Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

Abstract

The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.

Evolutionary Genetic Signatures of Selection on Bone-Related Variation within Human and Chimpanzee Populations

“…Given the absence of COL1A1 protein coding-length polymorphisms in humans—at least unlinked to severe disease—the observation of one at high frequency in chimpanzees could reveal interesting information about the origin of COL1A1 functional variation. Using PCR analyses, we identified the exon 35 variant in two of the six pluripotent stem cell (iPSC) lines derived from western chimpanzees ( Supplementary File S1 ) [ 83 ]. However, our follow-up qPCR analyses of RNA extracted from the two iPSC lines suggest that the exon 35 variant is not incorporated into the COL1A1 RNA transcript, nor does it appear that it affects COL1A1 gene expression levels.…”