UC Santa Cruz

The cerebral cortex has undergone rapid changes in size and complexity in the primate lineage, yet the molecular processes underlying primate brain development are poorly understood. I have developed a common protocol for generating cortical organoids from human, chimpanzee, orangutan, and rhesus pluripotent stem cells that recapitulates early events in cortical development and enables comparative molecular analysis of this process. Here I focus on long non-coding RNAs (lncRNAs), which as a class have been implicated in gene regulation, differentiation of pluripotent cells into specific tissues, and can play a role in the fine-tuning of developmental processes. Despite their potential importance in driving the development of tissues, studies focusing on lncRNAs have been impeded by the low sequence conservation and extremely tissue- specific expression patterns of functionally relevant lncRNAs. For this reason, we developed a new approach focusing on the sequence, gene structure, and expression conservation of lncRNAs in equivalent tissues among closely related primate species. To use these aspects of conservation in concert, we collected RNA for high throughput total transcriptome sequencing at weekly time points during the differentiation protocol and identified thousands of multi-exonic lncRNAs in each species. Of the 2,975 expressed multi-exonic lncRNAs in human, 2,143 were conserved in gene structure to chimpanzee, 1,731 to orangutan, and 1,290 to rhesus. Among these were 386 human transiently expressed (TrEx) lncRNAs that were primarily induced at one time point during differentiation and off by week 5. This pattern was observed to be well conserved in 60-68% of transcripts among great apes but significantly diminished in rhesus macaque with only 39% of human TrEx lncRNAs with conserved structure retaining a transiently expressed pattern in that species. Many of these transiently expressed transcripts were also associated with specific cell subtypes in single cell RNA-sequencing and 8 were found to influence key transcription factors in correlated gene networks by endogenous locus activation via CRISPRa. Identification of lncRNAs expressed during cortical development and this initial functional analysis is a first step towards mechanistic studies that will evaluate the full extent of their importance during neurogenesis, provide insight to primate-specific and human-specific features of cortical development, and give insight to the role of many disparate genetic lesions that contribute to human neurological diseases. This study provides a framework for identifying new potentially functional transcripts by their expression conservation in closely related species.