Larkin, Kerry et al. (2021) International Worm Meeting "Modeling Timothy Syndrome in Caenorhabditis elegans"

Larkin, Kerry, Golden , Andy, Smith, Harold, Bauer, Rosemary

[International Worm Meeting, 2021]

Timothy Syndrome (TS) is a rare genetic disease arising from mutations the gene CACNA1C, which codes for a subunit in calcium channel Cav1.2 expressed throughout the body. Because of the gene's wide-ranging expression, TS patients have a variety of symptoms in different organ systems including arrhythmias, neurodevelopmental delays, hypoglycemia, and syndactyly. Like many rare genetic diseases, treatment options for patients are limited and there is no cure. It is not clear how the mechanism of the disease functions to cause a complex array of symptoms in the many organ systems. The nematode Caenorhabditis elegans provides an excellent model system in which to study the genetic underpinnings of TS further as it contains an orthologous gene to CACNA1C: egl-19. We have used the CRISPR/Cas-9 system to create missense alleles in egl-19 corresponding to TS patient CACNA1C alleles. While CRISPR is a modern gene editing technique with few off-target editing effects, a previous egl-19 missense allele with mutation G365R was historically generated by random mutagenesis with ethyl methanesulfonate (EMS). We observed significant differences between strains with the same missense mutation created by CRISPR versus EMS, with the EMS strain demonstrating far milder phenotypes. Our CRISPR generated G365R allele is embryonic lethal, while the EMS-derived strain is viable. Through further analysis, we hypothesized this EMS strain also contains a suppressor mutation that is repressing the phenotype of the egl-19 mutation. Here, we use a molecular mapping strategy and whole genome sequencing techniques to find both the location and identity of this unknown suppressor. We then aim to characterize this gene and uncover how it functions as a regulator of the orthologous TS phenotype. This regulator could provide insight into new molecular targets for TS therapeutics to better treat the syndrome at the cellular level.

Mondoux, Michelle et al. (2021) International Worm Meeting "A High-Glucose Diet Reduces Male Fertility and Sperm Quality in C. elegans"

Larkin, Kerry, Mondoux, Michelle, Garcia-Chope, Jose, Mastroianni, Michael, Davis, Caroline, Erichsen, Jason, Liggett, Marjorie

[International Worm Meeting, 2021]

Infertility affects ~15% of Americans of reproductive age. High-sugar diets, obesity, and type 2 diabetes have all been associated with infertility, and have been correlated with decreases in sperm and oocyte quality and viability. Despite the burden of infertility and the prevalence of high-sugar diets, the cellular and molecular mechanisms that link diet to fertility are unknown. As in humans, a high-glucose diet leads to reduced fertility in C. elegans hermaphrodites. We found that high-glucose diet also reduces male fertility in a dose-dependent manner. Concentrations of glucose that have no effect on hermaphrodite self-fertility disrupted mated fertility, which allows us to separate the effects of glucose on males from the effects on hermaphrodites or fog-2 females. We tested several aspects of male fertilization success and find multiple defects on a high-glucose diet. First, a high-glucose diet reduces male sperm competitiveness. On a control diet, male sperm is used almost exclusively when males are mated to hermaphrodites. However, on a high-glucose diet, we find that although male sperm are still used preferentially, there was a dose-dependent reduction in the percentage of offspring dervied from male sperm, suggesting a reduction in sperm quality. We then measured sperm size, which is known to correlate with sperm competitiveness, and found that male sperm size is significantly decreased on a high-glucose diet. We also find a decrease in male spermatid production on a high-glucose diet. However, a high-glucose diet had no effect on spermatid transfer during mating or in mating behavior. We are currently testing other known indicators of sperm quality to determine whether glucose affects other facets of male fertility, and whether the fertility defects we observe are separabale phenotypes. Understanding how a high-glucose diet affects male gametes contributes to our understanding of how diet affects fertility in C. elegans and can provide insight into the range of cell biological responses to excess glucose.

Sawa H et al. (1995) International C. elegans Meeting "lin-17 ENCODES A PROTEIN SIMILAR TO THE PRODUCT OF THE Drosophila TISSUE-POLARITY GENE Frizzled"

Horvitz HR, Sawa H

[International C. elegans Meeting, 1995]

Asymmetric cell divisions are involved in many aspects of development. lin-17 mutations disrupt the asymmetry of a number of cell divisions in C. elegans, suggesting that lin-17 is required to establish these asymmetries. We genetically localized lin-17 0.2 map units right of the RFLP nP58, which was identified by Leslie Lobel. Using cosmids and phage clones in this region, we searched for RFLPs in lin-17 mutant strains. A phage clone isolated by Kerry Kornfeld can detect RFLPs in rh41 and sy277. We identified cDNAs that detect both RFLPs. Because genomic clones containing the whole cDNA sequences are not available, we ligated a fragment of the genomic phage clone to one of these cDNAs and to the 3' region from the unc-54 gene. This construct rescued the Lin-17 phenotype. We determined the sequence of this cDNA. The predicted product of a 1.7 kb ORF is 28% identical to the product of the tissue-polarity gene frizzled of Drosophila. As in Lin-17, the polarities of a number of cells are disrupted in frizzled animals, resulting in the disordered orientation of hair, bristles, ommatidia and tarsi. Because lin-17 and frizzled encode proteins each with seven putative transmembrane domains, lin-17 is likely to be involved in cell-cell interactions required for the asymmetry of certain cell divisions. The similarity of lin-17 to frizzled raises the possibility that other genes related to tissue-polarity genes are involved in asymmetric cell divisions. The genome sequencing project identified an another frizzled-like cDNA that is different from lin-17 cDNAs. Also, a cDNA related to the Drosophila tissue-polarity dishevelled gene has been identified by the cDNA project led by Yuji Kohara. We are currently analyzing these cDNAs.

Peter Swoboda et al. (2001) International C. elegans Meeting "The regulation of sensory cilia specific genes in C. elegans"

Peter Swoboda, James H Thomas

[International C. elegans Meeting, 2001]

Cilia are an important cellular differentiation of sensory neurons for receiving information from the environment. 60 of the 302 C. elegans neurons have ciliated endings, accounting for the bulk of informational input into the worm. Mutations in a large number of genes have been identified that affect the structure and function of these sensory cilia. One of these genes, daf-19 , encodes an RFX-type transcription factor. By acting through its target site in promoter regions, the x-box, DAF-19 appears to be the main regulator of a number of effector genes involved in sensory cilium formation. These effector genes, including che-2, osm-1, osm-5, osm-6 and daf-19 itself, are expressed in essentially all ciliated sensory neurons and, when mutated, cause morphological defects in sensory cilia. DAF-19 is also involved in the regulation of x-box containing genes that are expressed only in subsets of ciliated sensory neurons (e.g. odr-4 ). odr-4 is implicated in localizing odorant receptors to sensory cilia. Our results strongly suggest that DAF-19 regulates the structural and functional differentiation of sensory cilia by activating the transcription of a battery of genes whose products form the cilium and endow this structure with sensory functions. We initiated the identification of many more ciliary components by analyzing genes that have an appropriately spaced x-box promoter element, which we call xbx genes. In repeated searches through the C. elegans genome sequence (with Kerry Bubb, Genetics, UW), using an algorithm designed to find promoter elements, we uncovered hundreds of candidate xbx genes, several of which had C. briggsae and Drosophila homologues that also had an appropriately positioned x-box promoter element. Initial expression analyses of a subgroup of these xbx genes showed that some of them were specifically expressed in ciliated sensory neurons in a daf-19 dependent manner. Presently we are attempting to generate cilium specific phenotypes for some of the xbx genes using gene specific RNA interference techniques. To further characterize the set of genes that comprise the cilium formation module we are searching through promoter sequences of selected subsets of cilium specific and xbx genes for other conserved promoter elements (with Martin Tompa, Computer Sciences and Engineering, UW). In combination with previous data about sensory cilia, these approaches have the potential to reveal all the genes required for sensory cilium structure and function.