The nematode worm Caenorhabditis elegans has become a model system for the analysis of the genetics of aging. Previously, mutations in four genes, age-1, clk-1, daf-2 and daf-28 had been shown to lengthen adult life span. Based on the molecular genetic analysis of these genes, the sole function of the dauer genes age-1, daf-2 and possibly daf-28 is to regulate the activity of the forkhead-like transcription factor daf-16. daf-16 may determine life span by regulating the transcription of genes that are necessary for resistance to stresses, especially oxidative stress. Mutations in clk-1 affect behavioral and developmental timing as well as increasing mean and maximum life span. I show that mutations in the genes clk-2, clk-3 and gro-1 affect many of the same processes as clk-1 and that these four genes interact to determine the length of development and adult life span. These four Clock genes lengthen life span in a manner that is distinct from that of the dauer genes. clk-1 has been cloned and has been implicated in the regulation of metabolism. This suggests that Clock mutants may live long because they have reduced metabolic rates. I also show that mutations in 7 genes that affect feeding behavior, eat-1, eat-2, eat-3, eat-6, eat-13, eat-18 and unc-26 lengthen life span. This effect is presumably due to reduced caloric intake (caloric restriction) which has been shown to lengthen the life span of a wide variety of animals. eat-2 lengthens life span by a mechanism that is distinct from that of the dauer mutants but may be similar to that of the Clock mutants. This suggests that caloric restriction may also reduce metabolic rates, possibly through down-regulation of the Clock genes. These results indicate that life span in C. elegans is a polygenic trait, influenced by many different physiological processes. The study of genes that affect aging in C. elegans provides support for the antagonistic pleiotropy and free radical theories of aging.

Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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.

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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.

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