Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata)

Abstract

Primordial germ cells (PGCs) are undifferentiated gametes with heterogeneity, an evolutionarily conserved characteristic across various organisms. Although dynamic selection at the level of early germ cell populations is an important biological feature linked to fertility, the heterogeneity of PGCs in avian species has not been characterized. In this study, we sought to evaluate PGC heterogeneity in zebra finch using a single-cell RNA sequencing (scRNA-seq) approach. Using scRNA-seq of embryonic gonadal cells from male and female zebra finches at Hamburger and Hamilton (HH) stage 28, we annotated nine cell types from 20 cell clusters. We found that PGCs previously considered a single population can be separated into three subtypes showing differences in apoptosis, proliferation, and other biological processes. The three PGC subtypes were specifically enriched for genes showing expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Additionally, we discovered a novel biomarker, SMC1B, for gonadal PGCs in zebra finch. The results provide the first evidence of substantial heterogeneity in PGCs previously considered a single population in birds. This discovery expands our understanding of PGCs to avian species, and provides a basis for further research.

Comparative single‐cell transcriptomic analysis reveals differences in signaling pathways in gonadal primordial germ cells between chicken ( Gallus gallus ) and zebra finch ( Taeniopygia guttata )

“…We obtained gene expression signatures for eight types of gonadal cells in both species. Although our research group has previously identified nine types of gonadal cells in zebra finch, 19 some of the clusters assigned to supporting cells are shared with clusters assigned to steroidogenic cells. Steroidogenic lineages may be derived from supporting cells of the chicken embryonic gonad, as evidenced by the detection of cells expressing both supporting and steroidogenic cell markers among chicken gonadal cells 25 .…”

“…We newly generated 10× single‐cell transcriptome data for chicken embryonic gonads for the identification of major cell types. These data were combined with our previously reported zebra finch data 19 to investigate differences in signaling pathways (Figure 1A). After quality control, 12 611 and 10 778 cells were sequenced from pooled male and female gonads ( n = 6 each), respectively (Figure 1B and Figure S1).…”

“…We performed a clustering analysis based on gene expression levels in 22 389 cells, resulting in 19 clusters (referred to as c1–c19) using the UMAP algorithm (Figure S2A,B). After identifying biomarkers that were specifically upregulated in each of the 19 clusters (Figure S2C,D and Table S2), we annotated the clusters according to previously established cell type classification criteria for chicken and zebra finch 19,25 . Based on the expression levels of the somatic cell markers KRT18 , DCN , and DMRT1 and the germ cell marker DAZL (Figure 1C and Figure S3), somatic cells and germ cells could be clearly distinguished.…”

“…In total, 813 950 052 reads were generated for male chickens and 829 528 994 for female chickens. For zebra finch, scRNA‐seq data generated in a previous study were applied, 19 including 720 503 190 reads generated for the male library and 785 997 522 reads generated for the female library.…”

“…After identifying biomarkers that were specifically upregulated in each of the 19 clusters (Figure S2C,D and Table S2), we annotated the clusters according to previously established cell type classification criteria for chicken and zebra finch. 19,25 Based on the expression levels of the somatic cell markers KRT18, DCN, and DMRT1 and the germ cell marker DAZL (Figure 1C and Figure S3), somatic cells and germ cells could be clearly distinguished. In chickens, the germ cell counts were similar between sexes, accounting for 1.87% of all sequenced gonadal cells in males and 2.28% in females.…”