Adult stem cells provide foundation for the homeostasis, growth, and regeneration of most tissues in mammals. Stem cells rely on extrinsic signals from their specialized niches to balance self-renewal and lineage commitment (Jones and Wagers, 2008). The interactions between stem cells and niches also influence stem cell behaviors in aging and pathological conditions (Lukjanenko et al., 2016; Mesa et al., 2015). Identification and functional characterization of niche components and regulatory mechanisms are essential for elucidating the molecular basis of stem cell fate decisions.Spermatogenesis is a classic stem cell-supported process that requires the activities of spermatogonial stem cells (SSCs). SSCs self-renew to maintain an undifferentiated state and differentiate to produce transient amplifying progenitors that are committed to enter meiosis after several rounds of mitotic divisions (De Rooij, 1988; Oatley and Brinster, 2008). Extrinsic cues from the SSC niche work in concert with intrinsic programs to dictate SSC proliferation, differentiation, and apoptosis (De Rooij, 2017). Sertoli cells serve as the major cellular component of the SSC niche by providing growth factors, extracellular matrix, and structural support (Griswold, 1998; Meng et al., 2000). Sertoli cells also regulate spermatogonial differentiation, meiosis progression, and spermatid development (Griswold, 2018). These specified functions of Sertoli cells are directed by precise gene expression regulation at the transcriptional, post-transcriptional, and translational levels; however, limited knowledge exists about how these regulations are conducted. Elucidating gene regulation programs mediating Sertoli cell-SSC interaction will enhance our understanding of stem cell niche formation and maintenance.
N6-methyladenosine (m6A), one of the most widespread modifications in eukaryotic mRNA, has been linked to mRNA stability, structure, splicing, processing, and translational efficiency (TE) (Gilbert et al., 2016; Zhao et al., 2017). The m6A modification is catalyzed by a multiprotein methyltransferase complex composed of methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor 1-associated protein (WTAP) (Liu et al., 2014). METTL3 functions as the major catalytic subunit, while METTL14 forms a stable heterodimer with METTL3 to enhance methyltransferase activity (Wang et al., 2016). WTAP recruits METTL3 and METTL14 to mRNA targets (Ping et al., 2014). Findings from knockout mouse models uncovered pivotal roles of METTL3- and METTL14-mediated RNA methylation in embryogenesis (Horiuchi et al., 2006), neurogenesis (Wang et al., 2018), and adipogenesis (Kobayashi et al., 2018). In stem cell-dependent tissues, m6A modification has critical functions in controlling lineage specification and fate choice of stem and progenitor cells. METTL3-deficient hematopoietic stem cells fail to differentiate properly (Cheng et al., 2019) and Mettl14 deletion in neural progenitors causes defects in cell cycle progression (Yoon et al., 2017). Moreover, conditional deletion of Mettl3 or Mettl14 in germ cells using Ddx4-Cre leads to SSC depletion and male sterility (Lin et al., 2017). Despite these key findings, the roles of m6A modification in Sertoli cells are unknown and the functions of WTAP in establishing and maintaining the SSC niche remain to be determined.
Here, using spermatogenesis as a model system, we investigated features and functions of m6A modification in controlling the establishment and maintenance of stem cell niche. We illustrated the m6A methylome and examined the function of WTAP-dependent m6A in Sertoli cells. We showed that WTAP is essential for SSC maintenance and spermatogonial differentiation. Mechanistically, WTAP-mediated m6A modification controlled transcription and translation of a list of genes in Sertoli cells to sustain SSC niche and govern normal spermatogenesis.
We firstly examined the expression of m6A methyltransferases WTAP, METTL3, and METTL14 in mouse testis. Immunofluorescent staining revealed that METTL3 or METTL14 was co-localized with WTAP in germ cells and Sertoli cells within seminiferous tubules (Figure S1A). Interestingly, we noticed that immunoreactive signal for WTAP appeared to be strong in Sertoli cells. Co-staining of WTAP, METTL3, or METTL14 with Sertoli cell markers showed that all these proteins were expressed in Sertoli cells and WTAP indeed was strongly enriched in Sertoli cells (Figure 1A). We then examined relative abundances of Wtap, Mettl3, and Mettl14 in fluorescence-activated cell sorting isolated Sertoli cells from Sox9Gfp transgenic mice. Wtap and Mettl3 transcript concentrations were increased by 5.23-fold and 3.47-fold in SOX9-GFP+ cells compared with those in Sertoli cell-depleted testicular cells (Figure S1B). Together, these data indicated that all three methyltransferases co-existed and likely catalyzed m6A modification in Sertoli cells.
This result was published in Stem Cell Reports with the title of "WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche".
The link below will guide you to the reading:
https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(20)30376-3