Developmental Biology

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

Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College; Beijing, 100005, China
Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee; Dundee, DD1 5EH, UK

https://doi.org/10.7554/eLife.85380.1

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Figures and data

Identification of the FOXC2⁺-SSCs in adult mouse and human testis.
(A) Schematic illustration of the single-cell analysis workflow. (B) t-SNE plot and developmental trajectory of all uSPG, colored by cluster. (C) Heatmap of the Top10 DEGs in Cluster1. (D) Immunostaining for ZBTB16 (red), FOXC2 (green), and DAPI (blue) in testicular paraffin sections from wild-type adult C57 mice. Scale bar, 50 μm; C57, C57BL/6J. (E) The proportion of FOXC2⁺ cells in different uSPG subtypes. (F) Immunostainings for MKI67 (red), FOXC2 (green), and DAPI (blue) in adult mice testis and the proportion of MKI67⁺ cells in FOXC2⁺ population (n=10). Scale bar, 50 μm; values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (****p-value < 0.0001). (G) The co-expression proportion between the FOXC2 and differential known SSCs makers (n=10). (H) t-SNE plot of germ cells in adult human testis (GSE112013), colored by germ cell type. Feature plot showing the expression patterns of FOXC2 and MKI67 in human germ cells. (I) The developmental trajectory of the human germ cells, colored by germ cell type, FOXC2 expression cells (red), or MKI67 expression cells (red). (J) Immunostaining for ZBTB16/MKI67 (red), FOXC2 (green), and DAPI (blue) in testicular paraffin sections from adult humans.

Lineage tracing and functional validation of FOXC2⁺-SSCs in Foxc2^CRE/+;R26T/G^f/f mice.
(A) Schematic illustration of the lineage tracing workflow for FOXC2⁺ cells. (B) Immunostainings for DAPI (blue) and FOXC2 (red) at day 3 post TAM induction. Scale bar, 50 μm; d, day. (C) FACS analysis of GFP⁺ populations derived from R26T/G^f/f or Foxc2^CRE/+;R26T/G^f/f mice at day 3 post TAM induction. (D, E) The recipient mice testes (D) and colony numbers (E) 2 months after transplantation (n=10) of the FACS-sorted GFP⁺ cells from the Foxc2^CRE/+;R26T/G^f/f mice 3 days after TAM diet and the MACS-sorted THY1⁺ cells from adult mice. Scale bar, 1 mm; values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (****p-value < 0.0001). (F) Immunostaining for DAPI (blue), ZBTB16/FOXC2 (red), and GFP (green) at week 1 post TAM induction (scale bar, 50 μm). (G) Seminiferous tubules of Foxc2^CRE/+;R26T/G^f/f mice 2, 4, and 6 weeks post TAM induction. Scale bar, 50 μm. (H) Testes (scale bar, 1 mm), seminiferous tubules, and epididymis (scale bar, 50 μm) at month 4, 7, and 12 post TAM induction in Foxc2^CRE/+;R26T/G^f/f mice. (I, J) The GFP⁺ patches (I) and progeny (J) population dynamics (n=10). Values, mean ± s.e.m. (K) Immunostainings for DAPI (blue), EOMES (red), GFRA1 (red), or PAX7 (red) in GFP⁺ population at week 2 post TAM induction. Scale bar, 50 μm.

Specific ablation of FOXC2⁺-SSCs and phenotypic validation in Foxc2^Cre/+;R26^DTA/+ mice.
(A) Schematic illustration of the lineage tracing workflow for FOXC2⁺ cells. (B-D) Phenotypic validation of the R26^DTA/+ and Foxc2^Cre/+;R26^DTA/+ mice (n=5) for testes size (B), testis weight and body weight (C), and HE-staining of the testes (D). Scale bars in (B), 1 mm; in (D), 50 μm; d, day; values were mean ± s.e.m.; p-values were obtained using two-tailed t-tests (ns > 0.05, *p-value < 0.05, **p-value < 0.01). (E) ZBTB16⁺, GFRA1⁺, LIN28A⁺, and FOXC2⁺ SPG populations dynamics. Values, mean ± s.e.m. (n=10); p-values were obtained using one-way ANOVA followed by Tukey test (ns > 0.05, *p-value < 0.05, **p-value < 0.01, ****p-value < 0.0001). (F) Immunostainings for DAPI (blue), SYCP3 (green), and LIN28A (magenta) at day 14 post TAM induction. d, day; scale bar, 50 μm. (G) Quantitative RT-PCR analysis of SPG markers expression in the testes of the R26^DTA/+ and Foxc2^Cre/+;R26^DTA/+ mice (n=3). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (***p-value < 0.001, ****p-value < 0.0001).

FOXC2⁺-SSCs are critical for germline regeneration.
(A) Co-immunostaining of FOXC2 (green) with ZBTB16 (red) in seminiferous tubules of the adult testes at day 20 post busulfan treatment. Scale bar, 50 μm. (B) ZBTB16⁺, GFRA1⁺, and FOXC2⁺ population dynamics after busulfan treatment (20 mg/kg, n=10). (C) Co-immunostaining of FOXC2 (green) with MKI67 (red) in seminiferous tubules of the adult testes at day 20 post busulfan treatment. Scale bar, 50 μm. (D) MKI67⁺FOXC2⁺ proportions in relation to the whole FOXC2⁺ population at different time points after busulfan treatment (n=4). (E) Schematic illustration for lineage tracing of FOXC2⁺ cell after busulfan treatment. (F) Lineage tracing of the GFP⁺ cells at day 20 and month 2 after busulfan treatment (scale bar, 50 μm). (G) The testes (scale bar, 1 mm), seminiferous tubules, and epididymis (scale bar, 50 μm) at month 4, 7, and 12 post TAM induction and busulfan injection. m, month. (H, I) The proportion dynamics of GFP patches (H) and GFP⁺ progenies (I). Values, mean ± s.e.m. (n=10). w, week; m, month.

Spermatogenesis exhaustion in the adult Foxc2^f/-;Ddx4-cre mice.
(A) Construction of the Foxc2^f/-;Ddx4-cre mice. (B) The testes size of the Foxc2^f/-;Ddx4-cre mice. Scale bar, 1 mm; M, month. (C) Body weight and testis weight of the Foxc2^f/-;Ddx4-cre mice at different age (n=5). M, month; values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (ns > 0.05, ****p-value < 0.0001). (D) HE-staining of the testis and epididymis. Scale bar, 50 μm; M, month. (E) Estimation of degenerative tubules and sperm counts in cauda epididymis of the Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice with age (n=5). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (**p-value < 0.01, ****p-value < 0.0001). (F) Immunostainings for DAPI (blue), ZBTB16 (green), FOXC2 (magenta), and DDX4 (white) in the seminiferous tubules of the Foxc2^f/+ and Foxc2^f/-; Ddx4-cre mice. Scale bar, 50 μm. (G) Estimation of ZBTB16⁺ uSPG number in the Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice with age (n=5). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (ns > 0.05, ***p-value < 0.001, ****p-value < 0.0001). (H) Quantitative RT-PCR analysis of the uSPG and germ cell markers expressed in the testis of the Foxc2^f/+ and Foxc2^f/-; Ddx4-cre mice (n=3). M, month; values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (**p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001).

scRNA-seq analysis of THY1⁺ uSPG in Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice.
(A) Schematic illustration of the scRNA-seq workflow. (B) t-SNE plot and developmental trajectory of uSPG from Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice respectively, colored by cluster. (C) Developmental trajectories of uSPG from Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice, colored by sample or derivation. (D) Developmental trajectories of the cells in Cluster1 from Foxc2^f/+ (CON) and Foxc2^f/-;Ddx4-cre (KO) mice, colored by derivation or developmental state. (E) The Cluster1 cells proportion of each state in CON and KO mice. (F) Heatmap showing the DEGs in the Cluster1 cells from the Foxc2^f/-;Ddx4- cre mice compared with the Foxc2^f/+ mice. (G) Top GO terms enrichment by the down-regulated DEGs in KO mice. (H) Gene set enrichment analysis (GSEA) of the Cluster1 cells (Foxc2^f/-;Ddx4- cre v.s. Foxc2^f/+ mice). NOM, nominal; FDR, false discovery rate; ES, enrichment score; NES, normalized enrichment score.

FOXC2 is essential for sustaining the primitive SSCs via regulating cell cycle.
(A) Workflow schematic illustration of the CUT&Tag_FOXC2 analysis on the FACS-sorted FOXC2⁺ cells. (B) Pie chart for CUT&Tag_FOXC2 peaks genome distribution. (C) Profiling of CUT&Tag_FOXC2 peaks in proximity to transcriptional starting site (TSS). The distance to TSS within 1000 was highlighted in the purple box. (D) Top GO terms enrichment by genes annotated by CUT&Tag_FOXC2 peaks. (E) Venn diagram of FOXC2 target genes defined by overlapping the CUT&Tag sequencing and scRNA-seq datasets. (F) GO terms enrichment by the FOXC2 target genes related to cell cycle regulation. (G) Chromatin landscapes of CUT&Tag_FOXC2 peaks of the candidates associated with negative cell cycle regulation. (H) The DNA-binding motif for FOXC2 (predicted with HOMER). (I) The cell cycle-related candidates possessing high binding potential (>0.8, predicted with JASPAR SCAN). (J) CUT&Tag-qPCR validation of the cell cycle arrest regulatory genes. (n=3). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (****p-value < 0.0001). (K) The model for the maintenance of the FOXC2+ SSCs subpopulation in adult testis.

Validation and characterization of the MACS-sorted THY1⁺ uSPG from wild-type adult C57 mice.
(A) Immunostainings of DAPI (blue), THY1 (green), and ZBTB16 (red) in the MACS-sorted THY1⁺ cells (n=5). Scale bar, 50 μm. (B) Quantitative RT-PCR analysis of uSPG and dSPG markers expressed in the MACS-sorted THY1⁺ cells (n=3). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (ns > 0.05, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001). (C) Feature plots showing the expression pattern of classic SPG markers (stemness and differentiation). (D) Heatmap showing the expression pattern of markers for SPG in different clusters. (E) Expression pattern dynamics of the SPG markers with pseudotime progression. (F) Heatmap showing the expression pattern of markers for cell cycle phase in different clusters. (G) The developmental trajectory of the overall SPG, colored by pseudotime.

Expression of top10 DEGs of Cluster1 in Figure 1B and classic SSC and SPG markers in adult human germ cells.
(A) Feature plots and violin plots of the Top10 DEGs of Cluster1. (B) Immunostainings for LIN28A (red), DAPI (blue), and newly-found markers (green) in testicular paraffin sections from adult mice. Scale bar, 50 μm. (C) Feature plots showing the expression pattern of classic SSCs and SPG markers in adult human germ cells.

Construction of the Foxc2^iCreERt2 mice and depletion of uSPG pool in Foxc2^Cre/+;R26^DTA/+ mice 14 days after specific ablation of FOXC2⁺-SSCs.
(A) Construction of the Foxc2^iCreERt2 mice. (B) Immunostainings for DAPI (blue), DDX4 (white), ZBTB16 (green), and FOXC2 (magenta) at day 3 and day 14 post TAM induction in Foxc2C^re/+;R26^DTA/+ mice (scale bar, 50 μm).

Phenotypic validation of the Foxc2^f/-;Ddx4-cre mice.
(A) Immunostainings for DAPI (blue), ZBTB16 (green), LIN28A (magenta), and TRA98 (white) in seminiferous tubules of 1-week-old Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice. Scale bar, 50 μm. (B) Immunostainings for DAPI (blue), LIN28A (green), and Cleaved-CASP3 (red) in seminiferous of the Foxc2^f/+ and Foxc2^f/-; Ddx4-cre mice (2-month-old). M, month; scale bar, 50 μm.

scRNA-seq analysis of THY1⁺ uSPG in adult Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice.
(A) Feature plots of classic SPG markers for uSPG in adult Foxc2^f/+ or Foxc2^f/-;Ddx4-cre mice. (B) Expression dynamics of SPG markers with pseudotime progression for uSPG from Foxc2^f/+ or Foxc2^f/-;Ddx4-cre mice respectively. (C) Expression dynamics of SPG markers with pseudotime progression for overall uSPG from Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice.

Re-cluster and developmental trajectory analysis of cells in Cluster1 derived from adult Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice.
(A) The t-SNE plot of the Cluster1 cells aggregated from the Foxc2^f/+ and Foxc2^f/-;Ddx4-cre mice colored by sample or subcluster. (B) The cell proportion of each sample in each subcluster. (C) Feature plots of SPG markers expression. (D) Developmental trajectory of the aggregated Cluster1 cells colored by subcluster or pseudotime. (E) Expression dynamics of SPG markers with pseudotime progression.

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