Date of Submission
1-2023
Document Type
Thesis
Degree Name
Master of Science in Cellular and Molecular Biology
Department
Biology and Environmental Sciences
Advisor
Carter Takacs, Ph.D.
Committee Member
Anna Kloc, Ph.D.
Committee Member
Ellen J. Hoffman, MD, Ph.D.
Keywords
adnp Gene, Genotyping, Morphological Phenotype, Double Homozygous Mutants
MeSH
Autism Spectrum Disorder, Zebrafish, Genotype
LCSH
Autism spectrum disorders, Zebra danio
Abstract
Autism spectrum disorder is a complex neurodevelopmental disorder characterized by impaired social
skills, restricted/ repetitive behaviors, and developmental delay. The disease has a genetic component,
and mutations in the adnp gene have been found in 0.17% of non-inherited genetic cases of the disorder. The exact molecular mechanism by which adnp is involved in the development of autism is not known. This study aimed to use zebrafish to determine the phenotypic effects of adnp mutations using two adnp paralogs- adnpA and adnpB to understand the underlying mechanism. Novel zygotic adnp double heterozygous mutants (A+/- B+/-) were previously generated using CRISPR-Cas9. These mutants were crossed to create embryos with varying genotypes. These embryos were closely observed, genotyped using a specifically designed genotyping strategy, and used to establish stock lines. It was found that not only do zygotic double homozygous mutants (A-/- B-/-) not exhibit any gross morphological phenotype, but they can also successfully survive to adulthood. They exhibit daytime hypoactivity and heightened startle response to visual stimuli. Maternal-zygotic mutants born to a double homozygous mother had spinal curvature and a novel cardiac edema phenotype, due to a lack of maternal adnp. An attempt was made to optimize CRISPR-Cas13d knockdown for adnpA in wild-type zebrafish to eliminate maternal mRNA expression and evaluate its effects. This attempt yielded inconclusive results and knocking down maternal adnpA and adnpB after redesigning sgRNAs will be the focus of future studies. This study will lead to the establishment of a zebrafish model to study the behavioral and morphological changes associated with adnp mutations in autism spectrum disorder. It also uncovered the important role of maternal adnp in embryonic development and opened up the possibility of investigating if CRISPR-Cas13d knockdown of maternal adnp can recapitulate the observed phenotype.
Recommended Citation
Balaji, Vaishnavi, "Characterizing the Developmental Role of Autism-Risk Gene adnp in a Zebrafish Model for Autism" (2023). Master's Theses. 198.
https://digitalcommons.newhaven.edu/masterstheses/198