Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†

Abstract

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

Muscarinic receptor M3 contributes to intestinal stem cell maintenance via EphB/ephrin-B signaling

“…EphB2 correlates with activity of the MAPK/ERK kinase (MEK) signaling pathway, which is involved in self-renewal and proliferation of germline stem and progenitor cells ( N’Tumba-Byn et al, 2020 ). Because deletion of M3 from the small intestine results in up-regulation of the MAPK signaling pathway ( Fig 4 ), we examined the consequence of M3 deletion on activation of the MAPK/ERK signaling pathway using a MEK inhibitor (U0126).…”

FOSL1 modulates Schwann cell responses in the wound microenvironment and regulates peripheral nerve regeneration

“…The expression of Sox2 in Schwann cells, however, is not significantly altered after FOSL1 knockdown or overexpression according to our sequencing data, proposing that FOSL1 may not regulate the movement speed of Schwann cells via the stemness factor Sox2. In addition to cellular movement, our data indicate that similar to cutaneous squamous cell carcinoma cells ( 27 ), cervical cancer cells ( 28 ), and somatic stem cells ( 29 ), EPHB2 enhances the proliferation rate of Schwann cells, supplying new knowledge about the biological activity of EPHB2.…”

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline

“…Such NFRs typically act rapidly to prevent pathway hyperactivation and also are transcriptionally induced by the same upstream signals [ 65 ]. For these experiments, we employed SSC lines derived from adult mice as described previously [ 51 ]. Among the genes tested, Spry4 and Dusp6 were significantly induced by FGF2 stimulation in a cell-autonomous manner (i.e., in the absence of feeder cells; Figure 1A ).…”

“…Lentivirus transduction was performed in feeder-free SSC cultures overnight in growth medium containing polybrene (5 μg/ml; TR-1003-G, Millipore), after which cells were transferred onto fresh feeder cells for expansion. CRISPR/Cas9-mediated targeting of SSCs was performed as described previously [ 51 ]. The following lentiviruses were purchased from Vectorbuilder, Inc. and handled as per manufacturer’s instructions: CRISPR/Cas9 lentivirus targeting mSpry4 (pLV-U6-sgRNA [ mSpry4 1673]-EFS-Cas9-2A-puro and pLV-hCas9:T2A:Neo-sgRNA [ mSpry4 1673]), scramble controls (pLV-hCas9:T2A:Neo-sgRNA [scramble]), mSpry4 cDNA (pLV-Puro-hPGK> mSpry4 [NM_011898.2]), and Stra8-H2B-BFP2 (pLV-Neo-Stra8-hH2BC3 [NM_021062.3] * linker/TagBFP2).…”