Birsoy, Bilge et al. (2009) International Worm Meeting "Right Way to Have Sex."

Bloss, Tim, Birsoy, Bilge, Rothman, Joel H., Downes, Joanna C., Choi, Shin Sik

[International Worm Meeting, 2009]

While the mechanism that breaks left-right (L-R) anatomical asymmetry (handedness) has been described in C. elegans, we have found that at least one additional mechanism must exist to establish other handedness cues that generates L-R differences in the deployment of alternative cell death pathways (see abstract from our lab by Choi et al.). To assess whether such a system might affect a behavior in the worm, we investigated whether male mating behavior exhibits L-R handedness. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite''s body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites'' body (away from the agar surface) and >55% of turns when made under the hermaphrodites'' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays (Choi et al. abstract), we examined the turning bias in egl-1(n1084n3082) mutants. We were surprised to find that, although wild-type males lack rays more frequently on the right, the right-hand turning bias is actually increased in the egl-1 mutant, in which no rays are lost and therefore rays are always symmetrically arranged. Thus, the intrinsic turning bias is even more apparent in males with symmetric mating structures. To assess whether this L-R behavioral bias is dependent on anatomical handedness, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that a handedness-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. These findings raise the possibility that, analogous to brain laterality in humans, functionally L-R asymmetry in the C. elegans motor nervous system may be independent of anatomical handedness.

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. .

Jeong, Pan-Young et al. (2009) International Worm Meeting "CED-4-dependent regulation of germ cell proliferation in dauer larvae by protein phosphatase 2A."

Joshi, Pradeep M., Birsoy, Bilge, Jeong, Pan-Young, Rothman, Joel H.

[International Worm Meeting, 2009]

When C. elegans senses inappropriate growth conditions (high temperature, limited food supply and dauer pheromone), it enters the long-lived dauer larva state. It has been previously reported that the germ cells in dauers remain in G2 arrest until exit from the dauer state. To investigate the mechanisms that promote this arrest, we analyzed the function of genes that when inactivated by RNAi result in supernumerary germ cells (i.e., substantially more than the ~42 present in normal dauers) in dauer-constitutive daf-2 and daf-7 mutants. The gene set analyzed included those that are strongly upregulated during dauer development based on microarray data (P.-Y. Jeong et al., PlosOne 2009) and previously known cell cycle regulators. This screen identified wee-1.3, cki-1, ngp-1, nst-1, paa-1 and let-92, as suppressors of germ cell proliferation during the dauer stage. We found that RNAi of paa-1, which encodes a structural subunit of protein phosphatase 2A, and let-92, which encodes the catalytic subunit of this enzyme, results in an ~50% increase in germ cell number in dauer larvae. Prolonged culture of daf-2; paa-1(RNAi) and daf-2; let-92(RNAi) dauers did not result in a further increase in germ cell number, revealing that additional regulatory mechanisms must exist in dauers to inhibit germ cell proliferation. Many of the arrested germ cells showed a condensed nuclear morphology and stain positively for phospho-histone H3, suggesting that they are arrested at M-phase; in some animals, virtually all of the germ cells show such morphology. Thus, PP2A regulates G2 arrest in dauer larvae, and in its absence, other factors cause cells to accumulate in mitosis. Remarkably, we found that the increase in germ cell number is partially dependent on the pro-apoptotic regulator CED-4 but is independent of CED-3 and CED-9 function. Thus, in addition to other activities recently ascribed to CED-4, these findings implicate CED-4 in germ cell arrest during dauer diapause apparently independent of its role in regulating programmed cell death.

Terry, Eric et al. (2015) International Worm Meeting "Development of early C. elegans embryos in a temperature gradient provides evidence for a cell-non-autonomous coordination system."

Meinhart, Carl, Sigurdson, Marin, Birsoy, Bilge, Bothman, Dave, Rothman, Joel, Terry, Eric

[International Worm Meeting, 2015]

C. elegans embryos can develop normally over a wide range of temperatures, with differences in developmental rates that vary correspondingly with temperature. Although each embryonic founder cell lineage possesses a distinct clock, all cells must respond coordinately to changes in temperature over the entire viable range to maintain the stereotyped geometry characteristic of early embryos. Each cell might independently assess temperature and intrinsically respond accordingly or, alternatively, different cells might compare their progress with that of their neighbors and adjust to events that shift other cells out of register. Such a compensation system might be used to correct errors that inevitably result from intrinsic or environmental noise that embryos experience. To assess these alternatives, we are asking whether cells are able to alter their division timing based not on the conditions they experience, but on the conditions of surrounding cells. To this end, we have designed, built, and employed a microfluidics device that establishes a temperature gradient of approximately 5oC across the long axis of the developing embryo. Exposing embryos to the gradient at the two-cell stage creates a local environment for each cell that, in the absence of communication and compensation, would be expected to result in discordant development, with aberrant cell division patterns and embryonic geometry, inevitably leading to lethality. In contrast, we found that embryos can develop normally in a 5oC gradient, suggesting that cells communicate and compensate for the discordant conditions of the temperature gradient. Moreover, observations of cell cycle timings provide direct evidence that cells adjust their cell cycle clocks in response to deviations in the relative division timing of other cells. These findings and our microfluidics device will make it possible to investigate a previously unexplored biological error correction and compensation system that appears to function in the early embryo.

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. .

Suzuki, Taiga et al. (2009) International Worm Meeting "Role of the nuclear hormone receptor DPR-1 in dauer development."

Newman-Smith, Erin, Broitman-Maduro, Gina, Birsoy, Bilge, Suzuki, Taiga, Rothman, Joel H.

[International Worm Meeting, 2009]

The dauer larva is an alternative stage of C. elegans development induced by a combination of low food abundance and a high concentration of dauer pheromone, a mixture of small lipophilic compounds. The mechanism of reception of dauer pheromone and response to starvation are not fully understood. We previously isolated a gene encoding an apparent nuclear hormone receptor (NHR) dpr-1 (dauer pheromone responsive) that modulates dauer larva formation. Immunoreactive DPR-1 is expressed in gut cells, and apparently some neurons, of embryos and L1 larvae. Surprisingly, DPR-1 protein is cytoplasmically localized in sparsely grown worms, but appears to relocalize to the plasma membrane of gut cells in worms exposed to high concentrations of dauer pheromone. Overexpression of dpr-1 from the elt-2 promoter leads to an increase in the number of dauer larvae when worms are grown at high density. This led us to hypothesize that DPR-1 may regulate dauer development and might be a component of the pheromone reception system, perhaps acting through a non-canonical NHR signaling system. Consistent with a possible role for DPR-1 in regulating dauer formation, we found that in both liquid and plate assays, the dpr-1(tm898) mutation partially suppresses the formation of dauer larvae induced by pheromone, indicating that dpr-1 is a partial daf-d gene. Further, we found that the daf-c phenotypes of daf-11(m47) and daf-7(e1372) were partially suppressed by dpr-1(tm898) mutations, suggesting that dpr-1 may function downstream of these genes in the dauer pathway. Further studies are focused on elucidating the function of DPR-1 and its unusual intracellular distribution during dauer signaling.