<p> ABSTRACT The nervous system comprises more diverse and intricately specialized cell types than any other tissue in the body. Understanding the developmental mechanisms that generate cellular diversity in the nervous system is a major challenge in neuroscience. The nematode <i>C. elegans</i> offers the opportunity to study neuronal development at the molecular level with extraordinary resolution.</p><p> My dissertation focuses on the elucidation of genetic mechanisms required for the proper development of the chemosensory BAG neurons, which are specialized for detecting the respiratory gas carbon dioxide (CO₂). Analogs of these neurons play diverse roles in animals from different phyla. CO₂-sensing neurons in the mammalian brainstem are critical regulators of the respiratory motor program, and their dysfunction has been linked to fatal apneas such as Sudden Infant Death Syndrome. In nematodes, CO₂-sensing neurons mediate an avoidance behavior, but their ethological function was not known.</p><p> In my initial studies of BAG neuron development, I demonstrated that a conserved ETS-family transcription factor directly regulates genes required for CO₂-sensing, including the receptor-type guanylate cyclase, GCY-9, which likely functions as a CO₂ receptor. To uncover other genes that function together with <i>ets-5,</i> I carried out a large-scale chemical mutagenesis screen for mutants with improper BAG neuron differentiation. From this screen I identified two new genes required for BAG neuron development: the Pax6 homolog <i>vab-3</i> and the p38 Mitogen-Activated Protein (MAP) kinase <i>pmk-3</i>.</p><p> VAB-3 likely acts during embryonic development to pattern the expression of ETS-5 in head neurons of <i>C. elegans</i>. In loss of function <i> vab-3</i> mutants, ETS-5 protein is misexpressed in hypodermal cells and a motor neuron, in addition to its expression in BAG. VAB-3 likely represses transcription of ETS-5 in some lineages, such as those that give rise to hypodermal cells.</p><p> I next demonstrated that the p38 MAPK PMK-3 functions in a Toll-like receptor (TLR) signaling pathway. This discovery revealed an unexpected role for TLR signaling in neuronal differentiation. Because TLR signaling was known to be required for behavioral responses to microbes, I tested whether BAG neurons were required for pathogen avoidance. I found that this was the case and propose that TLR signaling functions in pathogen avoidance by promoting the development and function of chemosensory neurons that surveil the metabolic activity of environmental microbes.</p><p> Because ETS-5, VAB-3 and TOL-1 are members of gene families that are conserved between nematodes and vertebrates, a similar mechanism might act in the specification and differentiation of CO₂-sensing neurons in other phyla.</p>

<p> Na⁺/Ca²⁺ exchangers are low affinity/high capacity transporters that mediate Ca²⁺ extrusion by coupling Ca²⁺ efflux to the influx of Na⁺ ions. Their primary function is to maintain Ca²⁺ homeostasis in cells of all organisms and they play a particularly important role in excitable cells that experience transient Ca²⁺ fluxes. While their functions have been studied extensively in muscle cells, much is still unknown about their contributions to the nervous system. Data suggests that Na⁺/Ca²⁺ exchangers play a key role in neuronal processes such as memory formation, learning, oligodendrocyte differentiation and axon guidance. They are also implicated in pathologies such as Alzheimer&rsquo;s Disease, Parkinson&rsquo;s Disease, Multiple Sclerosis and Epilepsy. While they are implicated in critical neuronal processes, a clear understanding of their mechanism remains unknown. This dissertation examines the role of Na⁺/Ca²⁺ exchangers in the invertebrate model organism <i>Caenorhabditis elegans </i>. There are ten identified Na⁺/Ca²⁺ exchanger genes in <i>C. elegans</i> (labeled <i>ncx-1</i> to <i>ncx</i>-10). Data presented here is the first comprehensive description of their genetics and function in <i>C. elegans</i>. The expression pattern of all 10 Na⁺/Ca²⁺ exchanger genes is described and their phylogeny is examined comparatively across humans and flies. Analysis of <i>ncx-2</i> and <i>ncx-8</i> mutants shows important roles for Na⁺/Ca²⁺ exchanger genes in egg-laying, lipid storage and longevity, suggesting a role in diverse biological functions for Na⁺/Ca²⁺ exchangers in <i>C. elegans</i>. The function of an NCLX type Na⁺/Ca²⁺ exchanger NCX-9 is also detailed comprehensively. Analysis of <i> ncx-9</i> mutants shows that NCX-9 is required for asymmetrical axon guidance choices made by the DD and VD GABAergic motor neuron circuit. Pathway analysis shows that NCX-9 regulates asymmetric circuit patterning through RAC-dependent UNC-6/Netrin signaling and LON-2/Glypican Heparan Sulfate signaling. <i> In vitro</i> analysis of NCX-9 physiology in HEK cells shows that NCX-9 is a mitochondrial Na⁺/Ca²⁺ exchanger, similar to NCLX, which is its homolog in humans.</p>

<p>We propose to establish a free-living, fresh water flatworm species from the superphylum Lophotrochozoa, Schmidtea mediterranea, to be a genetic model system. S. mediterranea has been vigorously investigated as a powerful system to study adult stem cells and organ regeneration. Its sexual biotype has also been established as a system to understand the inductive mode of germ cell formation that is broadly shared by a lot of species including mammals. However, little is known about the sexual reproduction and genetics in this organism, which limited the availability of genetic approaches. As the sexual planarian is found scattered but with relative abundance in Sardinia, its natural history also presents us the opportunity to study inbreeding?s effect on genetic variability and species survival. Therefore, to study sexual reproduction and genetic inheritance in S. mediterranea will provide us unique opportunities to understand whole body regeneration, inductive germ cell formation, and inbreeding. In this dissertation, progresses in the establishment of the foundation for genetic analysis in S. mediterranea were presented. Though a simultaneous hermaphrodite, the anatomical and genotyping studies concluded that S. mediterranea cross-fertilize. One worm (line S2) was inbred for 10 generations by taking one progeny from each generation and crossing this individual to its regenerated clones. Whole genome sequencing of four different generations in this inbreeding pedigree revealed ~300Mb of the genome maintained their heterozygosity. Further sequencing analysis of the male and female gametes found these regions had low recombination rates, and maintained as two haplotypes (J-/V- haplotypes). Failure of gametes of the same haplotype to form progeny is unlikely due to embryonic lethality as the arrested embryos were significantly less than hatchlings. Additional analysis of two lines (D5D/D5I) with 90% of these regions homozygous as the J-haplotype suggested failure in fertilization between gametes of the same haplotype. Hence, we propose that haplotype incompatibility is the driving mechanisms to maintain genome heterozygosity in the planarian genome. Understanding of the genetic strategies in S. mediterranea will help the development of genetic approaches to study regeneration and germ cell specification. Our findings also suggest S. mediterranea can be a model system to study the evolution of sex and gamete incompatibility.

<p> Skeletal muscle generates contractile forces that allow the body to execute movements for walking, speaking and breathing. Although we understand a great deal about the steps of muscle formation, the mechanisms that control muscle size are poorly understood. Even less is known about how muscles interact with skeletal elements, including connective tissue, tendon and bone. This dissertation describes a novel role for Abelson tyrosine-protein kinase 2, a non-receptor tyrosine kinase, during muscle development. First, I characterize the defects in skeletal muscle of <i>abl2</i> mutant mice and show that muscle fibers in the diaphragm and other muscles are extraordinarily long in <i>abl2</i> mutant mice. As a consequence of expansion of the diaphragm muscle, the central tendon of the diaphragm is proportionally reduced in size. Second, I demonstrate that <i>abl2</i> controls muscle size by regulating myoblast proliferation. Third, I show that Abl2 acts in myoblasts to attenuate their proliferation, thereby limiting myoblast fusion and muscle fiber size. Fourth, I show that the exercise endurance of <i> abl2</i> mutant mice is diminished, likely due to the compensatory reduction in size of the diaphragm central tendon. Finally, I provide evidence for signaling between muscle cells and tendon cells that induces tendon cell differentiation. </p><p>

One of the many ways that the ubiquitously expressed Polycomb Group protein, Yin-Yang1 (Yy1), is believed to regulate gene expression is through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and the Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, Yy1 was deleted in the epiblast of mouse embryos using Sox2-Cre. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but the primitive streak fails to extend anteriorly. Migration through the streak is severely impaired, and streak descendants fail to downregulate E-Cadherin resulting in an aberrant accumulation of streak cells. Intriguingly, we find an accumulation of Nodal and a concomitant reduction of Nodal antagonists suggesting that YY1 is normally required for proper Nodal regulation. We have observed that definitive endoderm is specified but fails to properly delaminate into the outer layer and mutant embryos also fail to accumulate any axial midline structure. Although anterior neuroectoderm is clearly specified, mesoderm specification is severely restricted. Our results reveal critical requirements of YY1 in several important developmental processes, including epithelial to mesenchymal transition (EMT), Nodal regulation and PRC2 mediated H3K27Me3 of the inactive X-chromosome. Despite the localization of Oct4 and Sox2 transcripts in the neuroectoderm of the Yy1 epiblast cKO and the presence of stable transcripts of both genes in the Yy1 RNAi knock down blastocyst, both proteins are void in these models and Oct4 protein is absent in the peri-implantion Yy1 KO mouse. We believe YY1 is required for stabilization of the Oct4/Sox2 pluripotency complex in vivo. We have identified two endogenous forms of YY1 and we believe these posttranslational modifications of YY1 permit the protein to perform the diverse activities it performs in vivo. For example, in addition to the roles in transcriptional regulation and protein complex stabilization, we have also observed a role in YY1 in epigenetic regulation in vivo, including deposition of histone 3 lysine 27 trimethylation (H3K27Me3) on the inactive X-chromosome in female embryos and a role in imprinted gene expression of the Dlk1/Dio3 locus. Detailed analysis of the peri-implantation lethal Yy1 KO mouse in utero revealed unexpected novel developmental events. A large scale follow up examination of wildtype implantation primarily through analysis of immunohistochemical data and gene expression profiling at the cellular level. We analyzed expression patterns of important developmental genes including Oct4, Sox2, Nanog, Cdx2, Gata6 and Sox17, as well as markers of epithelial biogenesis including ZO1, E-Cadherin and Laminin. Interestingly we identified consistent variances in cell populations within the ICM as well as likely primitive endoderm progenitors that produce Laminin and first appear at the periphery of the ICM. We also identified a novel upregulation of Sox17 specifically at the site of implantation. With these data we compose a staging diagram of peri-implantation embryonic and maternal changes during the elusive window of development. These results are the first to elucidate the role of YY1 during gastrulation and peri- implantation, providing potential in vivo targets of YY1 and highlighting the diversity of function of YY1 in the early embryo. Additionally we have been able to advance molecular knowledge of peri-implantation development, in order to provide a platform from which to analyze other peri-implantation lethal KO mice, as well as to aid biomedical understanding of implantation and implantation failure in mammals.

<p> Jun N-terminal kinase (JNK) signaling is a key modulator of development and disease in all multicellular organisms. One process in which the consequences of both gain and loss of JNK signaling can be monitored is embryonic dorsal closure (DC) in the fruit fly, <i>Drosophila melanogaster.</i> DC occurs midway through embryogenesis; it is the process by which the lateral epidermis expands bilaterally to meet and fuse at the dorsal midline, thereby encasing the entire embryo in epidermis. JNK signaling in leading edge (LE) cells (the dorsal-most row of epidermis) initiates closure. My studies of a novel but conserved JNK signaling antagonist, Raw, have provided several unique insights into: 1) Jun function as a component of the AP-1 transcription factor, and 2) the role of the epidermis as a signaling template mediating development of the epidermis and adjacent tissues.</p><p> My graduate work has built upon the demonstration that <i>raw</i> is required to prevent promiscuous JNK signaling in the embryonic epidermis just prior to DC. I have shown that <i>raw</i> is necessary for proper accumulation of Jun in LE cells required to define the LE, which functions as a signaling center required for epidermal closure as well as for underlying heart development. I have gone on to show that Jun accumulates at previously unrecognized sites in the embryonic epidermis, including tracheal pits and solitary epidermal cells lying directly above the peripheral nervous system (PNS). Jun activity is required for tracheal and nervous system defects observed in mutants of two JNK signaling antagonists, <i>raw</i> and <i> rib,</i> and indicates that cell signals within and to an adjacent tissue are integral to proper development. I have found that the epidermis plays an instructive role during development, and results from my work have led to insights into how JNK signaling centers in the epidermis coordinate morphological processes.</p><p> As Raw is a novel but conserved JNK signaling antagonist, I have built and tested models of its molecular mechanism of action as well. Bolstering conclusions of previous studies of mammalian c-Jun in cell culture, my data indicate that N-terminal phosphorylation is not an on/off switch, but rather it increases Jun stability for its activity as a component of the AP-1 transcription factor. <i>raw</i> mutants exhibit both high levels of Jun protein and an accumulation of phosphorylated Jun (P-Jun), and my data point to a role for Raw in effecting the Jun:P-Jun ratio via mediation of Jun degradation. In deciphering the mechanism of Raw function, we are gaining significant new insights into previously unrecognized mechanisms of JNK signaling regulation. Understanding these mechanisms will be important for dissecting the etiology of developmental abnormalities and diseases, such as cancer, which hinge on the Goldilocks effect, having just the right amount of signaling at just the right time.</p>

Complex multicellular organisms are composed of at least 200 cell types, which contain the same DNA "black box" of genetic information. It is the precise regime according to which they express their genes, exquisitely controlled by gene regulatory circuits, that defines their cellular identity, morphology and function. We have developed an in vivo biotinylation method that uses genetically encoded components in zebrafish, termed biotagging, for genome-wide regulatory analysis of defined embryonic cell populations. By labelling selected proteins in specific cell types, biotagging eliminates background inherent to analyses of complex embryonic environments via highly stringent biochemical procedures and targeting of specific interactions without the need for cell sorting. We utilised biotagging to characterise the in vivo translational landscape on polysomes as well as the transcriptional regulatory landscape in nuclei of migratory neural crest cells, which intermix with environing tissues during their migration. Our migratory neural crest translatome presented both known and novel players of the neural crest gene regulatory network. An in depth look into the active nuclear transcriptome uncovered a complex world of non-coding regulatory RNAs that potentially specify migratory neural crest identity and present evidence of active bidirectional transcription on regions of open chromatin that include putative cis-regulatory elements. Analysis of our transcribed cis-regulatory modules functionally links these elements to known genes that are key to migratory neural crest function and its derivatives. We also identified a novel cohort of circular RNAs enriched at regions of tandem duplicated genes. Last but not least, we recovered developmentally regulated long non-coding RNAs and transcribed transposable elements. To functionally dissect the biological roles of these factors, we have built two Ac/Ds-mediated in vivo toolkits for efficient screening of putative enhancers and for CRISPR/Cas9-based transcriptional modulation. Overall, our methods and findings present a comprehensive view of the active coding and non-coding landscapes of migratory neural crest on a genome-wide scale that refine the current regulatory architecture underlying neural crest identity.