Organismal development relies on precise temporal and spatial regulation of numerous processes to ensure proper formation of the body plan including the enforcement of symmetric and asymmetric systems. Gene regulatory networks (GRNs) are organized in such a way as to ensure fidelity of each process, or to ensure a precise and accurate phenotypic outcome despite the noise inherent in biological systems. This leads to nontrivial questions, including what genetic mechanisms, if any, are in place to ensure fidelity and how is a GRN as a whole affected by genetic variation in its individual components. Further, how does such variation lead to phenotypic variation, especially with complex or multigenic traits that rely on multiple inputs. Here I describe the application of quantitative genetic methods to identify loci associated with fidelity of two separate but important developmental processes in the nematode C. elegans: 1) Left-right (L/R) asymmetry of the gut-gonad orientation, and 2) proper implementation of programmed cell death (PCD) events that typically sculpt bilaterally symmetric sensory rays of the male tail, which are necessary for copulation. We found significant phenotypic variation for both phenotypes in males from C. elegans isotypes and closely related gonochoristic and hermaphroditic species, suggesting that variation in these traits is not solely attributable to lab domestication or differences in selective pressure (s) on males (Chapters 3 and 4). For association mapping, we use two sources of genetic and phenotypic variation – a globally diverse collection of C. elegans isotypes and recombinant inbred lines (RILs) generated from isotypes with either consistently high or low phenotypic variance for additional traits. We apply genome wide association studies (using linear mixed model design for highly inbred model organisms) and quantitative trait locus (QTL) mapping to identify causal genetic regions. We report that the missing rays or ray defects originally noted by Sulston were nearly suppressed in canonical programmed cell death mutants in the reference strain in N2 (Chapter 2); this was also the case in some C. elegans isotypes with a high propensity for defects when the endogenous pro-apoptotic factor EGL-1, a BH3-only domain protein, was knocked down using RNAi (Chapter 3), suggesting there is standing genetic variation in mis-regulation of the PCD pathway. GWAS and linkage disequilibrium analysis of significant SNPs suggest a ~3 Mb region on chromosome II is associated with stochastic cell death in 87 unique C. elegans isotypes. QTL mapping with RILs made from isolates QX1211 and AB4 further identified a much larger region on chromosome II, in addition to QTL on chromosomes III and X, that explains ~ 55% of natural variation in this trait (Chapter 3). In the case of the observed heterotaxy – the improper arrangement of at least one but not all visceral organs, GWAS identified two QTL on chromosomes II and III that affect only males, suggesting that different QTLs and other genetic/cellular mechanisms mediate natural variation in males compared to those affecting hermaphrodite heterotaxy (Chapter 4). 95% confidence intervals and linkage disequilibrium analysis indicate that these QTL for both traits are relatively large (compared to the length of the chromosome (Chapters 3 and 4)), and near-isogenic lines (NILs) excluded a small ~1.5 mb region on chromosome II. We applied variant-effect prediction (VEP) analysis of the remaining QTL on chromosomes II, III and X as an alternative to identified possible candidates (Chapter 3).

In parallel to the standard approaches, we have also applied a machine learning pipeline using ElasticNet Regression as a complementary approach for identifying genetic loci that may be missed as a result of small effect size, population structure, or low statistical power for naturally varying traits in C. elegans isotypes (Chapter 5). Our approach was similar to its use in multi-parental inbred mice lines but was combined with, and enhanced by, the genetic toolkit of C. elegans. For example, C. elegans isotypes can be used to screen a large amount of candidate genes for functional studies in polygenic backgrounds. We used the previously described, naturally varying requirement for SKN-1 – a maternally-loaded transcription factor necessary for endoderm specification - to test the performance of ElasticNet regression in identifying biologically confirmed regions from other standard approaches like QTL mapping and GWAS. ElasticNet regression was able to identify all loci identified by GWAS and QTL mapping with a set of recombinant inbred lines made with isotypes N2 and MY16 (Spearman’s rho, R2 = 0.55, p = 6.5x10-5), in addition to novel loci on chromosomes V and X. While this model is significant statistically, isogenic line testing will be necessary to validate this method as a valid alternative to more traditional association mapping methods (Chapter 5).

Cellular reprogramming has the potential to treat numerous degenerative diseases and repair injured organs, but safe and effective therapeutic applications require deeper understanding of the molecular and cellular consequences of forced cell fate changes. The model organism Caenorhabditis elegans can be used to study cellular reprogramming in vivo and features diverse reprogramming-related events, including: the multipotency-to-commitment transition during embryogenesis, naturally occurring transdifferentiation, and forced reprogramming of the germline and differentiated somatic cells. A remarkable finding from a previous study showed that ectopic expression of the transcription factor ELT-7 alone is capable of triggering transdifferentiation of the pharynx into intestine-like tissue, and “transorganogenesis” of the entire uterus into a well-formed intestine-like organ. However, the molecular mechanisms involved in this process are largely unknown.

In this work, I primarily utilize reverse genetic screening and time course mRNA-sequencing methods to probe the ability of ELT-7 to initiate reprogramming and to identify biological processes involved in somatic gonad-to-intestine transorganogenesis. I find that reprogramming to intestine mirrors embryonic endoderm development, engaging most, if not all, of the endoderm gene regulatory network (GRN). Three chromatin-associated factors are identified that appear to be playing important roles in ELT-7-directed transorganogenesis, suggesting that establishment of proper chromatin architecture may define a permissive cellular context for reprogramming into intestine. Bioinformatics analysis of the ELT-7-directed reprogramming temporal transcriptional profile shows that widespread gene expression changes occur even in some non-reprogrammed tissues, indicating significant cell fate elasticity and suggesting cell type-specific modes for cell fate maintenance. Finally, and somewhat surprisingly, gene expression changes following ectopic ELT-7 expression reveal the activation of an intracellular pathogen response (IPR) that correlates both spatially and temporally with cellular reprogramming and may be promoting somatic gonad-to-intestine transorganogenesis.

This work presents the gene expression dynamics of in vivo cellular reprogramming and characterizes the distinct biological processes involved. These findings suggest that the cell type-specific reprogramming ability of ELT-7 may rely on the robust endoderm GRN and require a cellular context that is capable of activating the IPR. These data will enable further targeted research into the mechanisms of transorganogenesis and may lead to the discovery of similar phenomena in C. elegans and other organisms.

All life on Earth, from the smallest microbe to the largest blue whale, is subject to Earth’s gravitational pull. This force may be the only environmental variable that has remained constant for all organisms since the origin of life. Because of this, the ability to sense gravity is present in many species and is often critical for survival. Plants use gravity as a cue for directing root growth. Animals, including humans, sense gravity to facilitate movements and to build spatial awareness. Additionally, gravity sensation is one of many modalities that are integrated by cells and nervous systems to make decisions about behavior. Little is known about gravity sensation compared with other sensory systems. Likewise, polymodality and sensory integration are relatively new and understudied areas of research within sensory biology. To investigate gravity sensation, I developed a novel, large-scale assay for observing gravitactic behavior in C. elegans. I found that the worms negatively gravitax — a behavior that has not previously been observed in this species — and that gravitaxis is altered in the presence of light and electromagnetic fields. A screen of known DEG/ENaC mechanosensory components revealed that MEC-7 and MEC-12, which form specialized microtubules required for gentle touch, are required for negative gravitaxis. However, mutations affecting MEC-4 and MEC-10 — the subunits of gentle-touch transducing ion channels — did not impede worms’ ability to gravitax. Instead, I found that negative gravitaxis depends on the polymodal TRPA-1 channel protein. These findings suggest a previously unidentified connection between DEG/ENaC and TRPA-1 in mechanosensation. I then assayed worms that, through genetic ablation, lacked either the gentle-touch sensitive touch receptor neurons (TRNs) or a pair of proprioceptive PVD neurons, which express MEC-7/12 and TRPA-1. While TRN- worms exhibited behavior similar to N2 controls, worms lacking PVD neurons failed to show negative gravitactic preference. This work contribute to an understanding of gravity sensation and sensory integration in C. elegans that can provide insight into vestibular, auditory, and cognitive disorders.

Animal development is a complex process, shepherded by systems of robustness to ensure its success. To date, such systems that have been experimentally identified, largely have been found to ensure specific and correct cell identities in terms of gene and protein expression and spatial position. Little is known about systems that supervise, guide, and compensate for variability in the timing of events within development. The developing embryo of the nematode C. elegans follows a highly-stereotyped sequence of events starting at the two-cell stage where an asymmetric first division results in two cells differing in size, genetic and proteomic identity, and lineage dependent rates of division. These first two cells stereotypically divide at different times, with the larger dividing before the smaller. This sequence will later result in a number of key cell-cell interactions necessary for successful development. The rigid stereotype of this sequence and the critical nature of the dependent later events, suggests that the sequence itself may be under the influence of a system of robustness. At this stage such a system would necessarily include communication between the two cells to ensure their coordination in time. The work presented here establishes a method of challenging this system, by placing the C. elegans embryo in a temperature gradient sufficient to push the temperature dependent rate of division of the two cells away from their stereotyped temporal relationship. To achieve this we built and characterized a novel microfluidic temperature gradient device that can establish a 7.5 OC temperature gradient across the ~ 50 μm long developing embryo within biologically permissive temperatures. This temperature gradient establishes a condition that would be considered aberrant by the embryo at this stage, if and only if the two cells are monitoring each other's behavior. We have found that within a temperature gradient, the two cells of the embryo identify the existence of an aberrant condition and compensate for it by slowing their division rates. We find that the fold change in division timing of the two cells is dependent on their orientation in the temperature gradient, and that embryos that survive this condition to hatching generally slow down more than those that do not. We are able to reverse the sequence of divisions between the two cells and although they do not hatch, a surprising percentage undergo morphogenesis and result in a product that looks “wormlike” suggesting even later checkpoints and compensation. We also found that in a fraction of the embryos loaded into the gradient after the first division, the cleavage between the two cells reverses and the two nuclei of the two cells migrate back toward each other.

The behavior of the two-cell embryos in the temperature gradient: 1) surviving a high percentage of time in lower gradients, and even a fraction of the time in higher gradients, 2) the slowing down of each cell relative to its expected behavior at the temperature it is experiencing with greater slowing resulting in a greater likelihood of survival, and 3) the entry of embryos into morphogenesis even after violation of the stereotypical sequence of division at the two-cell stage, constitutes evidence for one and possibly two previously unidentified compensation and coordination mechanisms that act to ensure robustness against variation in the timing of events in the development of the early C. elegans embryo.