represent 10 μm.

Fig. 5

Three regulatory levels of GSCs and CPCs. See text for detail. In (C), the blue inhibitory lines represent CPCs competing with GSCs for the niche; the black inhibitory lines represent GSCs negatively regulating CPCs proliferation.