Poster abstracts
Poster number 52 submitted by Anthony Caputo
Disruption of CLP1 alters neuronal lineage specification and alternative polyadenylation landscapes in human cortical organoids
Anthony Caputo (Case Western Reserve University School of Medicine, Department of Genetics and Genome Sciences), Jingyi Liu (Case Western Reserve University School of Medicine, Department of Genetics and Genome Sciences), Anthony Wynshaw-Boris (Case Western Reserve University School of Medicine, Department of Genetics and Genome Sciences), Ashleigh Schaffer (Case Western Reserve University School of Medicine, Department of Genetics and Genome Sciences and Oregon Health & Science University School of Medicine, Department of Medical and Molecular Genetics )
Abstract:
Homozygous mutations in CLP1, a core constituent of the cleavage and polyadenylation (CPA) complex, cause a severe neurodevelopmental disorder known as pontocerebellar hypoplasia type 10 (PCH10). In cultured neurons, CLP1 knockout causes transcriptome-wide 3’ UTR shortening, also known as alternative polyadenylation (APA). It is well-known that APA can change the regulatory potential of affected transcripts, affecting cellular level processes. However, the extent to which APA can impact cell fate determination in differentiating progenitor cells remains unexplored.
Our hypothesis is that CLP1-dependent APA disrupts lineage specification in the developing human cortex, causing the cortical dysgenesis phenotype observed in PCH10 patients. To test this hypothesis, we depleted CLP1 at key developmental time points during differentiation of H9 human embryonic stem cells (hESCs) into physiologically relevant cortical organoids. We subjected immature and mature organoids to 10X single cell 3’ end RNA sequencing and simultaneously assessed gene expression and APA at cellular resolution. On a molecular level, we found that CLP1 perturbations alter the 3’ UTR landscape of hundreds of genes, many of which are key players in neurodifferentiation. On a cellular level, we found that organoids subjected to early CLP1 perturbations had increased proportions of progenitors and decreased proportions of inhibitory neurons compared to control organoids or those in which CLP1 was depleted at later developmental stages. Taken together, our results indicate that CLP1 disruptions that occur early in development change APA across a plethora of genes important for neurogenesis in the developing cerebral cortex. These changes likely affect the timing, efficiency, or cell fate specification during neuronal lineage commitment, which may explain the altered cell type proportions observed in CLP1 perturbation organoids and the cortical dysgenesis observed in PCH10 patients.
Keywords: Alternative polyadenylation, cortical organoids, neurodevelopment