Transcription and imprinting dynamics in developing postnatal male germline stem cells

Abstract

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1(+); high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1(+); moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT(+)). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles.

Keywords: DNA methylation; germline; imprinting; monoallelic; spermatogonia; stem cells.

Figure 1.

Transcriptional changes accompanying SSC development. (A) Graphical summary of the biology of germline stem cell specification, transitions, and data sets generated in this study. (B) Multidimensional scaling (MDS) plot comparing transcriptional profiles of PGCs, undifferentiated SSCs (THY1⁺, high-ID4, OCT4, or VASA), and differentiating SSCs (KIT⁺) from all tested developmental stages. (C) Pairwise RNA sequencing (RNA-seq) correlation matrix plot of all data sets generated. The color intensity and the size of the circle reflect the correlation between the data sets. (D) RNA-seq hierarchical clustering of developing THY1⁺-enriched SSCs, with enriched gene ontology terms at the right. Note that all cell purifications were performed using MACS or FACS. (E,F) Line plots depicting the dynamics of genes involved in germline THY1⁺ SSC maintenance or self-renewal (E) or embryonic stem cell pluripotency (F). The X-axis is the chronological developmental time course, and the Y-axis is log₂ (FPKM [fragments per kilobase per million mapped fragments] + 1). (G) Expression heat map summarizing signaling pathways involved in self-renewal or maintenance. Scale is log₂ FPKM.

Figure 2.

Postnatal SSC subtypes can resemble stem-like or mesenchymal-like states. (A) MDS plot comparing transcriptional profiles of SSC populations at P7. (B–D) Line plots depicting the dynamics of genes involved in SSC maintenance self renewal (B) and epithelial–mesenchymal transition (D). The X-axis is the chronological developmental time course, and the Y-axis is log₂ (FPKM + 1). (C) RNA-seq heat map of all transitioning THY1⁺ SSCs, with enriched GO terms at the right. Note that all cell purifications were performed using either FACS or MACS.

Figure 3.

DNAme and chromatin dynamics in SSC subtypes. (A) K-means clustering (n = 6) of DNAme (mean fraction CG methylation) at TSS regions (±1 kb) of promoters with ≥30% change in methylation. Pairwise comparisons of all germ cell stages (summed) yielded differentially methylated promoters (DMRs; criteria: three or more CpGs, eight or more reads per C, ≥30% change in fraction CG methylation). Enriched GO terms are in the middle column. At the right, HOMER motif analysis reveals distinctive transcription factors for clusters 1–8. P-value < 1/100. Due to low to moderate sequencing coverage, the high-ID4 data set was removed from the differential analysis but is included in subsequent snapshots. (B) DNA hypomethylation of meiotic and spermatogenic genes is completed by P14. Piwil1 (left) and Stra8 (right) genomic snapshots (mouse ESC and E16.5 methylation data were obtained from Stadler et al. 2011; Seisenberger et al. 2012, respectively). (C–F) Genes with known neural monoallelic expression (e.g., Olfr and Pchd) lose methylation during germ cell development and acquire H3K27me3 in round spermatids. Genomic snapshots of Olfr, PchdA, PchdB, and PchdG clusters (adult SSC and round spermatid data are from prior work [Hammoud et al. 2014]).

Figure 4.

THY1⁺-enriched SSCs have improper imprinting (high DNAme) at most paternally expressed imprinted loci. (A) Heat map summarizing the fraction DNAme of the DMR at all known paternal imprinting control regions (ICRs) and paternally expressed imprinted loci. Grey boxes with ND (not determined) within are regions with insufficient sequencing coverage in high-ID4 data sets. (B) Single-cell DNAme validation of 16 loci in P7 THY1⁺-enriched SSCs and in spermatocytes using the Fluidigm Biomark system. Genomic loci analyzed include known methylated (M) and unmethylated (U) control loci, paternally imprinted ICRs (highlighted in dark blue), paternally expressed imprinted loci (highlighted in light blue), and maternally expressed imprinted loci (highlighted in pink). (C) DNAme genomic snapshots of paternally imprinted ICRs (e.g., H19/Igf2 and Dlk1/Gtl2). The dark-blue bar depicts the ICR. (Y-axis) Fraction CG DNAme. (D) DNAme genomic snapshots of paternally expressed imprinted loci (e.g., Xist [left] and Peg10 [right]). (Y-axis) Fraction CG DNAme in ESCs, PGCs, and prepubertal and adult SSCs. The blue bar depicts previously defined imprinted loci.

Figure 5.

Summary schematic depicting changes in DNAme and transcription during germ cell development. (Top panel) Whereas postnatal SSCs with high OCT4/ID4 (epithelial-like SSCs) display normal imprinting patterns, THY1⁺-enriched SSCs (mesenchymal-like SSCs) at P0–P7 display imprinting defects (high DNAme) at paternally expressed imprinted genes and certain monoallelically expressed genes but resolve to normal/expected patterns before puberty. THY1⁺-enriched SSCs transcriptomes enrich for particular GO categories during development, aligned with needed processes and the germ cell–niche codevelopment.