Function of the adult nervous system relies on the appropriate establishment of neural circuits during embryogenesis. In vertebrates, the neurons that make up motor circuits form in distinct domains along the dorsoventral (DV) axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing the fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. We used CRISPR/Cas9 genome editing technology to generate the first germline mutants for the prdm12 gene and used this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal Mauthner cell-dependent escape response. Using cell culture-based luciferase reporter assays, we demonstrate that Prdm12b acts as transcriptional repressor, most likely by recruiting EHMT2/G9a. We also show that the Bhlhe22 TF binds to the Prdm12b zinc finger domain to form a Bhlhe22:Prdm12b complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate for its loss during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in the formation of the p1 domain – suggesting that prdm12b is not solely required to repress non-p1 fates, but is also needed to promote p1 fates.

Synapse development and injury-induced reorganization in the nervous system have been extensively characterized morphologically, although, relatively little is known regarding the molecular and biochemical events that underlie these processes. In an attempt to better understand, at the molecular level, the role of the expression of synaptic proteins during synapse establishment and regeneration, this dissertation examines the dynamics of expression of the neuron-specific gene synapsin I during development and in response to selective lesions of the rat central nervous system. Synapsin I is the best characterized member of a family of nerve-terminal specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies by Lu et al., (1992) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during development, we have used in situhybridization histochemistry to localize synapsin I mRNA in the rat central and peripheral nervous systems throughout embryonic and postnatal development, and into the adult period. During development, from the earliest embryonic time point examined (E12), the expression of the synapsin I gene was detectable in both the rat central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To precisely determine the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of the cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. In a second phase, in accord with prior analyses, synapsin I gene expression increases to a maximum for a given neuronal population during synapse formation. In the adult rat brain, our data demonstrates a widespread yet regionally variable pattern of expression of synapsin I mRNA similar to that seen at earlier time points, with noteworthy exceptions. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer n neurons of the piriform and entorhinal cortices, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25. Then, to determine specifically how synapsin I mRNA levels are related to levels of synapsin I protein in the adult rat brain, we employed in situhybridization histochemistry and immunohistochemistry to examine in detail the local distribution of both synapsin I mRNA and protein in the hippocampus. In short, these data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus. Based on these data we hypothesized that locally high levels of synapsin I mRNA in neuronal somata may reflect the ability of the nervous system to respond to select enviromental stimuli and/or injury by producing longterm changes in synaptic circuitry. To test this hypothesis and to better understand the regulation and putative role of synapsin I gene expression in the development of functional synapses in the central nervous system, we first examined the developmental pattern of expression of the synapsin I gene; in dentate granule neurons of the dentate gyrus and their accompaning mossy fibers during the main period of synaptogenic differentiation in the rat hippocampus. The results of these studies indicate a significant difference between the temporal expression of synapsin I mRNA in dentate granule cell somata and the appearence of protein in their mossy fiber terminals during the posmatal development of these neurons. Next, to investigate the regulation and putative role of synapsin I gene expression during the restoration of synaptic contacts in the central nervous system, we examined the expression of the synapsin I mRNA and protein following lesions of hippocampal circuitry. These studies show marked changes in the pattern and intensity of synapsin I immunoreactivity in the dendritic fields of dentate granule cell neurons following perforant pathway transection. In contrast, changes in synapsin I mRNA expression in target neurons, and in those neurons responsible for the reinnervation of this region of the hippocampus, were not found to accompany new synapse formation. On a molecular level, both developmental and lesion data suggest that the expression of the synapsin I gene is tightly regulated in the central nervous system, and that considerable changes in synapsin I protein may occur in neurons without concommitant changes in the levels of its mRNA. From a functional standpoint, our results suggest that the appearance of detectable levels of synapsin I protein in developing and sprouting synapses does not reflect simply synaptogenesis, but coincides with the acquisition of function by those central synapses.

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term.

The Hedgehog (Hh) signaling pathway plays many important roles throughout embryonic development, including the regulation of tissue patterning, cell differentiation, proliferation, and apoptosis. The loss of SHH signaling in human development has been shown to cause holoprosencephaly. Conversely, inappropriately activated Shh signaling in adults has been implicated in many cancers. Furthermore, Shh has been found to be a key regulator of neural stem cells in the mammalian brain. To further study the roles of Hh, I have developed a transgenic zebrafish line as a tool to monitor tissues that respond to Hh signaling throughout the vertebrate life-cycle. A number of genes have been identified that are transcriptionally up-regulated by Hh signaling. Transcription of these genes is initiated through binding of activated Gli transcription factors to an identified Gli binding site (GBS) in the cis-regulatory region. This Gli binding site is largely conserved across vertebrate species. I have generated transgene constructs in which 12 GBSs have been placed upstream of a minimum promoter that drives GFP, RFP, or Kaede fluorescent proteins. These plasmid constructs are activated in embryonic regions known to be Hh responsive, such as the ventral CNS. Treatment with cyclopamine eliminates this expression, confirming that these transgenes accurately report an active Hh response. These transgenic lines will be extremely powerful tools for research into the mechanisms by which Hh signaling regulates adult cell types such as neural stem cells. These lines will also be important tools that will help understand how misregulation of Hh signaling can lead to cancer.

The dissertation consists of three major projects with the focus on the immunotoxicity of chromium and the behavior disorders caused by early ETOH exposure respectively. Hexavalent chromium [Cr(VI)] is widely used in various industrial processes and has been recognized as a carcinogen. As the first line of host defense system, the immune system can be a primary target of Cr(VI). T cell population represents a major arm of the immune system that plays a critical role in host anti-tumor immunity. Dysfunction of T cells compromises host anti-tumor immunity resulting in oncogenesis. Using mouse splenic T cells as an in vitro model system, the present study assessed the effects of Cr(VI) on T cell functions, as the first step of our investigation of the mechanism underlying Cr(VI)-inhibited immunosurveillance and carcinogenesis. Our results showed that Cr(VI) decreased the viability of CD4+ and CD8+ T cells, inhibited T cell activation, functions, including cytokine release, and degranulation. Fetal ethanol (ETOH) exposure can damage the developing central nervous system and lead to cognitive and behavioral deficits, known as fetal alcohol spectrum disorders (FASD). The use of animal models, especially mouse models is essential for investigating the neurogenetic mechanism of fetal ETOH effects and screening pharmacotherapies against it, due to the extensive knowledge of mouse genetics. However, the availability of mouse model is limited. Via adopting various dosage, timing and administration routes of ETOH exposure, we developed two mouse models to assess behavioral or cognitive changes caused by fetal ETOH exposure in pre-weaning and adolescent period. Our results show that high dosage of ETOH exposure (4 g/kg) during PD 4-10 resulted in hyperactivity, disinhibition, and deficits in learning and memory in mouse offspring, which lays the groundwork for the future FASD research.

Every year over 2.5 million traumatic brain injuries (TBI) occur and are the leading cause of death and disability among adolescents. There are no approved treatments for TBI. Survivors suffer from persistent cognitive impairment due to posttraumatic tissue damage and disruption of neural networks which significantly detract from their quality of life. Posttraumatic cognitive impairment depends in part on the brain's limited ability to repair or replace damaged cells. Immature neurons in the hippocampus dentate gyrus, a brain region required for learning and memory, are particularly vulnerable to TBI. Insulin-like growth factor-1 (IGF1) is a potential therapeutic for TBI because it is a potent neurotrophic factor capable of mediating neuroprotection, neuro-repair, and neurogenesis. We hypothesized that conditional IGF1 overexpression in the mouse hippocampus following experimental controlled cortical impact injury (CCI) would enhance posttraumatic neurogenesis chronically. To this end, conditional astrocyte-specific IGF1 overexpressing mice (IGFtg) and wild-type (WT) mice received CCI or sham injury. The proliferation marker BrdU was used to label neurons born the first week after injury. Six weeks after injury, when surviving posttrauma-born neurons would be fully developed, we counted proliferated cells (BrdU+) and the subset expressing a mature neuronal marker (NeuN+/BrdU+) in the hippocampus. We also assessed cognitive performance during radial arm water-maze reversal (RAWM-R) testing, a neurogenesis-sensitive assay. IGF1 promoted end-stage maturity and decreased mis-migration of neurons born after trauma. These effects coincide with IGF1 induced improvements in performance on neurogenesis sensitive cognition following TBI. Mammalian target of rapamycin (mTOR), an early signaling molecule downstream of IGF1, has been identified as a potential target for TBI interventions because of its regulatory role in neuronal plasticity and neurogenesis. However, recent studies have also reported maladaptive plasticity and recovery associated with posttraumatic mTOR activation. It is imperative to elucidate the mechanism of action of IGF1 during pre-clinical evaluations. We hypothesized that IGF1 mediates posttraumatic neurogenic effects through IGF1 induction of mTOR activation. We injured cohorts of IGFtg and WT mice and harvested their brains for immunohistochemistry to assess IGF1 overexpression effects on posttraumatic mTOR activation at 1, 3, and 10 days post-injury (dpi). We found that IGF1 upregulated mTOR activation following TBI in a region-specific manner at 1 and 3dpi. To determine if IGF1 regulated differentiation and arborization through the mTOR pathway, injured WT and IGFtg mice received daily i.p. injections of rapamycin (10mg/kg), the inhibitor of mTOR, or its vehicle for 7 days. Vehicle and rapamycin administration began 3dpi, after the cells dividing at the peak of posttraumatic proliferation were labeled with BrdU. IGF1 enhancement of posttraumatic neurogenesis was not dependent on mTOR activation. In summary, IGF1 directs newborn neuron localization, promotes end-stage maturation, and chronically improves cognition. IGF1 can stimulate posttraumatic neurogenesis and plasticity independent of mTOR activation. These data suggest that IGF1 can stimulate neuron replacement following trauma-induced hippocampal neuron loss and cognitive improvement. Further studies should investigate IGF1 and mTOR inhibition as a combination therapy for neurorehabilitation.

Dexter Morgan is a serial killer, but he may not be blameworthy for his actions There are two possible explanations that could absolve Dexter of moral responsibility: (1) His inability to empathize with others makes it so that he cannot make appropriate moral decisions. Or (2) his serial killing tendencies are determined in nature, set off by events of which he had no control. I conclude that Dexter is, in fact, morally responsible for his actions because he is capable of second order desires

The present study characterized two fiber pathways important for language, the superior longitudinal fasciculus/arcuate fasciculus (SLF/AF) and the frontal aslant tract (FAT), and related these tracts to speech, language, and literacy skill in children five to eight years old. We used Diffusion Tensor Imaging (DTI) to characterize the fiber pathways and administered several language assessments. The FAT was identified for the first time in children. Results showed no age-related change in integrity of the FAT, but did show age-related change in the left (but not right) SLF/AF. Moreover, only the integrity of the right FAT was related to phonology but not audiovisual speech perception, articulation, language, or literacy. Both the left and right SLF/AF related to language measures, specifically receptive and expressive language, and language content. These findings are important for understanding the neurobiology of language in the developing brain, and can be incorporated within contemporary dorsal-ventral-motor models for language.

Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in gametogenesis and embryogenesis remain to be fully understood. Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the one-cell stage for several hours before dying by 10 hours post fertilization. We demonstrate that dusp6 is expressed in the gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults. The fact that offspring of homozygous dusp6 mutants stall at the one-cell stage, prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for gametogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other feedback regulators of the ERK pathway compensate for loss of dusp6 at later stages.

Recent developments in neurobiology make it clear that glia play fundamental and active roles, in the adult and in development. Many hereditary cognitive disorders have been linked to developmental defects, and in at least two cases, Rett Syndrome and Fragile X Mental Retardation, glia are important in pathogenesis. However, most studies of developmental disorders, in particular intellectual disability, focus on neuronal defects. An example is intellectual disability caused by mutations in ACSL4, a metabolic enzyme that conjugates long-chain fatty acids to Coenzyme A (CoA). Depleting ACSL4 in neurons is associated with defects in dendritic spines, a finding replicated in patient tissue, but the etiology of this disorder remains unclear. In a genetic screen to discover genes necessary for visual function, I identified the Drosophila homolog of ACSL4, Acsl, as a gene important for the magnitude of neuronal transmission, and found that it is required in glia. I determined that Acsl is required in a specific subtype of glia in the Drosophila optic lobe, and that depletion of Acsl from this population causes morphological defects. I demonstrated that Acsl is required in development, and that the phenotype can be rescued by human ACSL4. Finally, I discovered that ACSL4 is expressed in astrocytes in the mouse hippocampus. This study is highly significant for understanding glial biology and neurodevelopment. It provides information on the role of glia in development, substantiates a novel role for Acsl in glia, and advances our understanding of the potential role that glia play in the pathogenesis of intellectual disability.