Richard Dorsky - US grants

The goal of this project is to investigate the role of the Wnt signaling pathway in zebrafish neural cres development. Because the neural crest has been shown to be critical for normal embryogenesis, understanding the molecular control of cell determination in this cell type is important in the analysis of particular human diseases as well as general control of cell determination. The expression of a downstream component of the pathway, beta-catenin, will be analyzed at a single cell lived by in immunohistochemistry to determine whether neural crest cells respond to Wnt signals in vivo. Following this analysis, gain and loss of function experiments will be carried out in neural crest cells by using single-cell mRNA injection to produce targeted gene misexpression. An analysis of the fate of individual misexpressing cells will be used to ascertain the effects of the Wnt pathway on their development.

The vertebrate central nervous system (CNS) is patterned by environmental signals that control the specification of cell fate. Understanding the mechanism of cell fate specification in the nervous system is vital for our ability to diagnose disease and design regenerative therapeutic treatments. One important signal in the developing CNS is produced by Wnts, which regulate transcription through the downstream effectors (i-catenin and Tcf. Wnt/B-catenin signaling plays an important role in spinal cord patterning, but the cellular and molecular targets of Wnt signaling in this tissue are unknown, and it is unclear whether Wnts act only to promote cell division or also directly assign cell fate. The zebrafish now gives us an ideal model system to address this problem. In this study, we will test the hypothesis that Tcf- mediated transcription directly regulates progenitor cell fate specification in the spinal cord. First, we will test whether Tcf7 and Tcf3 are required for the expression of spinal progenitor domain-specific genes. Our preliminary data suggest that at least one intermediate domain is mis-specified when Tcf3 activity is lost. We will now examine whether other progenitor domain markers also require Tcf3, whether dorsal progenitors specifically require Tcf7 activity, and whether these molecules function independently of cell-cycle control. Second, we will test whether Tcf3 normally acts as a transcriptional activator, represser, or both. We will examine whether Tcf3 overlaps with and is required for endogenous B-catenin activity, determine whether mutant forms of Tcf can phenocopy or rescue Tcf3 loss-of-function phenotypes, and ask whether Tcf3 functions synergistically or antagonistically to canonical Wnt signaling. These experiments will support either a model in which Tcf3 functions exclusively as a represser, or one in which it also activates target genes. Third, candidate target genes will be tested as direct transcriptional targets of Tcf3 signaling by chromatin immunoprecipitation (ChIP) analysis. We will use a combination of known genes, computational genomic analysis, and an unbiased screen to identify candidates. These experiments will yield a picture of Tcf3 targets in vivo, and the roles of the B-catenin signaling pathway in spinal cord progenitor specification. Overall, these studies will allow us to understand how genes are regulated during spinal cord development, ultimately resulting in the correct placement and wiring of spinal neurons. This understanding will help in the treatment and repair of spinal cord injuries and disease.

DESCRIPTION (provided by applicant): The vertebrate central nervous system (CNS) is patterned by environmental signals that regulate neuronal phenotype and function. Understanding the mechanism of cell differentiation in the nervous system is vital for our ability to diagnose disease and design regenerative therapeutic treatments. One important signal in the developing CNS is produced by Wnts, which regulate transcription through the downstream effectors ?-catenin and Lef/Tcf. We have identified a specific role for Wnt signaling, acting through Lef1, in the development of zebrafish GABAergic hypothalamic neurons. Our data indicate that Lef1 continues to be expressed in these neurons after they become postmitotic, suggesting that Wnt signaling pathway may control the expression of particular genes critical for their function. In this study we will test the hypothesis that Lef1 regulates the development of posterior hypothalamic neurons by activating genes that regulate their specification, differentiation, and survival. In the process we will establish the zebrafish hypothalamus as a model for vertebrate neurogenesis, and we will discover new targets of Lef1 function in the developing CNS. First, we will test whether Wnt signaling and Lef/Tcf-mediated transcription are required for the proper differentiation of postmitotic GABAergic neurons in the hypothalamus. Using transgenically-expressed pathway inhibitors we will assess the temporal and spatial requirements for Wnts and Lef/Tcf factors during the period of neurogenesis. These experiments will determine whether target gene regulation by these molecules participates in a conserved pathway of GABAergic neurogenesis and determines the phenotype of a specific hypothalamic cell population. Second, candidate target genes will be tested as direct transcriptional targets of Lef1 signaling by chromatin immunoprecipitation analysis. We will also screen for novel Lef1 targets by constructing a library of precipitated DNA fragments from hypothalamic tissue, followed by secondary confirmation of specificity. The expression of potential target genes will then be tested for in vivo regulation by Lef1 using in situ hybridization. These experiments will yield a picture of Lef1 targets in vivo in the hypothalamus and will help characterize the role of the ?-catenin signaling pathway in hypothalamic neurogenesis. Overall, these studies will allow us to understand how genes are regulated by environmental signals during hypothalamic neurogenesis, ultimately resulting in the correct function of this structure. The mechanisms we uncover will help in the treatment of CNS disorders and diseases. PUBLIC HEALTH RELEVANCE: The hypothalamus is an important regulatory center in the brain that controls hormone release and behavior. Little is understood about the developmental signals governing the production of hypothalamic neurons. In this proposal we will determine the role of the Wnt signaling pathway in hypothalamic neurogenesis, and identify target genes of the Wnt effector Lef1 that underlie this process.

DESCRIPTION (provided by applicant): Animal behavior depends on the function of a large collection of overlapping neural circuits. To fully under- stand the circuit underlying a particular behavior, one must identify the neurons involved, determine what synaptic connections they make with each other, and measure their electrical responses during activation of the circuit. The zebrafish larva is an excellent system to study circuits: it has well-established behaviors, can be manipulated genetically, and most importantly, is transparent. By genetically expressing fluorescent reporters or light-activated channels, one can optically image neurons' morphology, connectivity, and activity, and even optically control their electrical activity, in an intact, living animal (Scott, 2009). What has been largely missing, until recently, are methods to express genes in particular neurons of interest. A powerful solution to this problem is provided by Gal4 enhancer trap screens in zebrafish (Scott et al., 2007; Asakawa et al., 2008). The Gal4 gene, which acts as a genetic trigger, is integrated randomly into the zebrafish genome; depending on where it lands, it will be turned on in a different set of cells (often including specific neuronal types), controlled by the regulatory elements of nearby genes. By screening through many Gal4 mutant lines, one can find lines that express in one's favorite neurons, then cross these to UAS responder lines, so that fluorescent reporters or other genes are turned on in those neurons. This proposal will carry out a second-generation Gal4 enhancer trap screen with several improvements. (1) A new DNA trapping construct that not only expresses Gal4, but can be converted to instead express a different genetic switch, Cre recombinase. This will allow expression of genes in even more specific sets of cells by intersecting a Gal4 pattern with a Cre pattern. (2) An online database of Gal4 expression patterns, including three-dimensional views. This will allow collaborators, and eventually the zebrafish community at large, to quickly determine which lines may express in the tissues or neurons that they study. (3) A new public- domain 3D visualization package, FluoRender, that has been optimized for confocal microscopy data. This will improve and speed up documentation of expression patterns. (4) A toolkit of UAS responder lines, validated for uniform expression levels, to visualize neuronal shape and connectivity. Diencephalic dopaminergic neurons, for which no specific enhancer is yet known, will be analyzed as a test case. In summary, then, this project will generate a large number of well-characterized Gal4 enhancer trap lines and UAS responder lines, which will allow zebrafish neurobiologists as well as other zebrafish researchers to express genes of interest specifically in many different neuron classes and nonneural tissues. Techniques developed for intersectional gene expression, generation of UAS responders, and 3D visualization will also be widely applicable in the field. The project will significantly increase the utility of the Gal4-UAS method in zebrafish, aiding analysis of the development and function of many organs, in addition to neuronal circuits.

The vertebrate central nervous system (CNS) forms from a population of multipotent progenitor cells that generate all mature neurons and glia. These cells are particularly important for the treatment of injury and disease, but in most cases they are not maintained in adult CNS tissues. The mechanisms responsible for CNS progenitor maintenance are poorly understood, and the goal of this research proposal is to define and examine a new molecular pathway underlying this process. This work will investigate the role of a specific transcriptional regulator, Tcf3, in maintenance of zebrafish spinal cord progenitors. Tcf3 regulates progenitor and stem cell state in other epithelial tissues, but its function in the CNS is unknown. Specifically the proposed experiments will test the hypothesis that Tcf3 acts both transcriptionally and epigenetically as a master regulator of the progenitor state in the embryonic spinal cord. First, the capacity of Tcf3 to maintain spinal progenitor multipotency, self-renewal, and gene expression will be tested. Second, specific genes obtained from a microarray analysis in tcf3 mutants will be assayed to determine which have altered expression in spinal progenitors, and their function in spinal progenitor maintenance will be determined. Third, the function of DNA methylation in progenitor maintenance downstream of Tcf3 will be assayed. Together, the work proposed here will lead to a new model for CNS progenitor maintenance, by identifying novel genes and molecular mechanisms that preserve multipotency and self-renewal. Our findings will help exploit CNS progenitors for the future treatment of neurological injury and disease.

DESCRIPTION (provided by applicant): The specific regulatory pathways regulating adult neurogenesis and the behavior of neural stem cells are poorly understood. Radial glial stem cells in the vertebrate brain can continuously produce neurons during homeostasis and regenerate lost cells following injury. Yet the molecular mechanisms underlying neural stem cell regulation are poorly understood. Our laboratory has discovered that Wnt signaling regulates neurogenesis in the post-embryonic zebrafish hypothalamus via the transcriptional effector Lef1. In contrast the formation of radial glia in the zebrafish hypothalamus does not require Wnt signaling and ectopic Wnt activity reduces their number. This work will test the specific hypotheses that Wnt signaling functions to convert radial glia into neural progenitors, and to promote their proliferative response following injury. First, we will determine whether Wnt signaling is necessary and sufficient to drive radial glia into a neural progenitor state. Second, we will test whether Lef1 promotes neural progenitor formation through downstream targets. Third, we will determine whether Wnt signaling is required for radial glial proliferation after injry. Together, this will characterize the regulation of a novel population of neural progenitors in the vertebrate hypothalamus, and establish a new model for Wnt signaling in CNS neurogenesis.

The genetic and molecular mechanisms that establish innate behaviors across diverse species are largely unknown. We have discovered an evolutionarily conserved role for Lef1-mediated Wnt signaling in the regulation of anxiety and in the differentiation of anxiolytic neurons in the zebrafish posterior hypothalamus. However mechanistic links between Lef1 targets, neurogenesis, and neuronal function in behavior have not yet been established. We also know that new neurons are continually added to this brain region, but it is not clear whether they contribute to anxiety-related behavior. This proposal will test the hypothesis that Lef1-mediated target genes regulate hypothalamic neurogenesis to control anxiety-related behavior throughout life. Three specific aims will determine which Lef1 targets are required for the formation of anxiolytic neurons, whether those neurons regulate stress hormone levels through the HPI axis, and whether postembryonic Lef1-dependent neurogenesis can mediate behavior. Together this work will define a novel mechanism for the regulation of an innate behavior through neurogenesis.