FOXC2 marks and maintains the primitive spermatogonial stem 1 cells subpopulation in the adult testis 2

W.S., Z.W. and C.J


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Through spermatogenesis, spermatozoa are generated from spermatogenic cells that are 49 originated from spermatogonial stem cells (SSCs). It is critical for this process to be continuous and 50 successful that SSCs are maintained in a homeostatic balance between self-renewal and 51 differentiation (1). The SSCs, as the least differentiated spermatogonia, belong to a subgroup of 52 undifferentiated spermatogonia (uSPG) that are morphologically categorized into three subtypes, 53 i.e., Asingle (As), Apaired (Apr), and Aaligned (Aal) cells (2). So far, three models have been proposed for 54 the mechanism underlying SSCs' self-renew based on the dynamic transitions among subgroups.

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In the 'As model', As spermatogonia serve as SSCs that are capable of both self-renew and further 56 transformation into Apr and Aal that eventually give rise to spermatogonia (3, 4). Later, based on the 57 discovery of Ngn3 and Gfrα1 as SSCs markers, the 'fragmentation model' suggests all three 58 subgroups with stem cell potential and the SSCs renewal is achieved through the fragmentation of 59 pairs and chains (5). Further work on SSCs markers such as ID4 and PAX7 inspired the 60 'hierarchical As model', in which only specific As spermatogonia possess the potential for long-term 61 self-renewal whereas the majority are restricted in their capacity (6, 7). Though their standing points 62 of view differ, each model seems well supported by the respective collection of evidence, which to 63 some extent reflects the nature of heterogeneity and dynamics among SSCs subpopulations.

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In recent years, great insights into SSCs behaviors and regulations have been provided by a

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We next analyzed the expression of FOXC2 in adult human testis using the published scRNA-120 seq dataset (17) (GSE112013). As expected, FOXC2 was also specifically expressed in the human 121 SSCs, most of which were MKI67 - (Fig. 1H, Fig.S2C). Pseudotime analysis showed that the 122 FOXC2 + cells located at the start of the developmental trajectory with a proportion of about 90% 123 that were MKI67 - (Fig. 1I). Immunofluorescence staining confirmed that FOXC2 + cells were a subset 124 of ZBTB16 + spermatogonia in adult human testis, and most of them were MKI67 - (Fig. 1J), possibly 125 representing the Adark SSCs also known as the reserve stem cells or 'true SSC' in human testis(29-126 33). These results suggested that FOXC2 was similarly expressed in the SSCs of adult human and 127 mouse testis and may possess a conserved function.

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Specific ablation of the FOXC2 + -SSC results in depletion of the uSPG pool. We then prepared     predominantly GFP + (Fig. 4F). Over 68.5% of the total length of the seminiferous tubules were GFP + 185 at m2, and this proportion rose to 95.43%, 98.41%, and 99.27% at m4, m7, and m12 respectively 186 ( Fig. 4G, H), which was comparable to the proportion by tamoxifen induction alone (Fig. 2I). From      Table S3) (41-50). More specifically, significant 241 peaks enrichment at the promoter region were observed for these candidate genes (Fig. 7G).

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Meanwhile, as predicted using the JASPAR Scan function (binding potential >0.8), there showed 243 strong binding potential of FOXC2 towards these candidate genes (Fig. 7I) via the binding motif of 244 FOXC2 (Fig. 7H), which was further confirmed by the results from the CUT&Tag qPCR (Fig. 7J).

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Overall results implied that FOXC2 may function as a gatekeeper that ensures the quiescent state 246 of the primitive SSCs by impeding cell cycle progression.

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In this work, a comprehensive analysis of uSPG populations with scRNA-seq and the following

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All mice were randomly assigned to experiments and no statistical methods were used to

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Single-cell RNA-seq data processing 686 Raw sequencing reads were processed using the Cell Ranger v.3.0.1 pipeline of the 10x Genomics 687 platform. In brief, reads from each sample were demultiplexed and aligned to the mouse mm10 688 genome, and UMI counts were quantified for each gene per cell to generate a gene-barcode matrix.

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Default parameters were used. The UMI counts were analyzed using the Seurat R Package (58)

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(v.3.0.1) following the Seurat pipeline. Cells with more than 200 detected genes or less than 10% 691 mitochondria reads were retained. Genes not detected in at least 10 cells were removed from 692 subsequent analysis. The resulting matrix was normalized, and the most variable genes were found 693 using Seurat's default settings, then the matrix was scaled with regression against the mitochondria