INVESTIGATION OF RHOTEKIN-2: A CANDIDATE GENE FOR THE MUTANT LOCUS IN ZEBRAFISH BRAIN VENTRICLE MUTANT SCHNITTER

Abstract

Brain ventricles are known to have a number of crucial roles in the brain, yet the molecular mechanisms underlying brain ventricle formation in vertebrates remain poorly understood. Zebrafish have been increasingly utilized as a model organism to study neurodevelopment, and mutants generated in large-scale mutagenesis screens allow for identification of genes and mechanisms involved in ventricle morphogenesis. This study aimed to characterize the zebrafish brain ventricle mutant strain schnitter by investigating a candidate gene for the mutant locus, rhotekin-2. Previous attempts to map the mutation have implicated rtkn2 because it is transcriptionally expressed at stages associated with ventricle formation and may encode for a Rho-GTPase effector protein crucial to several cellular processes. Immunofluorescence staining and RNA in situ hybridizations in wildtype and schnitter embryos at 18-24 hours post-fertilization allowed for visualization and comparison of rtkn2 and actin expression. Microinjections with rtkn2 mRNA and rtkn2 blocking morpholino were also conducted in attempts to rescue schnitter mutants and knock down rtkn2 in wildtype embryos, respectively. Results indicate that rtkn2 is differentially expressed in neuroepithelial cells of wildtype and schnitter zebrafish during ventricle formation, and actin organization is significantly disrupted at the midline in schnitter. However, repeated rescue and knockdown assays proved to be ineffective and inconclusive. In spite of this, RNA-Seq mapping has genetically confirmed a series of potentially adverse mutations in schnitter rtkn2 that continues to lend support to its candidacy. Further study of the genetic and cellular components affected in zebrafish brain ventricle mutants such as schnitter can elucidate mechanisms with crucial clinical applications for brain ventricle morphogenesis in humans and vertebrates.