Date of Award
August 2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biological Sciences
First Advisor
Jennifer H Gutzman
Committee Members
Claire de la Cova, Heather Owen, Michael Laiosa, Christopher Quinn
Keywords
Development, Disease, Genetics, Kidney, Non-muscle myosin, Zebrafish
Abstract
The MYH genes encode highly conserved actin-based molecular motor proteins, non- muscle myosins (NMIIs), which have essential roles in cell division, cell migration, and cell shape changes. Zebrafish have two myh9 genes (myh9a and myh9b) both of which encode for an NMIIA isoform, along with myh10 and myh14, which encode for the NMIIB and NMIIC isoforms, respectively. To understand the role of the myh genes in development, we obtained a null mutant for the myh9a gene and generated null mutants for the myh9b and myh10 genes using CRISPR/Cas genome editing. The myh14 gene was not examined due to low sequence homology. Through our studies we identified myh9b, not myh9a or myh10, as the critical NMII encoding myh gene required for normal zebrafish development and morphogenesis. Consistent with this finding, myh9a and myh10 homozygous mutants are viable through adulthood and do not develop any obvious phenotypes. However, myh9b homozygous mutants have a partially penetrant lethal phenotype and develop pericardial edema between 48 and 96 hours post fertilization, a phenotype consistent with kidney dysfunction in zebrafish. This study also established genetic interactions between the zebrafish myh9a, myh9b, and myh10 genes. We determined that myh9b is required for the expression of both myh9a and myh10, and myh10 is required for the expression of myh9b. Furthermore, our protein analyses suggest that enhanced NMII protein stability in some mutant backgrounds may play a role in compensation within the mutants. Finally, our examination of double mutants revealed more severe phenotypes develop at earlier timepoints than in our single mutants, suggesting roles for tissue specific genetic redundancy and in some genotypes haploinsufficiency. These zebrafish mutants are the first in vivo models allowing for the study of complete loss of the NMIIA and NMIIB proteins, establishing them as valuable tools for future studies of the NMII encoding myh genes.
Additional studies of the myh9a and myh9b zebrafish mutants explored their feasibility as model systems for MYH9-related disease (MYH9-RD). In humans, there are five clinical disorders resulting from mutations in the MYH9 gene that are classified as MYH9-RD, with many patients developing kidney dysfunction. However, the role of MYH9 in kidney development has not been clearly established. We examined myh9a and myh9b zebrafish mutants to identify the role of NMIIA in the kidney. These studies identified differential requirements for the myh9a and myh9b genes in kidney development, structure, and function. The myh9a and myh9b genes are both required for normal podocyte foot process size. However, only the myh9b gene is required for normal kidney function, slit diaphragm size, and basement membrane thickness within the zebrafish glomerulus. Furthermore, injection of human MYH9 mRNA rescued the zebrafish myh9b mutant phenotype, confirming the conservation of MYH9 between human and zebrafish. These results support the use of myh9b zebrafish mutants as a model to study the development and progression of MYH9-RD, specifically within the kidney.
Recommended Citation
Rolfs, Laura Anne, "THE ROLE OF NON-MUSCLE MYOSINS IN ZEBRAFISH DEVELOPMENT AND MYH9-RELATED DISEASE" (2024). Theses and Dissertations. 3618.
https://dc.uwm.edu/etd/3618