UC Davis

Elucidating the genetic modifications that contributed to the evolution of neurodevelopmental features unique to the human lineage has proved challenging. The rise in the availability of higher-quality primate genomes has allowed researchers to perform comparisons to identify genetic variation exclusively found in humans. Such analyses have highlighted different types of genetic variants as potential drivers in species differentiation within primates, where segmental duplications have gained particular attention given the overall increase in duplicated bases observed in primate genomes and the known potential of duplicated genes to gain novel functions. Therefore, recent surveys of human and non-human primate genomes have identified segmental duplications exclusively found in humans that contain genes with protein-coding gene paralogs that are clustered in genomic regions associated with human neurological conditions, making them top candidates for further functional studies. Since there are 30 gene families included in these human-specific segmental duplications, there is a need for higher-throughput and robust methods to functionally screen these genes to highlight candidates for more detailed molecular investigations. To address this need, we propose zebrafish as a model organism with ideal characteristics to screen candidate human-specific duplicated genes potentially impacting neurodevelopment. We first performed a thorough evaluation of current tools for gene editing in zebrafish and define a set of recommendations for general genetic screenings using zebrafish as a model, with a particular focus on the description of cost-efficient methods that provide consistent results across experiments. Then, we optimized a high-throughput phenotyping pipeline that includes rapid morphological and behavioral assessments, which we validated using generated mutants of known genes associated with several neurological conditions. Applying this information, we used zebrafish to functionally test a human-specific duplicated gene with comprehensive studies in mouse (SRGAP2) as a proof-of-concept. Our zebrafish srgap2 knockouts and SRGAP2C-humanized exhibited alterations in the expression of axogenesis-related genes and an imbalance in neuronal populations likely increasing their susceptibility to seizures, all results that are consistent with the findings in mice models. In addition, our data points to a potential novel function never-before-reported for this gene in visual system development. Building off the success of SRGAP2, we expanded our functional screen to an additional nine human-specific duplicated genes using a pipeline that combines CRISPR-based gene editing, temporal expression of human genes, phenotyping platforms, and cellular evaluations at single-cell resolution. This allowed us to highlight candidate genes consistently exhibiting alterations in zebrafish neurodevelopment, warranting follow-up with more detailed molecular functional studies to link these genes to the evolution of human-unique neurological features. Broadly speaking, this dissertation includes a framework to establish zebrafish as an effective model for functional evaluations of genes important in human neurodevelopment using optimized methods.