Comparative single cell analysis of transcriptional bursting reveals the role of genome organization on transcript origination.

Spermatogenesis is a key developmental process underlying the origination of newly evolved genes. However, rapid cell type-specific transcriptomic divergence of the germline has posed a significant technical barrier for comparative single-cell RNA-sequencing (scRNA-Seq) studies. By quantifying a surprisingly strong correlation between species- and cell type-specific divergence in three closely related species, we apply a new statistical procedure to identify a core set of 198 genes that are highly predictive of cell type identity while remaining robust to species-specific differences that span over 25-30 million years of evolution. We then utilize cell type classifications based on the 198-gene set to show how transcriptional divergence in cell type increases throughout spermatogenic developmental time. After validating these cross-species cell type classifications using RNA fluorescence in situ hybridization (FISH) and imaging, we then investigate the influence of genome organization on the molecular evolution of spermatogenesis vis-a-vis transcriptional bursting. We first demonstrate how mechanistic control of pre-meiotic transcription is achieved by altering transcriptional burst size while post-meiotic control is exerted via altered bursting frequency. We then report how global differences in autosomal vs. X chromosomal transcription likely arise in a developmental stage preceding full testis organogenesis by showing evolutionarily conserved decreases in X-linked transcription bursting kinetics in all examined somatic and germline cell types. Finally, we provide evidence supporting the cultivator model of gene origination by demonstrating how the appearance of newly evolved testis-specific transcripts potentially provides short-range regulation of neighboring genes' transcriptional bursting properties during key stages of spermatogenesis.