The regulation and evolution of the inverted repeat transposable element Tel, found in the nematode Caenorhabditis elegans, was studied. The stability of Tel elements in the N2 strain genome was investigated by cloning seventeen N2 Tel elements. To examine their structural integrity, sixteen cloned N2 Tel elements were restriction mapped and, in the case of some variants, their DNA was partially sequenced. Two restriction site variants, Tcl(Eco).12 and Tcl(Hpa-).9, were found. Tel(1.5).10b had lost 89 bp from one end, while Tcl(1.7).28 contained a 55 bp insertion. Two additional elements, Tcl(0.9).2 and Tcl(0.9).14, had different internal deletions. Each element was about 900 bp in length. The majority of Tel elements cloned from the N2 strain were found to have identical restriction maps. Somatic excision of Tel elements in the N2 genome was demonstrated. Tel elements in N2 are apparently both structurally and functionally intact. Nevertheless, mobilization of Tel elements in the N2 germline is restricted. Two new transposable element families, Barney (also known as TCbl) and TCb2, were discovered in a closely related nematode, Caenorhabditis briggsae due to Tel identity. These two families, distinguished through differential inter-element hybridization, showed multiple banding differences between strains. The open reading frames (ORFs) of Tel and Barney share 71% DNA sequence and 74% amino acid sequence identity. The putative terminus of Barney exhibits 68% identity with the 54 bp terminal repeat of Tel. Partial sequencing of TCb2 revealed that its ORF is equally diverged from Barney and Tel. The basis of the sequence heterogeneity observed in the C. briggsae transposons and not in the C. elegans transposons could be due to either horizontal transfer or alternate paths of divergence. Significant sequence identity was found between Tel, Barney, and HB1 (a transposable element from Drosophila melanogaster) within their coding regions and terminal repeats. These sequence similarities define a subclass of inverted repeat transposable elements inhabiting two different phylla, Arthropoda and Nematoda. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. elegans. Furthermore, these studies have begun to uncouple mechanisms of longevity and stress resistance.

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.

Many neuronal patterning genes are expressed in distinct populations of cells in the nervous system, leading researchers to analyze their function in specific isolated cellular contexts that often obscure broader, themes of gene function. In this thesis, I aim to make clearer those overlooked common functional themes. I show that the C. elegans homeobox gene unc-42 is expressed in 15 out of a total of 118 distinct sensory, inter, and motor neuron classes throughout the C. elegans nervous system. Of these 15 unc-42(+) synaptically interconnected neuron classes, I show the extent to which unc-42 controls their identities and assembly into functional circuitry. I find that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides and neuropeptide receptors. I also show that unc-42 controls the expression of molecules involved in axon pathfinding and cell-cell recognition. Consequently, I show how the loss of unc-42 has effects on axon pathfinding and chemical synaptic connectivity, as determined by electron microscopical reconstruction of serial sections of unc-42 mutants. I conclude that unc-42 plays a critical role in establishing functional circuitry by acting as a terminal selector of functionally connected neuron types. I speculate that in other parts of the nervous system “circuit transcription factors” may also control assembly of functional circuitry and propose that such organizational properties of transcription factors may be reflective of not only an ontogenetic, but perhaps also phylogenetic trajectory of neuronal circuit establishment.

Cytokinesis is the physical division of one cell into two driven by an actomyosin contractile ring and positioned by signals from microtubules. This process is highly regulated spatially and temporally to ensure accurate division into two daughter cells. Here, I present work that builds upon our understanding of cytokinesis, focusing on the spatial requirements for protein function during cell division and midbody assembly. In Chapter 1, I present an introduction to cytokinesis and the cell and molecular mechanisms that govern the process. In Chapter 2, I present work I contributed to on the use of Upconverting nanoparticles for co-alignment of visible and infrared light on a light microscope. In Chapter 3, I present work developing a new microscopy technology called FLIRT (Fast Local Infrared Thermogenetics) that uses infrared light to inactivate fast-acting temperature sensitive protein function with subcellular precision and validate its use to study cytokinesis and cell fate signaling in the nematode Caenorhabditis elegans. In Chapter 4, I improve upon FLIRT technology by increasing its precision and demonstrate its use in studying the spatial regulation of key cytokinesis proteins including the actomyosin cytoskeleton in contractile ring constriction. The central spindle is an array of antiparallel overlapping microtubules that forms between the separating chromosomes in anaphase and is thought to serve as a signaling hub for cytokinesis. The central spindle is thought to become compacted during contractile ring constriction to form the dense midbody at the end of cell division. In Chapter 5, I investigate the requirements for central spindle microtubules in assembling midbodies in the C. elegans one-cell embryo. I present evidence that the CENP-F-like protein HCP-1 plays a primary role relative to its paralog HCP-2 in assembling the central spindle, and that the midbody can form independently of central spindle assembly. In Chapter 6, I discuss future directions for my work on both technology development and the mechanisms of cytokinesis. Through this work, I develop new technologies and hypotheses for how cytokinesis is spatially regulated within a cell, adding new complexity to our understanding of cell division.

Intercellular communication is crucial during animal development and tissue maintenance to ensure that correct patterns of cell types are generated to meet the needs of the organism. During lateral specification, intercellular communication resolves cell fate decisions between equipotent cells, creating fate patterns that are biased by external factors in some contexts, but appear stochastic in others. The Notch signaling pathway mediates lateral specification; small differences in Notch activity are amplified by regulatory feedback loops to robustly differentiate cell fates based on relative levels of Notch activity. It is often unclear how noise in the environment is processed by cells to generate differences in Notch activity that can be translated into stochastic, but robust, cell fate outcomes. The nematode Caenorhabditis elegans contains a simple, Notch-mediated, stochastic lateral specification event; a small, random difference in Notch activity between two cells, the α cells, is amplified so that one α cell assumes Anchor Cell (AC) fate and the other assumes Ventral Uterine precursor cell (VU) fate. Two upstream factors bias the outcome of the AC/VU decision depending on the length of the time interval between the births of the α cells: the relative birth order of the α cells and the onset of expression of the transcription factor HLH-2. It is unknown how these factors create a difference in the relative Notch activity level between the two α cells, and limitations of existing Notch reporters have prevented the direct observation of Notch activity levels required for determining the relationships. In this thesis, I describe a genetically-encoded Sensor Able to detect Lateral Signaling Activity, or SALSA, which uses changes in nuclear Red:Green fluorescence to indicate Notch activity. I demonstrated that SALSA captures expected Notch activity patterns in four paradigms in C. elegans, encompassing both Notch homologs, and reports low levels of Notch activity that were predicted but undetectable with other Notch activity reporters. Using SALSA, I showed that the first-born α cell is able to develop an advantage in Notch activity prior to the birth of the other α cell when the time interval between α cell births is long, but the α cell that gains the Notch activity advantage is random with respect to birth order when the time interval between α cell births is short. These results agree with the current model of the AC/VU decision. I also describe Flexon, a method for the conditional activation of strong gene expression in specific cell lineages using a lox-stop-lox cassette encoded into an artificial exon flanked by two artificial introns. A flexon can be placed into the coding region of a gene to prevent translation of a functional gene product; gene expression is restored to specific lineages through expression of a tissue-specific Cre driver that excises the flexon. I show that flexon can be used to make bright, long-lasting, tissue-specific fluorescent lineage markers. I also showed that the flexon could be used for conditional activation of an endogenous gene by inserting a flexon into rde-1 to severely reduce RNAi activity and restore gene function in specific tissues using Cre drivers.

LIN-12/Notch is a conserved transmembrane receptor that is required during animal development for proper cell-fate decisions and specification. In Caenorhabditis elegans, activation of LIN-12 occurs through binding to ligand expressed by an adjacent cell. This binding event triggers two cleavage steps and results in the release of the LIN-12 intracellular domain [LIN-12(intra)], which translocates to the nucleus to form a ternary complex with two other proteins: LAG-1/Su(H)/Cbf1 and SEL-8/Mastermind/Mastermind-like. This ternary complex will then transcriptionally activate target genes via LAG-1 Binding Sites (LBSs). LAG-1 is the sole DNA-binding component within the complex, and in the absence of LIN-12(intra), can act as a transcriptional repressor. LIN-12 signal transduction can be studied in the C. elegans Vulval Precursor Cells (VPCs), which exhibit precise spatiotemporal patterning regulated by LIN-12 activity. Here, I show that LAG-1 is positively autoregulated by LIN-12 activity in cells where LIN-12 activity is high. Autoregulation is mediated by an enhancer element that contains a cluster of 18 LBSs that are located within a conserved high occupancy target region, which is a span of DNA that is pulled down promiscuously in ChIP-Seq experiments. Mutation of the LBSs abrogates preferential expression mediated by the enhancer in cells with high LIN-12 signal transduction. When the HOT region is deleted from the endogenous lag-1 locus, expression in the VPCs is strongly reduced and no overt Lag phenotype occurs. Instead, cold-sensitive vulval and egg-laying defects, reminiscent of phenotypes seen in lin-12 hypomorphs, are found. Autoregulation of lag-1, therefore, appears to contribute to the robustness of LIN-12 cell fate specification in response to stochastic environmental and genetic perturbations. Under adverse environmental conditions, C. elegans enter a state of diapause in which they form dauer larvae, which are long-lived and stress-resistant. The VPCs of dauer larvae remain developmentally arrested indefinitely until favorable conditions are reintroduced. Experimentally, this arrest can be relieved by depletion of the Forkhead transcription factor DAF-16. I show that expression of the components of the LIN-12/SEL-8/LAG-1 ternary complex are downregulated during the L2d-dauer molt (prior to dauer entry) and that this downregulation is not relieved by DAF-16 depletion. Instead, DAF-16 depletion leads to resumption of LIN-12 signaling and expression of ternary complex only in completely formed dauer larvae. These observations suggest that DAF-16 is required for the maintenance but not the initiation of blocking LIN-12 signaling. The components of the ternary complex are required to effect LIN-12 signaling. This work contributes to better understanding how these components are regulated and how their expression can affect LIN-12 -mediated cell fate decisions.

The specification and development of nervous system diversity is a driving question in the field of Neurobiology. The overarching goals of the projects described in this thesis are to describe tools to aid in the description of nervous system development and to show the use of the described tools to study nervous system development in the nematode Caenorhabditis elegans. The first chapter of this thesis describes a complete map of the male C. elegans nervous system using a tool developed in the lab to uniquely label all neurons in the C. elegans nervous system, NeuroPAL. The second chapter of this thesis largely focuses on a well-studied homeobox gene, unc-86, and its role in fate transformations in dopaminergic and GABAergic neuron types. These two seemingly disparate projects are united in their effort to investigate nervous system development and neuronal fate determination. NeuroPAL is a multicolor transgene that uniquely labels all neurons of the C. elegans hermaphrodite nervous system and here I show it can be used to disambiguate all 93 neurons of the male-specific nervous system. I demonstrate the wide utility of NeuroPAL to visualize and characterize numerous features of the male-specific nervous system, including mapping the expression of gfp-tagged reporter genes and neuron fate analysis. NeuroPAL can be used in combination with any gfp-tagged reporters to unambiguously map the expression of any gene of interest in the male, or hermaphrodite, nervous system. Furthermore, NeuroPAL is used in mutants of several developmental patterning genes to confirm previously described defects in neuronal identity acquisition. Additionally, I show that NeuroPAL can be used to uncover novel neuronal fate losses and identity transformations in these mutants because of the unique labeling of every neuron. Lastly, we show that even though the male-specific neurons are generated throughout all four larval stages, the neurons only terminally differentiate in the fourth and final larval stage, termed ‘just-in-time’ differentiation. In the second part of this thesis, I describe a few examples of mutant analysis of homeobox gene family members and describe their function in the C. elegans nervous system. I focus largely on a couple potential examples of homeotic fate transformations in mutants of the POU homeobox gene, unc-86. In unc-86 mutants, I describe the ectopic expression of multiple GABAergic terminal identity features in one cell in the head of C. elegans. I raise the hypothesis that this cell may be a transformation of a non-GABAergic ring interneuron, RIH, into that of its GABAergic sister cell, AVL, in unc-86 mutants. While ectopic dopaminergic neurons were previously described in unc-86 mutants, I expand the study to show the ectopic expression of all dopaminergic synthesis and packaging genes. I show support that all non-dopaminergic anterior deirid neurons, ADA, AIZ, FLP, and RMG, lose the expression of some of their wild type terminal fate genes and transform to a fate like that of their dopaminergic sister cell, ADE, as assessed by NeuroPAL expression. Taken together, these studies describe tools and methods for studying nervous system development as well as describe many examples of cell fate transformations.

Cytokinesis is defined as the physical division of one cell into two and occurs at the end of the cell cycle. Gestation and development are defined by dividing cells; as an organism develops, cells must duplicate their genetic material, divide, and form two daughter cells. This process is fundamental to all life on our planet. Here, I present work that builds upon our understanding of cytokinesis, focusing on the differential requirements for cytokinesis in different cell types in the early C. elegans embryo, specifically, the P2 cell of the 4-cell embryo. The textbook view of cytokinesis is that all animal cells divide using the same molecular machinery. Yet, growing evidence supports both cell type-specific regulation of cytokinesis and cell type-specific consequences for cytokinesis failure. The 4-cell C. elegans embryo is a powerful model for studying cell type-specific differences in cytokinesis as the cells are already programmed to form distinct cell linages, and previously, we identified cell type-specific regulation of cytokinesis at the 4-cell stage. We weakened the contractile ring using a temperature sensitive (ts) diaphanous formin/CYK-1 mutant. Under this condition, the two anterior cells (ABa and ABp) always failed in cytokinesis, whereas the two posterior cells (EMS and P2) divided successfully at a high frequency, even without detectable F-actin in the cell division plane. Here we focus on the cell type-specific protection of cytokinesis in the P2 germ precursor cell, required to produce all gametes in the adult worm. Using a candidate-based RNAi mini-screen to identify genes required for protection of P2 cytokinesis in the formin(ts) embryos, we identified members of the CCCH Zn2+-finger protein family that are enriched in P2 and required for proper germ cell fate specification. Depletion of MEX-1, PIE-1, or POS-1 led to loss of cytokinetic protection and P2 cytokinesis failure in formin(ts) mutants, but not in control embryos. While depletion of MEX-1 affected multiple cell types, PIE-1 and POS-1 acted exclusively in the P2 cell. Further analysis revealed these germ fate regulators protect cytokinesis by preventing excessive accumulation of septin/UNC-59 and its binding partner, anillin/ANI-1, on the cell cortex in the P2 cell division plane, both negative regulators of actomyosin constriction during cytokinesis in many contexts. We further found that co-depletion of septin and PIE-1 was sufficient to both reduce anillin levels at the P2 division plane and restore cytokinetic protection of P2 in formin(ts) mutant embryos. Thus, germ fate specification protects the P2 germ precursor cell from cytokinesis failure upon damage to the actin cytoskeleton at least in part by controlling the levels of septin and anillin at the division plane.