Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes

Abstract

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

Chromosome-specific maturation of the epigenome in the Drosophila male germline

“…We downloaded snRNA-seq data generated by the Fly Cell Atlas project (Li et al, 2022) as a Seurat object linked in supplementary data of (Raz et al, 2023), summarizing gene expression data of single nuclei from dissociated Drosophila adult testes. We used the Seurat function AverageExpression() to get the average expression of all genes in each of 40 UMAP groups which represent distinct cell types of the testis.…”

“…The 18 germline groups and 22 somatic groups assigned in (Raz et al, 2023) We compiled a list of genes with male-germline-enriched expression as follows. We compiled a list of unique protein-coding mRNAs and lncRNA genes from the Drosophila dmel_r6.31 genome assembly (http://ftp.flybase.net/releases/FB2019_06/dmel_r6.31/gtf/), and matched FCA expression data for 40 testis cell types [Raz 2023] to each gene with a lookup table. There were 1,062 genes that are not represented in the expression dataset; these genes are listed with #N/A values for gene expression.…”

“…H3K4me2 signal around gene promoters (-200-+500 bp) with transcripts enriched in specific germline cell types. Transcript expression was derived from FCA snRNA-seq clustering [Raz et al, 2023]. (A) Expression (snRNA-seq) in wildtype testes and H3K4me2 enrichment in bam mutant, in aly mutant, and in wildtype testes in germline stages.…”

“…(B) Selected examples of promoters with germline-enriched expression. Expression z-scores from FCA snRNA-seq across 18 germline clusters (Raz et al 2023) and H3K4me2 enrichment in bam mutant, in aly mutant, and in wildtype testes. (C) Distribution of H3K4me2 around promoters with germline-enriched expression.…”

“…(E) Selected examples of genes with late germline expression and for protamines. Expression zscores from FCA snRNA-seq across 18 germline clusters(Raz et al, 2023) and RNAPIIS2p enrichment in isolated spermatocytes. (F) Enrichment of RNAPIIS2p in isolated spermatocytes and in wing imaginal discs across genes with germline-enriched transcripts.…”