Functional Analysis Of The Zebrafish Orthologs Of Host Cell Factor 1 Gene

Host Cell Factor C1 (HCFC1) is a transcriptional co-activator factor that regulates the expression of over 5000 different downstream target genes in human cells. Missense mutations in the HCFC1 gene cause methylmalonic acidemia homocysteinemia cblX type (cblX). cblX, a multiple congenital anomaly syndrome (MCA) characterized by abnormal brain development, craniofacial abnormalities, metabolic deficits, and intractable epilepsy. Published studies suggest that HCFC1 regulates neural precursor proliferation, number, and differentiation. However, these results were derived from studies that were performed with nonsense alleles or transient knockdown approaches. Although these results demonstrate the importance of HCFC1 in brain development, they do not replicate the same type of missense mutation present in cblX patients. Therefore, our laboratory created a missense homozygous viable hcfc1aco64/co64 (co64) germline mutation in developing zebrafish. The mutation is in the kelch protein interaction domain, which is the region mutated in cblX patients. The co64 allele carries a two amino acid missense substitution created by CRISPR/Cas9 technology. In my research, we characterized the cellular, behavioral, and molecular phenotypes in the co64 allele. We observed hypolocomotion and abnormal brain development characterized by increased neural precursor cells and reduced radial glial cells. To continue investigating the possible mechanisms associated with cblX and intellectual disability, we used CRISPR/Cas9 to induce mutations in the zebrafish hcfc1b gene. Zebrafish have two HCFC1 orthologs and previous studies have been limited to the hcfc1a paralog. Since HCFC1 is a multidomain protein we targeted multiple domains of the protein. We successfully identified 2 novel germline alleles in the hcfc1b gene and performed basic characterization of the expression of molecular markers associated with brain development. Collectively, our analysis provided strong evidence for a function for HCFC1 in brain development and our novel model alleles will help to fully characterize HCFC1 gene function. Keywords: zebrafish, multiple congenital anomalies, cblX, neural precursor cells, HCFC1, intellectual disability