For human autosomal recessive diseases in which the responsible gene is known, we are using C. elegans to study the function of that gene and to genetically identify other factors that act in the same pathway. There are a number of criteria that must be met in order for this strategy to work. First, there must be a convincing and clear C. elegans ortholog. Second, there would have to be a mutation or deletion in this gene that already exists. Towards this end, we are using CRISPR technology to generate mutant alleles analogous to those found in human diseases. Third, there would have to be a scorable phenotype. The more penetrant the phenotype, the better. If these criteria are met, genetic suppressor and enhancer screens could be performed to identify interacting factors that function with any given gene and the biological process in which it functions. In the past year, we have identified a number of C. elegans orthologs of human disease-causing genes. We have determined that many of these candidates satisfy all of the above criteria- there are mutations in these genes and they reveal very penetrant and scorable phenotypes. We are currently focusing on two type IV collagen genes, emb-9 and let-2, whose mutant phenotypes were previously characterized by Dr. James Kramer at Northwestern University. There are temperature-sensitive missense alleles of these genes, making them ideal for genetic suppression screens. Mutations in the human type IV collagens cause a number of diseases. We are also suppressing a ubiquitin activating enzyme, uba-1, which was originally identified and characterized by Dr. Harold Smith (Kulkarni and Smith, 2008), currently here in NIDDK. This allele is also temperature-sensitive. Mutations in the human UBA1 gene cause a specific form of Spinal muscular atrophy. In addition to these genes, we have begun to characterize the germline defects of a deletion mutation in the lpd-8 ortholog of C. elegans; this is a gene that, when mutated, causes Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1). Using the CRISPR/Cas9 genome-editing technology, we have to introduced the orthologous mutation into C. elegans to mimic the mutations found in humans. One of these alleles, so far, causes a very penetrant sterile phenotype. For each of these genes, mutations in them appear to reveal recessive, highly penetrant phenotypes. We are carrying out straightforward genetic suppression screens with these mutants. The phenotypes of the above genes range from embryonic and larval lethality to slow development and sterility. The uba-1 mutant has a number of distinct phenotypes and thus we will need to score our suppressors for their ability to suppress each of these phenotypes. So far, we have only isolated alleles that suppress the larval lethality of uba-1, but not the sperm defects. We are currently testing candidates for suppressors of emb-9 and uba-1 for which the candidates were molecularly identified with whole genome sequencing. Two suppressors have been confirmed with RNAi and we will continue to confirm these using targeted genome editing. We will also characterize the phenotypes of these suppressor mutations in otherwise wild-type backgrounds to see if they perturb development. In the future, we hope to test drugs that mimic the effects of suppressor mutations to determine whether they might be worthy new therapies of diseases in which recessive mutations are known to be responsible for suppression. In the absence of effective gene therapy or stem cell therapy, drugs that target specific proteins may be beneficial to patients with such diseases. Most recently, we have initiated a project to model human craniofacial syndromes in C. elegans. We were approached by colleagues to determine whether mutations in the sole C. elegans orthologs of the Twist basic helix-loop-helix (bHLH) transcription factor results in distinct phenotypes in C. elegans. There are two Twist genes in humans, Twist1 and Twist2. Twist mutations have already been implicated in other craniofacial disorders such as Saethre-Chotzen Syndrome. Interesting, our clinical colleagues have recently shown that mutations in a conserved glutamic acid residue in the conserved basic domain of Twist1 and Twist2 are implicated in three other distinct craniofacial syndromes. In each case, this conserved glutamic acid is altered to one of five other amino acid residues. Using CRISPR/Cas9 genome-editing technology, we have made the orthologous changes in this conserved glutamic acid in the hlh-8 gene, the sole ortholog of the Twist genes in humans. We were able to screen for our mutations by PCR, restriction digests, and sequencing and were able to generate all of the desired mutations. Each of our mutations resulted in a very visible phenotype; homozygous animals were egg-laying defective (Egl). Interestingly, only some of the mutations resulted in a constipated (Con) phenotype and displayed a very deformed tail. We have characterized these strains quantitatively to determine how penetrant each of these phenotypes are. We have also crossed these strains with GFP reporter strains to look at the expression of known HLH-8 targets. We have also examined the expression of an hlh-8::gfp reporter to learn more about the M lineage in the developing larvae. The M lineage is responsible for the generation of the muscles that make up the egg-laying and defecation systems in C. elegans. Using this marker, we can determine when the M descendants divide and migrate. This marker also allows us to assay the generation of the sex muscles (SMs). With all these markers, we are able to characterize the mutant phenotypes of these hlh-8 alleles at the cellular level and better group our alleles into distinct phenotypic classes. Interestingly, these mutants also display diverse male tail phenotypes, further allowing us to put these mutants into distinct classes. We plan to test the weakest alleles in genetic suppressor screens to determine whether we can isolate suppressor mutants that reverse the mutant phenotypes and restore them to a more wild-type-like condition.
