Lopez Santos, Agustin et al. (2017) International Worm Meeting "Distinct genetic mechanisms underlie natural variation in left-right arrangement of the major organs in males and hermaphrodites of C. elegans."

Callander, Davon, Rothman, Joel, Lopez Santos, Agustin, Alcorn, Melissa

[International Worm Meeting, 2017]

Establishing the proper arrangement of internal organs is critically important during development, and defects in this process can lead to serious and even lethal birth defects. While the left-right (L/R) anatomical handedness in C. elegans was originally found to be essentially invariant in the laboratory N2 strain, we have observed that the L/R arrangement of the gonad and gut is frequently reversed under some environmental conditions in N2 and in many of the ~100 wild isolates examined. In some strains, up to ~11% of males show complete reversal in L/R gut/gonad arrangement, while in others, reversals were never seen. GWAS analysis revealed at least two regions of the genome responsible for these reversals in males. We also found that hermaphrodites of some strains show partial L/R reversals ("heterotaxy") of only the anterior or only the posterior gonad arm, with a small fraction of these animals showing complete reversals involving both arms. Variation in this phenotype across the wild isolates ranged from 0% to ~10% total reversals of all three types in hermaphrodites. Surprisingly, we found that the propensity for reversals in males and hermaphrodites is not significantly correlated across the isolates, indicating that the genetic basis for L/R reversals is independent in the two sexes. Analysis of 99 RILs generated from two strains at the extreme ends of the distribution of the hermaphrodite phenotypes (N2, 0% reversals, MY16, 10% reversals) revealed that the phenotype is multigenic and provided evidence of transgressive segregation, with one RIL showing ~20% reversals. QTL analysis is being performed to assess the genetic complexity and causal basis for this variation in handedness. Preliminary evidence suggests that the reversals in hermaphrodites may in part be traced back to an embryonic event, during which the developing gut undergoes a L/R asymmetric twist in comma-stage embryos (*Hermann et al., 2000), consistent with our finding that lin-12 mutants, which do not undergo normal gut twist, show frequent L/R gut/gonad reversals. Thus, variations in the anatomical handedness among wild isolates may reflect varying fidelity in the mechanism controlling the rotation of gut cells during mid-embryogenesis.

Alcorn, Melissa et al. (2017) International Worm Meeting "Natural variation in stochastic programmed cell death in the adult male tail is associated with several QTL."

Alcorn, Melissa, Torres Cleuren, Yamila, Rothman, Joel, Callander, Davon

[International Worm Meeting, 2017]

Somatic programmed cell death in C. elegans is thought to occur by a largely invariant pattern. However, we have found an exception in the development of the male copulatory structure, which exhibits variability (reduced fidelity) in the total number of sensory rays. C. elegans males typically develop eighteen bilaterally symmetric rays, in which each ray is comprised of the dendritic processes of two neurons and a structural support cell. We have found that in the laboratory N2 strain, ~20% of males are missing one or more rays as a result of stochastic, inappropriate programmed cell death (PCD). This stochastic cell death varies widely across 94 unique wild isotype strains, ranging from 0 to ~35% of males with missing rays. The absence of rays in selected strains is the result of inappropriate PCD, as ray losses are suppressed in egl-1(RNAi) animals. A genome-wide association study identified one quantitative trait locus (QTL) on chromosome I underlying this variation in stochastic PCD. In addition, genetic mapping strategies using RILs identified several independent QTL on chromosomes II, III, and X that contribute ~55% of the phenotypic variation seen in the RILs and most likely other isotypes. Concurrent with in silico analysis, we are validating candidate regions in different genetic backgrounds to test their effect on the propensity for cell death and to identify causal genes that determine the molecular mechanism(s) of stochastic PCD.

Alcorn, Melissa et al. (2015) International Worm Meeting "Developmental fidelity in males varies widely across C. elegans isotypes."

Cieslak, Matthew, Callander, Davon, Rothman, Joel, Birsoy, Bilge, Alcorn, Melissa

[International Worm Meeting, 2015]

The development of C. elegans hermaphrodites is generally highly reproducible under laboratory conditions. For example, the pattern of somatic programmed cell death (PCD) and establishment of left-right (L/R) handedness in the arrangement of internal organs in hermaphrodites are virtually invariant across large numbers of animals. In contrast, we have found that both N2 and many wild isolates of C. elegans males show frequent "errors," indicating that that the fidelity of male development is often relaxed and varies widely between wild isolates. We have observed that particular sensory rays in the male tail are often missing as an apparent result of inappropriate PCD, indicating that repression of EGL-1-dependent PCD is defective in many natural isolates. Further, the L/R arrangement of the gut and gonad, while essentially invariant in hermaphrodites and in males of some wild isolates, shows frequent reversals in males of other isotypes, with a reversal rate as high as ~30% at 25oC. Defects in the normal trajectory of gonad migration were also seen in some, but not all of the isotypes; however, these migration errors do not correlate with the L/R organ handedness phenotype across different isotypes. These errors in PCD and L/R handedness are not characteristic of all representatives of the species, as some isolates, unlike N2, nearly always make "perfect" tails and show fully stereotyped organ handedness. Moreover, such defects in male development are not explained by the androdioecious nature of C. elegans, in which males are of low significance to propagation of the species, as we have observed both kinds of defects in gonochoristic (male/female) species. Analysis of RILs made from isotypes that are high-fidelity and low-fidelity for both the stochastic PCD and L/R reversal phenotypes indicate that both traits are multigenic. Finally, we found that a strain that is high fidelity for both developmental processes also shows low variation in two hermaphrodite characteristics: variance in germline stem cell number and in length of newly hatched L1s. In contrast, a strain that is low-fidelity for the two male traits shows high variation in these hermaphrodite parameters. These findings raise the possibility that global differences in the fidelity of development may vary between different isolates of the species. .

Alcorn, Melissa et al. (2015) International Worm Meeting "The propensity for stochastic PCD during male tail development varies across C. elegans isotypes."

Alcorn, Melissa, Rothman, Joel, Birsoy, Bilge, Cieslak, Matthew, Lopez Santos, Agustin, Callander, Davon

[International Worm Meeting, 2015]

While somatic programmed cell death (PCD) occurs in a largely invariant pattern in C. elegans, we have found that it arises sporadically and inappropriately during the formation of the 18 bilaterally symmetric sensory structures (rays) of the adult male tail. The rays, each consisting of the dendritic ends from two neurons and a structural support cell, arise during post-embryonic development. Wild-type N2 males frequently lack one or more rays. Males defective for components required for PCD contain few or no missing rays, suggesting that ray loss is the result of stochastic, inappropriate PCD. We found that the propensity for ray loss varies widely between 97 unique isotypes: while some isotypes virtually never show missing rays, in others, up to ~35% of males lack rays. As with N2, the loss of rays in these other strains appears to be the result of PCD, as they are restored by egl-1(RNAi). EMMA analysis applied to the full dataset of the 97 strains revealed that the variation is associated with a region on Chromosome I. We have also developed a complementary computational approach to analyzing this type of data by using a predictive regression model based on machine learning. This method applied to this and other datasets has identified regions of significance on Chromosomes II, III, V and X that were not predicted from standard EMMA analysis. Both approaches indicate that the sporadic PCD in the male is a polygenic trait. Ongoing analysis of RILs between two strains that represent phenotypic extremes is being used to identify the specific causal genes that control stochastic PCD during formation of the male rays. .