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Accumulating evidence demonstrates that mutations in ALDH1A3 (the aldehyde dehydrogenase 1 family, member A3) are associated with developmental defects. The ALDH1A3 enzyme catalyzes retinoic acid biosynthesis and is essential to patterning and neuronal differentiation in the development of embryonic nervous system. Several missense mutations in ALDH1A3 have been identified in family studies of autosomal recessive microphthalmia, autism spectrum disorder, and other neurological disorders. However, there has been no evidence from animal models that verify the functional consequence of missense mutations in ALDH1A3. Here, we introduced the equivalent of the ALDH1A3 C174Y variant into the Caenorhabditis elegans ortholog, alh-1, at the corresponding locus. Mutant animals with this missense mutation exhibited decreased fecundity by 50% compared to wild-type animals, indicating disrupted protein function. To our knowledge, this is the first ALDH1A3 C174Y missense model, which might be used to elucidate the effects of ALDH1A3 C174Y missense mutation in the retinoic acid signaling pathway during development.

The relatively simple nervous system and facile genetics of Caenorhabditis elegans makes it a potential model organism to study the genetic basis of complex neural diseases, such as autism spectrum disorder (ASD). Our previously published study identified conserved human ASD-missense variants in C. elegans orthologs that have a role on morphology, locomotion or fecundity, suggesting C. elegans as an efficient phenotypic model to study conserved ASD variants (Wong et al., 2019). One of the ASD-associated genes screened in this study is daf-18, an ortholog of PTEN. DAF-18/PTEN is a lipid phosphatase protein that dephosphorylates PIP3, a critical lipid second messenger that mediates downstream kinase cascade signaling of the insulin pathway, that in turn regulates expression of genes involved in C. elegans lifespan and dauer formation (Murphy and Hu, 2013). It is reported that the daf-18(e1375) strain, which has a 30–base pairs insertion in the fourth exon, has a dauer defective phenotype (Ogg and Ruvkun, 1998); however, the effect of daf-18/PTEN single amino acid substitution in dauer formation has not been evaluated before. Here, we investigate the dauer entry abilities of previously published ASD-associated daf-18/PTEN missense variants (Wong et al., 2019). We performed a dauer-entry assay using crude pheromone as a proxy for high population density and heat-killed bacteria as a limited food source. We first determined the crude pheromone concentration required to induce 50% of wild type (N2) strain to enter dauer stage (EC50). Using the calculated EC50value we evaluated each mutant strain in two independent experiments (Table 1). To control day-to-day variations caused by environmental conditions, N2 controls were used in every trail. Additionally, we used the previously reported daf-18 dauer-defective strain (e1375) as a control (Ogg and Ruvkun, 1998). To analyze the obtained data, we performed one-way ANOVA followed by multiple comparisons test (Dunnett test) (Figure 1A). Table 1 Evaluated ASD-associated daf-18/PTEN mutant strains. Strain name PS7439 PS7432 PS7436 PS7430 PS7434 C. elegans allele name daf-18(sy879) daf-18(sy887) daf-18(sy881) daf-18(sy885) daf-18(sy882) C. elegans protein change D66E L115V H138R H168Q T176I Human protein change D22E L70V H93R H123Q T131I As expected, we observed statistically significant difference between our wild type and dauer defective control (e1375). Our results show that H138R (PS7436) and T176I (PS7434) daf-18 mutant strains have a significant difference from the wild type control, and a non-significant difference from the dauer defective control, suggesting these missense alleles cause dauer defective phenotypes to the same level of the e1375 strain. In contrast, we found the D66E (PS7439), L115V (PS7432) and H168Q (PS7430) mutant strains are significantly different from the dauer defective control and not significantly different from the wild type control, suggesting these three missense alleles do not cause the same level of dauer defective phenotype as the defective control does. Altogether, our current data show that H138R and T176I are defective in dauer entry, while D66E, L115V and H168Q daf-18 mutant strains are not (Figure 1A). The Conserved Domain Database (Conserved Domain Search) of NCBI (ID: G5EE01) predicts that four of the five missense variations evaluated in this study are located in the phosphatase domain of DAF-18/PTEN(Figure 1B). It is predicted that amino acid Histidine 138 is located in the active site of the protein, suggesting that H138R (PS7436) amino acid change might have an impact on PIP3 substrate specificity, which could explain the dauer defect phenotype observed. Moreover, it is predicted that both amino acid Histidine 168 and Threonine 176 are located close to the catalytic site of the phosphatase domain. We found a dauer defective phenotype resulting from a T176I substitution (PS7434) but not from a H168Q amino acid substitution (PS7430). These data suggest that certain amino acids in the catalytic and active sites of DAF-18/PTEN decrease the function of the protein, in turn regulating C. elegans dauer development. Future evaluation of PIP3 phosphorylation levels could confirm the effect of these variations on the phosphatase activity. The results of this study contribute to the knowledge of the mechanisms controlling C. elegans dauer entry, and to the C. elegans phenotype-screening of conserved ASD variations.