Spon-1 and pxn-2 are essential basement membrane genes crucial to the elongation stage of C. elegans morphogenesis. However, the interactions of these proteins with other components of the basement membrane and the extracellular matrix are not very well known. In attempts to find factors that interact with spon-1 and pxn-2, EMS screens of both mutants were performed. A suppressor of spon-1 mutants, ju1185, was isolated from the spon-1 screen. We mapped this suppressor to chromosome II between -0.46 and +6.80 using two-point mapping with SNPs. Our rescue experiments suggests that this is a missense mutation in srap-1. We found that overexpression of this gene also suppresses spon-1 suggesting that this may be a gain-of-function mutation. In the pxn-2 mutant screen, a previously isolated suppressor ju1123 rescued embryonic and larval lethality, but has reduced locomotion due to defects in the synaptic release of acetylcholine. In initial observations of the mutant and suppressor nerve cords of C. elegans, we observed an abnormal separation of the dorsal nerve cord axon bundle and GABAergic synapses in commissures. With these observations, we hope to find the relationship between the separations of the nerve cord to pxn-2’s role in synaptic vesicle release. These results can potentially reveal additional roles played by these basement membrane genes, as well as discover additional genes essential in morphogenesis, applicable to the study of the epithelia in vertebrates.

The microtubule (MT) arrays in mature polarized cells comprise mostly of non-centrosomal MTs; their regulation is poorly understood. Recently, the MT minus end binding proteins CAMSAPs have been shown to be important regulators of non-centrosomal microtubule organization and dynamics. In this dissertation I describe my work on the C. elegans member of the CAMSAP family, PTRN-1, and its roles in the injury responses of two types of tissues, the neurons and the epidermis. First, I found that PTRN-1 is required for axon regeneration. Though PTRN-1 is largely dispensable for normal neuronal development, its proper regulation of neuronal microtubule dynamics is required for axons to regrow after injury. The second part of my work focuses on PTRN-1’s role in epidermal development and wound response by regulating epidermal microtubule stability. Mutations in the gene death associated protein kinase 1 (DAPK1)/dapk-1 results in both hyperactive innate immune response and enhanced wound closure in the epidermis. A genetic suppressor screen of the dapk-1 mutant identified PTRN-1 as a key player in dapk-1 mediated wound phenotypes. Further work uncovered DAPK-1 as a potential novel regulator of PTRN-1’s function in epidermal MT architecture and dynamics. My thesis work reveals new in vivo roles of PTRN-1 in regulating non-centrosomal MTs for normal tissue damage response.

Proper regulation of microtubule dynamics is critical for multiple events in embryogenesis, as microtubules are key cellular components that contribute to cell division, polarity, and migration. In C. elegans, the precise involvement and regulation of microtubules during embryonic development remains unclear. Our lab previously identified a gain-of-function (gf) mutation in an alpha-tubulin tba-1, which causes embryonic lethality at the restrictive temperature of 25°C. To identify the interactors of TBA-1 during embryo development, an EMS mutagenesis screen was performed and several lines that show suppression of tba-1(gf) embryonic lethality at 25°C were isolated. In this thesis, I analyzed the WGS data for all the obtained tba-1(gf) suppressor lines and focused on mapping the causative mutation for the ju1911 suppressor allele. By generating ju1911 recombinants, the genetic position of ju1911 is narrowed down to between -2.95 and +5.3 on chromosome III, containing function-altering mutations in two genes. I checked the candidate gene mapk-15 by introducing a loss-of-function allele in the tba-1(gf) background and CRISPR-Cas9 mediated gene knockout. I found that mapk-15(III, -1.42) is not responsible for the suppression. Overall, this study shows the disruptive effect of TBA-1(gf) in embryo development and narrows down the genetic locus of one of the tba-1(gf) suppressor alleles.