Cellular networks are required for a variety of processes in complex organisms. Caenorhabditis elegans is a useful model to gain insight into the gene regulatory networks that assemble cellular networks. Mutations in a variety of genes can affect the sinusoidal locomotive pattern of C. elegans. We isolated the mutant jd1500 from a standard genetic screen looking for mutants in C. elegans that exhibit asymmetric locomotive patterns. The two aims of this study were to: 1) identify the gene and characterize its role in the gene regulatory network and 2) characterize the cells affected by the mutation. We reasoned that jd1500 likely disrupts the proper balance between dorsal and ventral body wall muscle contractions. By using three-point genetic mapping, we predicted the locus of jd1500 between -9.42 and -11.73 centimorgans of the X chromosome. Our results implicate the embryonic, cholinergic DB motor neurons as likely cellular targets of the mutation.

Doctor of Philosophy / Department of Biology / Michael Herman / Soil nematode communities are important components of the micro fauna in grassland ecosystems and their interaction with soil microbes affects important ecological processes such as decomposition and nutrient recycling. To study genetic mechanisms underlying ecologically important traits involved in the response of nematode communities to soil microbes, we employed genomic tools available for the model nematode, Caenorhabditis elegans. Previous work identified 204 C. elegans genes that were differentially expressed in response to growth on four different bacteria: Bacillus megaterium, Pseudomonas sp., Micrococcus luteus and Escherichia coli. For many of the genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life-history traits in those environments. Mutations can have differential effects on fitness in variable environments, which can influence their maintenance in a population. Our fitness assays revealed that bacterial environments had varying magnitude of stress, defined as an environment in which the wild-type has a relatively low fitness. We performed fitness assays as part of a comprehensive analysis of life history traits on thirty five strains that contained mutations in genes involved in the C. elegans response to E. coli, B. megaterium, Pseudomonas sp. We found that many of the mutations had conditionally beneficial effects and led to increased fitness when nematodes bearing them were exposed to stressful bacteria. We compared the relative fitness of strains bearing these mutations across bacterial environments and found that the deleterious effects of many mutations were alleviated in the presence of stressful bacteria. Although transcriptional profiling studies can identify genes that are differentially regulated in response to environmental stimuli, how the expressed genes provide functional specificity to a particular environment remains largely unknown. We focused on defense and metabolism genes involved in C. elegans-bacterial interactions and measured the survivorship of loss-of-function mutants in these genes exposed to different bacteria. We found that genes had both bacteria-specific and bacteria-shared responses. We then analyzed double mutant strains and found bacteria-specific genetic interaction effects. Plasticity in gene interactions and their environment-specific modulation have important implications for host phenotypic differentiation and adaptation to changing environments.

A central question of developmental neurobiology is how the extraordinary variety of cell types in the nervous system is generated. A large body of evidence suggests that transcription factors acting as terminal selectors control cell fate determination by directly activating cell type-specific gene regulatory programs during neurogenesis. Neurons within the same class often further differentiate into subtypes that have distinct cellular morphology, axon projections, synaptic connections, and neuronal functions. The molecular mechanism that controls the subtype diversification of neurons sharing the same general fate is poorly understood, and only a few studies have addressed this question, notably the motor neuron subtype specification in developing vertebrate spinal cord and the segment-specific neuronal subtype specification of the peptidergic neurons in Drosophila embryonic ventral nerve cord. In this dissertation, I investigate the genetic basis of neuronal subtype specification using the Touch Receptor Neurons (TRNs) of Caenorhabditis elegans. The six TRNs are mechanosensory neurons that can be divided into four subtypes, which are located at various positions along the anterior-posterior (A-P) axis. All six neurons share the same TRN fate by expressing the POU-domain transcription factor UNC-86 and the LIM domain transcription factor MEC-3, the terminal selectors that activate a battery of genes (referred as TRN terminal differentiation genes) required for TRN functions. TRNs also have well-defined morphologies and synaptic connections, and therefore serve as a great model to study neuronal differentiation and subtype diversification at a single-cell resolution. This study primarily focuses on the two embryonically derived TRN subtypes, the anterior ALM and the posterior PLM neurons; each contains a pair of bilaterally symmetric cells. Both ALM and PLM neurons have a long anteriorly-directed neurite that branches at the distal end; the PLM, but not the ALM, neurons are bipolar, having also a posteriorly-directed neurite. ALM neurons form excitatory gap junctions with interneurons that control backward movement and inhibitory chemical synapses with interneurons that control forward movement, whereas PLM neurons do the reverse. Therefore, the clear differences between ALM and PLM neurons offer the opportunity to identify the mechanisms controlling subtype specification. Using the TRN subtypes along the A-P axis, I first found that the evolutionarily conserved Hox genes regulate TRN differentiation by both promoting the convergence of ALM and PLM neurons to the common TRN fate (Chapter II) and inducing posterior subtype differentiation that distinguishes PLM from the ALM neurons (Chapter III). First, distinct Hox proteins CEH-13/lab/Hox1 and EGL-5/Abd-B/Hox9-13, acting in ALM and PLM neurons respectively, promote the expression of the common TRN fate by facilitating the transcriptional activation of TRN terminal selector gene mec-3 by UNC-86. Hox proteins regulate mec-3 expression through a binary mechanism, and mutations in ceh-13 and egl-5 resulted in an “all or none” phenotype: ~35% of cells lost the TRN cell fate completely, whereas the rest ~65% of cells express the TRN markers at the wild-type level. Therefore, Hox proteins contribute to cell fate decisions during terminal neuronal differentiation by acting as reinforcing transcription factors to increase the probability of successful transcriptional activation. Second, Hox genes also control TRN subtype diversification through a “posterior induction” mechanism. The posterior Hox gene egl-5 induces morphological and transcriptional specification in the posterior PLM neurons, which distinguish them from the ALM. This subtype diversification requires EGL-5-induced repression of TALE cofactors, which antagonize EGL-5 functions, and the activation of rfip-1, a component of recycling endosomes, which mediates Hox activities by promoting subtype-specific neurite outgrowth. Thus, these results suggest that neuronal subtype diversification along the A-P axis is mainly driven by the posterior Hox genes, which induces the divergence of posterior subtypes away from the common state of the neuron type. I have also performed an RNAi screen to identify novel regulators of the TRN fate and identified the LIM domain-binding protein LDB-1 and the Zinc finger homeodomain transcription factor ZAG-1 as part of the regulatory network that determines TRN fate (Chapter IV). LDB-1 binds to and stabilizes MEC-3 and is also required for the activation of TRN terminal differentiation genes by MEC-3. ZAG-1 promotes TRN fate by preventing the expression other transcription factors EGL-44 and EGL-46, which inhibits the expression of TRN fate by competing for the cis-regulatory elements normally bound by the TRN fate selectors UNC-86/MEC-3. The mutual inhibition between ZAG-1 and EGL-44 establishes a bistable switch that regulates cell fate choice between TRNs and FLP neurons. I also investigated the genetic basis of neuronal morphogenesis using TRNs. By conducting a forward genetic screen searching for mutants with TRN neurite outgrowth defects, I identified a series of genes required for axonal outgrowth and guidance in TRNs. Following a few genes identified from the screen, genetic studies have revealed two novel mechanisms for neuritogenesis. First, Dishevelled protein DSH-1 attenuates the strength of Wnt signaling to allow the PLM posterior neurite to grow against the gradient of repulsive Wnt proteins, which are enriched at the posterior side of PLM cell body and normally repel the axons toward the anterior (Chapter V). Second, guanine nucleotide exchange factor UNC-73 and TIAM-1 promotes anteriorly and posteriorly directed neurite outgrowth, respectively; and outgrowth in different directions can suppress each other by competing for the limited neurite extension capacity (Chapter VI). As side projects, I performed mRNA expression profiling using isolated and separated populations of in vitro cultured ALM and PLM neurons and identified hundreds of genes differentially expressed between the two subtypes (Appendix I). I have also studied subtype differentiation of the VC motor neurons in the ventral nerve cord of C. elegans and discovered a mechanism by which histone modification patterns the expression of subtype-specific genes during terminal neuronal differentiation (Appendix II). In summary, my doctoral research established a framework for the study of neuronal subtype specification using the C. elegans TRNs and uncovered the genetic mechanisms for a variety of aspects of terminal neuronal differentiation. By investigating the generation of neuron type and subtype diversity in a well-defined model organism, my study provides novel insights for understanding the development of the nervous system.

The plasticity of cells in a multicellular organism is progressively lost during differentiation. This loss is reflected in studies involving the ectopic misexpression of fate-specifying or terminal selector transcription factors (TFs). These TFs can efficiently activate target genes in undifferentiated cells, but lose this ability as cells differentiate. While this phenomenon of cell fate restriction is widely observed, the mechanisms orchestrating it are poorly understood. In this thesis, I have used the ubiquitous overexpression of Zn-finger-TF CHE-1 as a tool to understand the mechanisms that restrict cell fate in Caenorhabditis elegans. When CHE-1 is ubiquitously expressed at embryonic stages, it activates target gene expression in many cell types, while in adults it can only act in a few neurons. To uncover factors that inhibit plasticity of all other adult cells, I first performed an RNAi screen against chromatin-associated factors. Using this approach I found that the removal of either the PRC2 complex, which deposits the H3K27me3 mark, or loss of proteins that indirectly regulate domains of H3K27me3, allows CHE-1 and two other terminal selector TFs to activate target genes in the germline. These data show that the correct distribution of H3K27me3 is crucial for the restriction of germ cell fate. I next took a candidate approach to identify genes that regulate fate restriction in other cell types. We hypothesized that terminal selector TFs themselves, in addition to specifying cellular identity by controlling large gene sets, may also act to inhibit plasticity. To test this, I first assayed the activity of CHE-1 in mutants of COE-TF unc-3, the terminal selector for a subset of cholinergic motor neurons (MNs). I found that in contrast to wildtype MNs, unc-3 mutant MNs remain plastic as CHE-1 can induce expression of target genes in these cells even at the adult stage. This phenotype is also observed in four of six additional terminal selector mutants tested. I further found that the removal of met-2, a protein required for H3K9 methylation, or mes-2, a PRC2 component, also makes differentiated cholinergic MNs amenable to the activity of CHE-1. Preliminary evidence suggests that met-2 may act in the same pathway as unc-3. These results raise the exciting possibility that selector TFs play a role in restricting cell fate by organizing the heterochromatin domains in differentiated cells. Overall, in this work I provide functional evidence to show that specific chromatin-modifying enzymes restrict the fate of germ cells and that both fate-specifying TFs and chromatin-modifying enzymes are required for the fate restriction in neurons.

A common organizational feature of any nervous system is the existence of groups of neurons that share a set of common traits but that can be further divided into individual neuron types and subtypes. Understanding the mechanistic basis of neuron type and subtype diversification processes will constitute a major step toward understanding brain development and evolution. In this dissertation, I have explored the mechanistic basis for the specification of motor neuron classes in the nematode C. elegans which serves as a paradigm for neuron diversification processes. Cholinergic motor neurons in the C. elegans ventral nerve cord share common traits, but are also comprised of many distinct classes, each characterized by unique patterns of effector gene expression (e.g. motor neuron class-specific ion channels, signaling molecules, and neurotransmitter receptors). Both the common as well as class-specific traits are directly activated by the terminal selector of cholinergic motor neuron identity, the EBF/COE-like transcription factor UNC-3. Via forward genetic screens to identify mutants that are defective in class specification, I have discovered that the diversification of UNC-3/EBF-dependent cholinergic motor neurons is controlled by distinct sets of phylogenetically conserved, motor neuron class-specific transcriptional repressors. One such repressor is in fact a novel gene previously uncharacterized in C. elegans or any nervous systems and is now named bnc-1. By molecularly dissecting the cis-regulatory region of effector genes, I found that the repressor proteins prevent UNC-3/EBF from activating class-specific effector genes in specific motor neuron subsets via discrete binding sites that are adjacent to those of UNC-3/EBF. And by using CRISPR/Cas9-mediated genome engineering to tag repressor proteins with inducible degrons, I demonstrate that these repressors share the important feature of being continuously required throughout the life of the animal to counteract, in a class-specific manner, the function of the UNC-3/EBF terminal selector that is active in all motor neuron classes. I propose that the strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a neuron subtype-specific manner may constitute a general principle of neuron subtype diversification.

Cellular quiescence, a reversible state of cell-cycle exit, and developmental potential, the ability to differentiate into appropriate cell types, are properties essential for normal development and stem cell function (reviewed in (Cheung and Rando, 2013; Fiore et al., 2018; Mihaylova et al., 2014). Understanding the mechanisms by which cells maintain quiescence has important implications for developmental biology, as this reversible state of cell-cycle exit is a key attribute of stem cells, as well as for cancer biology, as quiescence plays a key role in tumor dormancy and metastasis. Environmental conditions are key in regulating whether stem cells maintain quiescence or exit to resume divisions and developmentally progress. I aim to investigate how the properties of quiescence and developmental potential are retained over long periods of time and how they are appropriately regulated by external environmental inputs. The nematode Caenorhabditis elegans is an excellent model for investigating both of these questions because it is capable of entering and maintaining a developmentally arrested state for an unusually long time compared to the normal lifetime of the worm, and because the decision to enter this arrest is regulated entirely by external environmental inputs (Cassada and Russell, 1975). Upon encountering conditions unfavorable for growth, C. elegans enters an alternative, developmentally arrested state called dauer diapause in which precursor cells remain quiescent for months – a period many times the lifespan of a worm grown under favorable conditions (Cassada and Russell, 1975). Maintaining precursors in this arrested state is important in order for the worms to develop normally once conditions improve and requires components of the conserved Insulin/Insulin-like (IIS) signaling pathway (Karp and Greenwald 2013 and this work); of note, the IIS pathway also regulates mammalian quiescence (Eijkelenboom and Burgering, 2013). Canonical regulation of dauer diapause includes IIS, TGFß, and dafachronic acid (DA)/nuclear hormone receptor (NHR) signaling (reviewed in (Murphy and Hu, 2013a)). Here, I investigate how DAF-18, the sole C. elegans ortholog of the tumor suppressor PTEN (Phosphatase and tensin homolog) (Gil et al., 1999; Mihaylova et al., 1999; Ogg and Ruvkun, 1998; Rouault et al., 1999), maintains quiescence in dauer through regulation of these conserved signaling pathways using the C. elegans gonad as a model. The gonad is composed of somatic cells and the germline. Both the somatic gonad and germline develop post-embryonically from precursor cells present when dauer arrest occurs, and these precursor cells remain quiescent for the duration of dauer diapause (Cassada and Russell, 1975; Hong et al., 1998; Narbonne and Roy, 2006). After exit from dauer, division and differentiation resume. DAF-18/PTEN is required for germline quiescence during dauer diapause (Narbonne and Roy, 2006), and my results implicate DAF-18/PTEN in the control of quiescence of the somatic tissues as well, including the somatic gonad. In this role, DAF-18/PTEN activity in the somatic gonad non-autonomously coordinates both germline stem cell (GSC) and somatic gonad blast (SGB) quiescence. I have demonstrated this somatic gonad focus through mosaic analysis, tissue-specific rescue, and tissue-specific excision mosaics. We propose that DAF-18/PTEN mediates production of a signal promoting quiescence from the somatic gonad to the SGBs and GSCs and that this signal does not absolutely require or solely target the IIS, TGFß, or DA/NHR signaling pathways normally implicated in regulation of dauer diapause.

The nematode Caenorhabditis elegans is an alternative model used in biomedical research for the investigation of descriptive and mechanistic toxicity assessment of chemicals. There are considerable differences in published data, especially in terms of reproducibility and validation of toxicity endpoints, and the techniques used in the investigation of these endpoints. This thesis describes the evaluation of toxicological endpoints following the exposure of C. elegans to chemicals which include; zinc oxide nanoparticles (ZnONP), Diethylstilbestrol (DES) and derivatized target-specific anthelmintics. The results suggest that ZnONP prepared in anionic and cationic dispersants (AZNP and CZNP respectively) were the most toxic against the nematode resulting in the ‘bag of worms' (BOW) phenotype which can be exploited as a marker for reproductive toxicity. Also, worms treated with ZnONP prepared in 0.1% FBS (FZNP), molecular grade water (WZNP) or E. coli OP50 supernatant (SZNP) presented three-fold embryo elongation showing fully differentiated tissues encapsulated within the eggshell and still within the hermaphrodite gravid adult. The phenotype has been named accelerated embryonic development (AED) and could be used as a developmental toxicity endpoint. The results suggest that the AED endpoint is the most sensitive while lethality endpoint appears to be the least sensitive despite its extensive use in the literature. Also, microRNA microarray expression appears to be the most sensitive molecular endpoint while behavioural endpoints such as speed should be interpreted with caution, especially when performed manually. Importantly, good C. elegans culture practice (GCeCP) is required for reproducible chemical toxicity assessment and different endpoints may be required for different types of toxicity assessment. Additionally, the thesis describes a second but related study which explores a potential for enhanced anthelmintic targeting. Novel fluorophore-based asparagine-containing oligopeptide substrate probes were used to target the helminth protease, legumain. These probes were selectively cleaved by legumain in C. elegans, Haemonchus contortus and Teladorsagia circumcincta. The protease-specific probes could potentially be exploited to achieve protease-mediated prodrug activation and drug delivery.

Animals rely on the integration of a variety of external cues to understand and respond appropriately to their environment. The relative amounts of food and constitutively secreted pheromone detected by the nematode C. elegans determines how it will develop and grow. Starvation conditions cause the animal to enter a protective stage, termed dauer. Dauer animals are non-eating, long-lived and stress resistant. Yet, when these animals are introduced to food replete conditions they will recover from dauer and proceed into normal development. Furthermore, food restriction has been demonstrated to extend the lifespan of a wide-range of species including C. elegans. However, the exact mechanism by which food signals are detected and transduced by C. elegans to influence development and longevity remains unknown. Here, we identify a G protein-coupled receptor (GPCR) DCAR-1 that acts in two chemosensory neurons to mediate food signaling in an autophagy-related manner. The DCAR-1 ligand Dihydrocaffeic acid (DHCA) competes with dauer-inducing pheromone to promote growth. DHCA is a key intermediate in the shikimate pathway, which is required to synthesize folate and aromatic amino acids. We report that dcar-1 mutations influence dauer formation and extend wildtype lifespan via a mechanism of dietary restriction. Moreover, we show that the lifespan extension of dcar-1 mutants is completely dependent on autophagy gene atg- 18. Furthermore, our data suggests metabolites derived from shikimate are food signals that control aging and dauer development through GPCR signaling in C. elegans. These studies will contribute to the delineation of mechanisms behind the beneficial effects of dietary restriction in eukaryotic organisms. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Dauer larva formation in C. elegans is a life-history polyphenism that relies on the function of several pathways, including insulin, TGFβ and nuclear hormone receptor signaling. The downstream effectors of these pathways, DAF-16/FOXO, DAF-3/Co-Smad and DAF-12/VDR, are transcription factors (DAF TFs) with broad or ubiquitous expression patterns, null mutations in which result in the inability to form dauers regardless of environmental conditions. In preparation for the dauer diapause, all tissues of the worm undergo extensive morphological and functional remodeling in a coordinated manner. The broad goal of my thesis is to understand how these transcription factors act in different tissues of the worm to regulate the dauer-specific tissue remodeling and gene expression changes. In addition to characterizing dynamic expression pattern of chemosensory GPCR genes in dauer, which revealed an additional layer of plasticity and provided novel entry points to studying remodeling in distinct neuron classes and non- neuronal tissues, I have developed molecular tools – conditional alleles of the daf TFs – that allowed me to address the question of tissue-specificity and cell-autonomy of the DAF TFs in a previously inapproachable way. I have found that DAF TFs act in both cell-autonomous (DAF-16 in neurons, intestine, pharynx) and non-autonomous manner (DAF-16 in the pharynx) to control dauer tissue remodeling. Unlike DAF-16 and DAF-12, the function of DAF-3 in the dauer decision appears to be largely determined by its action in neurons, and specifically in sensory neurons. The three TFs also differ in their roles in pharynx remodeling: while DAF-16 controls dauer pharyngeal morphology and activity both cell-autonomously and non- autonomously, DAF-12 or DAF-3 depletion from pharyngeal muscle does not affect the dauer pharyngeal phenotypes. Yet, all three TFs are required continuously throughout all tissues to maintain the dauer state, once the decision to enter dauer has been made. This work is a first attempt to characterize tissue-specific roles of all transcriptional effectors of the dauer pathways in a systematic way, and contributes to a fundamental understanding of a polyphenic developmental switch regulated by highly conserved molecular pathways.