Supplementary MaterialsSUPPLEMENTARY MATERIAL 41598_2019_42630_MOESM1_ESM. and differentiating gonialblasts (GBs). The GBs are engulfed by RSL3 kinase activity assay two somatic cyst cells, generated from asymmetric CySC divisions. The GBs undergo four rounds of mitotic division with incomplete cytokinesis before differentiation. The somatic cyst cells grow without further division to encapsulate the germline cells with their cellular extensions throughout spermatogenesis12,13,17,26,27,40,41. CySCs are also critical for GSC maintenance, therefore, CySCs together with the hub define the niche for GSCs3,32,33,42. Bone Morphogenetic Protein (BMP) and Hedgehog (Hh) signaling play important functions in the maintenance of GSCs and CySCs20,21,23,24,29,30,43C46. The hub and the early cyst cells produce two BMP ligands, Glass bottom vessel (Gbb) and Dpp43,44. Short-range BMP signaling is critical for GSC maintenance and differentiation. BMP production and diffusion within the niche must be tightly controlled to ensure localized BMP signaling inside the niche, while ectopic BMP signaling outside of the niche leads to aberrant GSC proliferation and differentiation45,47C52. Our recent study found that Tkv functions as ligand sink to spatially restrict Dpp signaling within the testis niche53. However, it remains unknown whether ectopic Dpp signaling in CySCs has any role in stem cell regulation. CySCs and GSCs often compete for niche occupancy, making the testis an excellent model to study the underlying mechanisms controlling stem cell competition. Stem cell competition selects fittest stem cells for tissue homeostasis, and is potentially implicated in tumorigenesis1C5. Previous studies found that CySCs compete with each other and CASP3 with GSCs for niche occupancy. The mutant stem cell and its descendants with increased competitiveness will outcompete wild type stem cells4,6,15,16,19,24,46,54. In the testis, CySC-GSC competition is usually first revealed in mutant, the unfavorable regulator of JAK/STAT signaling16. Recent studies found that several signaling pathways, including Hh, Hippo (Hpo), and EGFR/Mitogen-activated protein kinase (MAPK), regulate stem cell competition15,19,24,46,54. However, the underlying mechanisms controlling stem cell competition are not fully comprehended. In this study, we investigate whether additional factors regulate stem cell competition in the testis niche. Interestingly, we find that ectopic expression of in CySCs results in competition between CySCs and GSCs for niche occupancy and GSC loss. We demonstrate that CySC-GSC competition observed in (in CySCs leads to CySC-GSC competition and GSC loss In order to search for new regulators of stem cell competition, we performed a large-scale screen using a driver (is strongly expressed in CySCs and somatic cyst cells of the testis (Fig.?1a). Our recent data show that Tkv acts as receptor trap to restrain Dpp signaling within the niche53. Surprisingly, we found that when a constitutively active form of (may cause CySC-GSC competition. The observed phenotype RSL3 kinase activity assay was resulted from systemic expression of in all CySCs, we wondered whether ectopic expression of in single CySC or only a portion of CySCs could cause the same defect. We explored this possibility by using MARCM technique to generate CySC clones expressing control CySC clones, we found that RSL3 kinase activity assay expression in CySCs causes stem cell competition. Open in a separate window Physique 1 Ectopic expression of in CySCs leads to GSC loss. (a) control testis. GSCs (white arrowheads) and CySCs (yellow arrowhead) are indicated. (b) testis. The hub is usually surrounded by CySCs (yellow arrowheads), and no germline cells can be observed (white arrowhead). (c) Quantification of the number of GSCs per testis in control and testes. testes. control. GSCs (white arrowheads) and GFP-marked CySCs (yellow arrowhead) are indicated. (f) CySCs (yellow arrowheads) tightly attach to the hub, and the number of GSCs per testis is usually greatly reduced (white arrowhead). (g) Quantification of the number of GSCs per testis in testes carrying control RSL3 kinase activity assay and MARCM clones. control and MARCM clones. induces ectopic Dpp signaling activation43,55, we found that Dpp signaling activation was greatly increased in the cyst cell lineage of testis, using pMAD as a readout (Supplementary Fig.?1). As Dpp signaling is usually highly activated upon ectopic expression of in CySCs (Supplementary Fig.?1), we examined whether.