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

Reviewer #1 (Public Review):

The expression and localization of Foxc2 strongly suggest that its role is mainly confined to As undifferentiated spermatogonia (uSPGs). Lineage tracing demonstrated that all germ cells were derived from the FOXC2+ uSPGs. Specific ablation of the FOXC2+ uSPGs led to the depletion of all uSPG populations. Full spermatogenesis can be achieved through the transplantation of Foxc2+ uSPGs. Male germ cell-specific ablation of Foxc2 caused Sertoli-only testes in mice. CUT&Tag sequencing revealed that FOXC2 regulates the factors that inhibit the mitotic cell cycle, consistent with its potential role in maintaining a quiescent state in As spermatogonia. These data made the authors conclude that the FOXC2+ uSPG may be the true SSCs, essential for maintaining spermatogenesis. The conclusion is largely supported by the data presented, but two concerns should be addressed: 1) terminology used is confusing: primitive SSCs, primitive uSPGs, transit amplifying SSCs... 2) the GFP+ cells used for germ cell transplantation should be better controlled using THY1+ cells.

Reviewer #2 (Public Review):

The authors found FOXC2 is mainly expressed in As of mouse undifferentiated spermatogonia (uSPG). About 60% of As uSPG were FOXC2+ MKI67-, indicating that FOXC2 uSPG were quiescent. Similar spermatogonia (ZBTB16+ FOXC2+ MKI67-) were also found in human testis.

The lineage tracing experiment using Foxc2CRE/+;R26T/Gf/f mice demonstrated that all germ cells were derived from the FOXC2+ uSPG. Furthermore, specific ablation of the FOXC2+ uSPGs using Foxc2Cre/+;R26DTA/+ mice resulted in the depletion of all uSPG population. In the regenerative condition created by busulfan injection, all FOXC2+ uSPG survived and began to proliferate at around 30 days after busulfan injection. The survived FOXC2+ uSPGs generated all germ cells eventually. To examine the role of FOXC2 in the adult testis, spermatogenesis of Foxc2f/-;Ddx4-cre mice was analyzed. From a 2-month-old, the degenerative seminiferous tubules were increased and became Sertoli cell-only seminiferous tubules, indicating FOXC2 is required to maintain normal spermatogenesis in adult testes. To get insight into the role of FOXC2 in the uSPG, CUT&Tag sequencing was performed in sorted FOXC2+ uSPG from Foxc2CRE/+;R26T/Gf/f mice 3 days after TAM diet feeding. The results showed some unique biological processes, including negative regulation of the mitotic cell cycle, were enriched, suggesting the FOXC2 maintains a quiescent state in spermatogonia.

Lineage tracing experiments using transgenic mice of the TAM-inducing system was well-designed and demonstrated interesting results. Based on all data presented, the authors concluded that the FOXC2+ uSPG are primitive SSCs, an indispensable subpopulation to maintain adult spermatogenesis.

The conclusion of the mouse study is mostly supported by the data presented, but to accept some of the authors' claims needs additional information and explanation. Several terminologies define cell populations used in the paper may mislead readers.

1. "primitive spermatogonial stem cell (SSC)" is confusing. SSCs are considered the most immature subpopulation of uSPG. Thus, primitive uSPGs are likely SSCs. The naming, primitive SSCs, and transit-amplifying SSCs (Fig. 7K) are weird. In general, the transit-amplifying cell is progenitor, not stem cell. In human and even mouse, there are several models for the classification of uSPG and SSCs, such as reserved stem cells and active stem cells. The area is highly controversial. The authors' definition of stem cells and progenitor cells should be clarified rigorously and should compare to existing models.

2. scRNA seq data analysis and an image of FOXC2+ ZBTB16+ MKI67- cells by fluorescent immunohistochemistry are not sufficient to conclude that they are human primitive SSCs as described in the Abstract. The identity of human SSCs is controversial. Although Adark spermatogonia are a candidate population of human SSCs, the molecular profile of the Adark spermatogonia seems to be heterogeneous. None of the molecular profiles was defined by a specific cell cycle phase. Thus, more rigorous analysis is required to demonstrate the identity of FOXC2+ ZBTB16+ MKI67- cells and Adark spermatogonia.

3. FACS-sorted GFP+ cells and MACS-THY1 cells were used for functional transplantation assay to evaluate SSC activity. In general, the purity of MACS is significantly lower than that of FACS. Therefore, FACS-sorted THY1 cells must be used for the comparative analysis. As uSPGs in adult testes express THY1, the percentage of GFP+ cells in THY1+ cells determined by flow cytometry is important information to support the transplantation data.

4. The lineage tracing experiments of FOXC2+-SSCs in Foxc2CRE/+;R26T/Gf/f showed ~95% of spermatogenic cells and 100% progeny were derived from the FOXC2+ (GFP+) spermatogonia (Fig. 2I, J) at month 4 post-TAM induction, although FOXC2+ uSPG were quiescent and a very small subpopulation (~ 60% of As, ~0.03% in all cells). This means that 40% of As spermatogonia and most of Apr/Aal spermatogonia, which were FOXC2 negative, did not contribute to spermatogenesis at all eventually. This is a striking result. There is a possibility that FOXC2CRE expresses more widely in the uSPG population although immunohistochemistry could not detect them.

5. The CUT&Tag_FOXC2 analysis on the FACS-sorted FOXC2+ showed functional enrichment in biological processes such as DNA repair and mitotic cell cycle regulation (Fig.7D). The cells sorted were induced Cre recombinase expression by TAM diet and cut the tdTomato cassette out. DNA repair process and negative regulation of the mitotic cell cycle could be induced by the Cre/lox recombination process. The cells analyzed were not FOXC2+ uSPG in a normal physiological state.

6. Wei et al (Stem Cells Dev 27, 624-636) have published that FOXC2 is expressed predominately in As and Apr spermatogonia and requires self-renewal of mouse SSCs; however, the authors did not mention this study in Introduction, but referred shortly this at the end of Discussion. Their finding should be referred to and evaluated in advance in the Introduction.

Reviewer #3 (Public Review):

By popular single-cell RNA-seq, the authors identified FOXC2 as an undifferentiated spermatogonia-specific expressed gene. The FOXC2+-SSCs can sufficiently initiate and sustain spermatogenesis, the ablation of this subgroup results in the depletion of the uSPG pool. The authors provide further evidence to show that this gene is essential for SSCs maintenance by negatively regulating the cell cycle in adult mice, thus well-established FOXC2 as a key regulator of SSCs quiescent state.

The experiments are well-designed and conducted, the overall conclusions are convincing. This work will be of interest to stem cell and reproductive biologists.