Hybrid incompatibility (HI) is frequently manifested as lethality or sterility in hybrid progeny between related species, and plays a key role in speciation. The genetic basis of HI has been intensively studied in model organisms such as yeast and fruit fly over decades, and "Two rules of speciation" have been observed across species. C. elegans as a nematode model organism contributes little to speciation research mainly due to lack of a close relative with which it can mate and produce viable progeny. Such limitation has recently been alleviated by identification of C. nigoni, a close relative, also termed as sister species, of C. briggsae. The two can make and produce a handful of viable hybrids. Both species are members of Elegans supergroup. Hybrid cross between the two species uncovered asymmetric hybrid incompatibilities, i.e. crossing direction-dependent hybrid male sterility and inviability. Asymmetry was also observed in F1 hybrids from reciprocal crosses exclusively in male but not female (Woodruff, Eke, Baird, Félix, & Haag, 2010). Asymmetry was also observed in backcrosses between the F1 female hybrids and the parental species. For example, F2 progeny fathered by C. briggsae suffered almost 100% embryonic lethality for both males and females, whereas those fathered by C. nigoni were partially viable and fertile. Further study of HI between these two species was initiated by investigating how C. briggsae chromosomal fragments in an otherwise pure C. nigoni genome affect fitness of hybrid worms. The hybrid worms were generated by repeatedly backcrossing C. briggsae genomic fragments each bearing a visible chromosomal-integrated marker to C. nigoni to produce introgression lines. Characterization of the introgression lines provided a detailed HI landscape of between the two species. Multiple intervals on the C. briggsae X chromosome were responsible for hybrid male inviability or sterility while most of the C. briggsae autosomes were not involved in these male phenotypes (Bi et al., 2015). RNA sequencing was performed in sterile male worms bearing independent introgressions, revealing a down-regulated gene expression pattern (Li et al., 2016). To uncover the HI mechanism underlying the asymmetric HI phenotypes exhibited in hybrids in F1 generation, I performed a genome-wide screening to identify HI loci that are responsible for the hybrid male inviability and sterility in F1 as well as hybrid breakdown in F2. By crossing between C. briggsae and C. nigoni introgression lines bearing a known C. briggsae fragment, I was able to construct hybrid animals homozygous or heterozygous for C. briggsae alleles on the introgression while those on counterpart of C. nigoni were absent. Contrasting the HI phenotypes here and those between two wild-type parents allows mapping of the loci responsible for the hybrid asymmetric phenotypes. The aggregated introgressions cover 94.6% of the C. briggsae genome, including 100% of the X chromosome. Surprisingly, I identified another two C. briggsae genomic intervals on chromosomes II and IV that can rescue the hybrid male inviability but not the male sterility in F1 fathered by C. nigoni, suggesting the involvement of differential epistatic interactions in the asymmetric hybrid male fertility and inviability. What's more, I observed that two independent C. briggsae X fragments that produce male sterility in C. nigoni as an introgression rescued hybrid male sterility in F1 fathered by C. briggsae. Backcrossing of the rescued sterile F1 male to its parental species showed that they can alleviate the F2 hybrid breakdown by a handful of viable F2 mothered by C. briggsae. Subsequent backcrossing of the rescued sterile males with C. nigoni led to the isolation of a 1.1-Mb genomic interval that specifically interacts with an X-linked introgression, which is essential for hybrid male fertility. In addition, I further identified three C. briggsae genomic intervals on chromosome I, II, and IV that produced inviability in all F1 progeny, dependent on or independent of the parent-of-origin. Taken together, I identified multiple independent interacting loci that are responsible for asymmetric HI phenotypes especially hybrid male sterility and inviability, which lays a foundation for their molecular characterization.

No description available.

Gene regulation in C. elegans germ cells depend on transgenerational chromatin modification and small RNA pathways. Germline silencing mechanisms evolved to repress foreign DNA from compromising the transfer of genetic information to progeny. Effective genetic tools that circumvent the silencing machinery will facilitate studies using this model organism. Specifically, translation of heat-shock inducible transgenes is inhibited in the germline making it challenging to transiently express enzymes to modify the genome. Here, we describe a genetic screen design that can be used to identify pathways that prevent germline expression of heat-shock induced transgenes. We use split-GFP (GFP1-10 and GFP11) to confine a genetic screen to germ cells. Stable transgenic lines with germline expression of single-copy integrated GFP11 were produced using MosSCI. The insertion lines will be used in RNAi or chemical mutagenesis screens for the germline de-repression of GFP1-10 expressed under heat-shock promoters. The screen is likely to identify candidate RNAi or chromatin factors involved in repressing heat-shock expression in the germline, particularly from extrachromosomal arrays. Inducible high-level expression in the germline from extrachromosomal arrays would be a valuable tool for large-scale genome engineering.

Large numbers of people suffer from nervous system injuries and neurodegenerative diseases each year, with little success in regaining lost neural functions. Attempts to successfully regenerate nervous tissue in the mammalian Central Nervous System have meet with limited success. Simpler models have thus been useful in determining conserved mechanisms in the enhancement of neural regeneration. One such mechanism is intracellular calcium signaling. We used <italic>Caenorhabditis elegans</italic> as a model system to study the effects of optogenetic stimulation on regeneration. Using a femtosecond laser we cut individual <italic>C. elegans</italic> axons <italic>in vivo</italic> and then periodically stimulated the neurons by activating the genetically encoded light activated channel, Channelrodopsin-2. We found that periodic photo-activation could increase regeneration over 24h by at least 31%. We repeated these experiments with dantrolene treatment and in <italic>unc-68(e540)</italic> mutants to assess the effects from a lack of internal calcium ion signaling in these worms. In both cases, we found a complete suppression of stimulated regeneration when calcium signaling was blocked. This indicates that intracellular calcium ion signaling is crucial in the initiation of neural regeneration in the first 24 hours and mediates the enhanced outgrowth we observe with periodic photo-activation. The importance of intracellular calcium ion signaling can lead to further studies to enhance the stimulation of neural regeneration, and improve therapies for patients with neural damage and loss of neural functions.

The basic helix-loop-helix (bHbH) family-of transcription factors is important in many developmental and regulatory pathways such as cellular proliferation and differentiation, lineage commitment, sex determination, neurogenesis, myogenesis, hematopoeisis and pancreatic development. The free-living nematode Caenorhabdits elegans is an important model organism. Genetics studies of a gene in nematodes help us to better understand the functioning of hornologs in more complex organisms. These studies investigate the nematode homolog of ABF-1, CeABF-1, and its potential role in the development of C. elegans. The BLAST Database (http://www.ncbi.nlm.nih.gov) predicted a bHLH protein in C. elegans, located on cosmid ZK682.4, of 170 amino acids with an overall 51% similarity to human ABF-1. Importantly, it had a 72% similarity to human ABF-1 within the bHLH domain. Genomic and eDNA clones of CeABF-1 were isolated using whole nematodes and PCR methods. Clones were constructed that would allow us to use green fluorescent protein (GFP) to localize CeABF-1 expression. We also used RNA interference to determine the function of CeABF-1. No obvious phenotype was observed in nematodes unable to produce the CeABF-1 protein. Preliminary studies suggest that CeABF-1 is not an essential gene for development in C. elegans. Constructs were also made for protein induction and antibody studies. Further studies are necessary to determine how CeABF-1 is involved in nematode development and its interaction with other proteins.

No description available.

No description available.

This thesis describes the use of a micropipette deflection technique to measure the viscous forces experienced by the millimeter sized undulatory swimmer and model organism C. elegans. Using a specialized pipette, we are able to simultaneously measure both the lateral and propulsive forces acting on the worm. We find that the measured force curves are well described by Resistive Force Theory, which is a low Reynolds number hydrodynamic model. This work constitutes the first justification of its applicability at Reynolds numbers of this magnitude (roughly 0.1). Through our comparison with Resistive Force Theory, we extract the worm's drag coefficients, which are in agreement with an existing theoretical prediction. Through a simple scaling argument, we obtain a relationship between the size of the worm and the typical viscous forces, which is in good agreement with our data. We also present a study aimed at measuring how the hydrodynamic forces on the worm change in proximity to solid boundaries. Using micropipette deflection, forces are measured at controlled distances from a single planar boundary and midway in between two parallel boundaries. We find the viscous forces and drag coefficients to increase significantly as the worm approaches a boundary. We find a constant value for the ratio of normal to tangential drag coefficients at all distances from a single boundary, but measure it to increase significantly as the worm is confined between two boundaries. In addition, the worm is seen to undergo a continuous gait modulation, primarily characterized by a decreased swimming amplitude, as it is subject to larger drag forces in confinement. Finally, the interactions between two worms swimming nearby one another are probed. Worms are held adjacent to one another using micropipettes, and are found to tangle with each other, rather than interact hydrodynamically. We develop simple models that well capture the onset and probability of tangles as a function of the separation distance between the worms. / Thesis / Master of Science (MSc)

Neurobiologists have sought an overarching logic of circuit assembly for decades. Canonically, piecemeal approaches have led to the discovery of many genetic pathways underlying discrete steps in nervous system development. These findings have cumulatively helped us understand how neurons extend axons, form neighborhoods, and choose synaptic partners to ultimately build sophisticated circuits. Today, advances in connectomics and transcriptomics have placed us in an exciting position to begin to tackle this systemically. This entails not only studying entire circuits and nervous systems, but also entire gene families which coordinate circuit assembly in space and time. The nematode C. elegans provides us with an opportunity to study circuit assembly on both genome-wide and nervous system-wide levels. In the past, C. elegans connectomics has relied heavily on the first wiring diagrams which were established in the 1980s. There is a growing need to scale this approach and study nervous systems across development, in different genetic backgrounds, and in various environmental paradigms. In this work, we first establish transgenic and in silico tools to facilitate interrogation of a previously understudied region of the C. elegans nervous system, the largest neuropil called the “nerve ring”. Our tools – WormPsyQi and AxoPAL - help study synapses and neuronal adjacencies in a precise and high-throughput manner, therefore overcoming constraints on sample size and phenotypic space. Next, we focus on the cadherin superfamily of cell adhesion molecules (CAMs) and its implications on nervous system structure and function. Across evolution, two families of CAMs have expanded significantly with increasing nervous system complexity: cadherins and immunoglobulins (IgSFs). While many studies have described the expression and function of IgSFs, many cadherins are relatively under-studied in most neuronal contexts. Here, we present an expression atlas of all cadherins encoded by the C. elegans genome. Expression patterns are described with neuron-type spatial resolution and across larval development to define the richness and diversity of the cadherin repertoire in an entire nervous system, which has never been previously done for any model organism. Our analysis reveals interesting temporal changes and a striking dichotomy between broad- and sparse-expressing cadherins. Some of the most well-conserved cadherin subfamilies - classical cadherin, calsyntenin, fat, and flamingo - are expressed in all neuron types in C. elegans. Furthermore, when analyzed in the context of the well-established C. elegans connectome, the expression atlas unfolds a putative molecular code underlying connectivity and selective adjacency. Altogether, by studying the expression of the entire cadherin family in neuronal and non-neuronal cell types, across several stages of development, this thesis highlights previously unknown salient themes of cadherin expression patterns which likely have functional implications. In addition to characterizing expression, we generated a collection of null mutants for all C. elegans cadherins, and proceeded to characterize them. To our surprise, most single mutants are viable and show minimal obvious phenotypes; we think this will favor studying neuronal functions of these genes since early lethality in other systems has often been a limitation. We also found that the C. elegans Fat cadherin homolog, cdh-4, has several structural and behavioral phenotypes. Studying neuronal structure defects in single and compound mutants of cadherins implicated by the expression and speculative molecular code will further help delineate the roles of this gene family in various aspects of circuit assembly; these include cell positioning, axodendritic patterning, synaptic partner choice, and downstream behavior. By addressing the question of circuit assembly from multiple directions and with new tools, this thesis provides a generic workflow; we hope that it will bring C. elegans neurobiologists a few steps closer to untangling complex circuit assembly in the context of entire gene families which orchestrate it.

Les cellules souches portent de grandes promesses envers la médecine régénératrice, tandis que leur perturbation peut mener au cancer. Ainsi, il est fondamental de définir les interactions qui prennent place au sein d'un organisme entre les cellules souches et leur micro- et macro-environnement. Chez le nématode Caenorhabditis elegans, les niveaux de prolifération des cellules souches germinales (CSG) sont régis par l'abondance de leur progéniture différenciée à travers une interaction entre la voie de signalisation ERK/MAPK et la kinase activée par l'AMP (AMPK). Cependant, l'intersection moléculaire entre AMPK et la voie MAPK demeure toujours inconnue. En regard aux déterminants embryonnaires précoces et PAR-4/LKB1, principale kinase activant AMPK, nos travaux ont démontré l'expression ubiquitaire de par-4 et l'enrichissement cytoplasmique et cortical de PAR-4 dans la lignée germinale et les embryons précoces, en plus d'une décroissance transitoire de l'enrichissement cortical de PAR-4 dans la zone pachytène. De plus, nos travaux suggèrent que par-4b est suffisant pour établir la polarité embryonnaire et maintenir la fonction essentielle de par-4. Par ailleurs, quant aux déterminants régissant la prolifération des CSG, nous avons constaté que chez les adultes porteurs d'ovocytes, LIN-3/EGF et LET-23/EGFR sont nécessaires pour promouvoir la prolifération des CSG. De surcroit, nous avons démontré que DAF-18/PTEN prévient l'ovulation d'ovocytes non-fécondés et contribue au mécanisme de rétroaction de la prolifération des CSG. Nos résultats indiquent aussi que l'état de prolifération des CSG est corrélé, de manière inversement proportionnelle, à la quantité d'ovocytes anovulés, et ne forme donc pas une régulation binaire, mais plutôt progressive. Finalement, nous avons démontré que DAF-18/PTEN prévient la prolifération des CSG en l'absence de sperme de façon strictement zygotique, potentiellement à travers son rôle dans la gonade somatique, car sa perte de fonction altère légèrement les contractions des cellules de la gaine. Ainsi, ces travaux approfondissent globalement les principales voies de signalisation physiologiquement impliquées dans la régulation homéostatique de la prolifération des cellules souches germinales chez le nématode C. elegans, et pourraient avoir des retombées envers la médecine humaine. / Stem cells hold great promises for regenerative medicine, whilst their disruption may lead to cancer. Therefore, it is fundamental to define the interactions between stem cells and their micro- and macro-environment. In the nematode Caenorhabditis elegans, retroactive control of germline stem cells (GSC) proliferation levels is governed by the abundance of their differentiated progeny through an interaction between ERK/MAPK signaling pathway and AMP-activated kinase (AMPK). However, the molecular intersection between AMPK and the MAPK pathway still remains unknown. With regard to early embryonic determinants and PAR-4/LKB1, the main kinase activating AMPK, our work has demonstrated the ubiquitous expression of par-4 and the cytoplasmic and cortical enrichment of PAR-4 in the germline and early embryos, in addition to a transient decrease in cortical PAR-4 enrichment in the pachytene zone. Furthermore, our work suggests that par-4b is sufficient to establish embryonic polarity and maintain essential par-4 function. Moreover, regarding the determinants governing GSC proliferation, we found that in oocyte-bearing adults, LIN-3/EGF and LET-23/EGFR are required to promote GSC proliferation. Additionally, we demonstrated that DAF-18/PTEN is involved in ovulation of fertilized oocytes and contributes to germline stem cell proliferation feedback mechanism. Our results also indicate that GSC proliferation status correlates, in a inversely proportional manner, to the amount of anovulated oocytes, and consequently does not form a binary regulation, but rather a progressive one. Finally, we demonstrated that the strictly zygotic effect of DAF-18/PTEN promotes GSC proliferation, potentially through its role in the somatic germline, since its loss of function slightly alters sheath cells contractions. Thus, this work broadly deepens the main signaling pathways physiologically involved in the homeostatic regulation of stem cell in the nematode C. elegans, but could have implications for human medicine.