Cecilia B. Moens, Ph.D. - US grants

DESCRIPTION (Adapted from the applicant's abstract): The establishment of functional neural circuits in the vertebrate central nervous system depends on the correct partial positioning of distinct neural cell types. In the hindbrain this is facilitated by the appearance of segments, or rhombomeres, that act as lineage-restricted compartments in which developmental programs are reiterated. Hindbrain neural crest contributes to the structural and neuronal components of the head and neck, and disruption of the early patterning of the hindbrain results both in neurological and craniofacial defects in humans. The long-term goal of this research is to understand how hindbrain segmentation is established, how segments acquire distinct identities and how these identities result in the specification of functionally distinct neuronal cell types. The zebrafish, suited to both genetic analysis and experimental embryology, is used as a model system in which to address these questions. Lazarus and valentino are zebrafish hindbrain segmentation genes that were identified in a screen for zebrafish mutants in which hindbrain patterning is disrupted. The proposed experiments use these mutants to address the genetic and molecular basis of head segmentation as follows: (1) Genetic analysis of lazarus will address whether its function is required within the hindbrain or in the periphery to bring about global segmentation in the head. Positional cloning will establish its molecular mechanism and its position in the hierarchy of hindbrain segmentation. (2) The role of valentino in hindbrain segmentation will be studied by identifying molecular partners with which it interacts in order to subdivide rhombomeres 5 and 6 from their common precursor in the presumptive hindbrain. (3) The hypothesized role of Eph receptors and their ligands in mediating repulsive cell-cell interactions in the hindbrain will be examined by assaying the effects of Eph and ephrin expression on the characteristic behaviors of valentino and lazarus mutant cells in genetic mosaics.

Moens, C; IBN #9816905 The enormous functional complexity of the vertebrate nervous system is built upon events that occur during early embryogenesis when an initially homogeneous field of cells, the neural plate, is divided and subdivided into progressively more sharply restricted domains. Within the hindbrain, these domains are overtly segmental in character, containing re-iterated sets of neurons and being separated by morphologically and molecularly distinct boundaries. These hindbrain segments, or rhombomeres, acquire their distinct anterior-posterior identities through the action of the hox genes, which also pattern the body segments of invertebrate animals. As such, the vertebrate hindbrain has an archetypal significance as being patterned by mechanisms that are conserved across animal phyla.
This project proposes to study the mechanism by which vertebrates establish anterior-posterior pattern within the hindbrain, and thereby co-opt the hox gens into their segmental body plan. The zebrafish is used as a model system, because its relatively simple nervous system allows the identification of individual neurons at particular anterior-posterior levels within the hindbrain. Thus, it is possible to study the specification and patterning of individual neurons both experimentally, by transplanting them to different anterior-posterior levels, and genetically, by studying mutants in which their organization is disrupted through gene deletions. Using this combination of experimental embryology, molecular biology and genetics, the experiments outlined in this proposal will address when and how individual neurons become committed to their region-specific identities, and what role candidate signalling molecules and candidate signalling sources play in this process.

DESCRIPTION (provided by applicant): The zebrafish has become the model system of choice for a growing number of investigators interested in understanding mechanisms of vertebrate development, disease and evolution. The three principal investigators on this multiple-Principal Investigator proposal have each established the Targeting Local Lesions IN Genomes (TILLING) methodology for identifying N-ethyl-N-nitrosourea (ENU)-induced mutations in specific genes of interest in zebrafish using Cel1 detection, and have used this approach to identify nonsense or splice site mutations predictive of strong or complete loss of function of 43 genes. The approach involves screening for unique mutations in a large library of randomly ENU-mutagenized, cryopreserved fish that has been built independently in each of our labs. We wish to make these valuable resources available to the zebrafish community. In this 3-year grant, we propose to identify loss-of-function mutations (defined for our purposes as mutations that create premature stop codons or that disrupt splice sites) in 120 genes of interest to the members of the zebrafish community and to make them available via submission to the Zebrafish International Resource Center (ZIRC). Establishing a TILLING consortium between the three groups reduces redundancy of effort and increases the chances of identifying deleterious mutations in target genes. Whereas separately, our libraries are predicted to contain loss-of-function mutations in between 20% and 58% of the genes we screen, combined, they are expected to contain loss-of-function mutations in over 80% of targets. In Aim 1 we propose to screen target genes sequentially at the three locations until one or more loss-of-function mutations are identified. Potential TILLING targets will be submitted by members of the community and will be ranked by a 10-member external advisory board according to criteria such as high biomedical relevance and inaccessibility to other reverse genetics methods. In Aim 2 we propose to recover these mutants, to do a preliminary phenotypic characterization and to provide them first to the requester and then, within six months of recovery, to the wider zebrafish community via ZIRC. Finally, in Aim 3 we propose to explore massively parallel sequencing of PCR-amplified targets from our ENU-mutagenized libraries as a higher-throughput alternative to our current TILLING methodology. PUBLIC HEALTH RELEVANCE: Vertebrate model organisms such as the zebrafish, in which gene function can be understood through the detailed analysis of mutant phenotypes, provide important insights into mechanisms of human development and disease. In the past grant period we adapted TILLING, a methodology to find mutations in genes of interest, to the zebrafish, and built resources that can allow us to find loss-of-function mutations in a large fraction of the genes in the zebrafish genome. We now propose to use TILLING to identify mutations in 120 genes that are of importance to biomedical research, and to make these mutants available to the zebrafish community as rapidly as possible.

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DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular pathways that regulate neural circuit and electrical synapse formation in vivo. Neural circuits are organized by synapses, which are specialized sites of adhesion and communication whose patterns and properties form the basis of all of brain function. Synapses can be either chemical, where signals are transmitted via neurotransmitter release and reception, or electrical, where signals pass directly through gap junctions between neurons. Of these, the chemical synapse has received more attention in recent years, however growing evidence suggests that electrical synapses are widespread in the brain where they modulate neural processing from vision to memory and learning. Underlying neural circuit and synapse formation are genetic mechanisms ensuring that neurons select appropriate targets and recruit the complex synaptic machinery to the sites of contact. However, the genes that regulate these processes are not well understood, especially in regard to electrical synapse formation. We propose to establish the zebrafish Mauthner (M) circuit as a model for understanding the genetic basis of neuronal target selection and electrical synapse formation. The well-characterized M circuit is simple and accessible, and is necessary for a stereotypical escape response behavior. These properties, in conjunction with genetic tools that specifically mark the cells of the neural circuit and their stereotyped chemical and electrical synapses, provide a unique opportunity to find mutations that affect electrical synaptogenesis, to understand the cellular basis of these defects, and to assess their behavioral consequences. The goal is to demonstrate that mutations affecting M electrical synapse formation can be identified using a forward genetic screen (Aim1), to demonstrate that these mutations specifically affect electrical synapse formation at the cell- biological level (Aim2) and that they have functional deficits that are evident at the level of the M-mediated escape response (Aim3). Overall this proposal will evaluate whether the M circuit is a suitable platform for studying vertebrate CNS electrical synapse formation; this will lay the groundwork for unraveling the underlying cellular and molecular mechanisms. Such knowledge is critical given that defects in synapse development or function are associated with a number of neurodevelopmental disorders, including autism and epilepsy, and also age-related diseases, such as Alzheimer

DESCRIPTION (provided by applicant): The zebrafish has emerged as a premier model organism for the study of human development and disease. Its greatest asset is that it is a genetic system in which mutant phenotypes can be effectively dissected at the organismal, cellular and subcellular levels. Many forward genetic screens have identified genes and subsequently delineated their roles in embryonic cell fate specification, patterning, morphogenesis and organogenesis. However with the sequencing of the zebrafish genome we now know that many genes have never been identified by forward genetics, so their role in development remains an open question. Furthermore, recent explosion in the identification of human disease-causing genes has the potential to re-purpose the zebrafish as a leading model for human genetic disease, provided the zebrafish homologs of these genes can be mutated within the next few years. A number of reverse genetics approaches are currently being pursued in the zebrafish, with the potential to generate knock-outs in many of the genes in the genome. However, these approaches are largely untargeted and therefore do not address the immediate and pressing needs of the scientific community. We have created a large library of 15,456 cryopreserved, ENU mutagenized fish and estimate that this library contains at least one nonsense allele in any average-sized zebrafish gene with 97% chance. We have developed methodologies to screen this library efficiently for mutations that are present in a single heterozygous fish, and using these methodologies we have identified one or more nonsense mutations in over 80 genes considered to be of high-priority by zebrafish researchers. We have distributed these mutants widely within the community, resulting in 18 publications in the past four years. We have now transitioned to innovative and high-throughput Illumina sequencing methodologies for mutation detection with which we propose to identify one or more loss-of-function mutation in over 300 zebrafish genes in three years, and to deliver them to the community via ZIRC. PUBLIC HEALTH RELEVANCE: The goal of this grant is to mutate zebrafish genes whose human homologs play important roles in development and disease, and to provide those mutants to the community. Using these mutants and the other attributes of this animal model (its fecundity, external development and optical qualities) researchers will be able to discover mechanisms underlying normal development and disease pathogenesis.

DESCRIPTION (provided by applicant): Almost all neurons in the vertebrate brain are foreign born - that is, they are born at or near the ventricular surface and then migrate to another location where they make their connections and carry out their specialized functions. Neuronal migrations can be radial, parallel to the api- co-basal axis of the neuroepithelium, or they can be tangential: orthogonal to the apical-basal axis - i.e., in the plane of the neuroepithelium. With te goal of discovering genetic mechanisms regulating directed neuronal migration, we have established the facial branchiomotor neurons (FBMNs; cranial nerve VII), which undergo a stereotyped and evolutionarily conserved tangen- tial migration from hindbrain rhombomere (r)4 to r6, as a model system in our lab. Forward ge- netic screens in our lab and others have identified multiple core components of the Planar Cell Polarity (PCP) pathway as being essential for FBMN migration. The PCP pathway is best under- stood as a cell contact-dependent molecular mechanism for generating and transmitting polarity between cells in the plane of an epithelium. However PCP has been implicated in a growing number of cell migrations during development and disease states. Currently no coherent model exists for how PCP regulates directional cell migration. This is mainly because the polarized cell- cell interactions that defin PCP are difficult to reconstitute in cell culture; we must therefore attack the problem in vivo. Th zebrafish model, with its exquisite live imaging, facile transgen- esis and powerful forward and reverse genetics tools, affords us this opportunity. We hypothe- size that direct PCP signaling between the planar-polarized cells of the neuroepithelium and the motile FBMNs polarizes FBMN protrusive activity in the direction of migration. We pro- pose to test the predictions of thi model first by identifying the cells in the migratory environ- ment that are responsible for directing migration (Aim 1), and then by elucidating how PCP sig- naling within the FBMNs affects their behavior in vivo (Aim 2). Furthermore in Aim 2 we will discover how interactions between migrating FBMNs and the surrounding neuroepithelium in- fluence PCP protein localization and protrusive activity in the FBMNs. Finally, we propose to enrich our understanding of PCP-dependent neuron migration through the cloning of FBMN migration mutants we have identified in a forward genetic screen (Aim 3).

? DESCRIPTION (provided by applicant): Melanoma is one of the deadliest forms of cancer and is poorly responsive to standard chemotherapeutics. Much of our understanding of tumor cell interactions with the microenvironment are either inferred from end-point assays and fixed tumor sections, or have only been visualized with high resolution in in vitro co-culture models. Cancer metastasis is a dynamic process; however, a major limitation to understanding cancer progression is the lack of genetically tractable in vivo model systems that are amenable to high-resolution imaging. We will overcome this obstacle by visualizing and manipulating tumor cells and their microenvironment directly in a human-in-zebrafish xenotransplant model. We will take advantage of the ease of expressing reporters in tumor cells in culture combined with imaging tumor cell and stromal cell interactions in zebrafish to understand heterotypic cell interactions and signaling pathways between the tumor cells and microenvironment that regulate malignant melanoma progression. We will then validate our key findings in mouse models with fixed imaging and/or end-point analyses. We have determined that zebrafish larvae can be injected with human melanoma cells and these xenotransplants exhibit a 14% rate of metastasis. Live cell imaging reveals that tumor cells at the periphery of the tumor mass respond to physical cell contact with macrophages by forming actin-rich protrusions. Depleting larvae of host macrophages resulted in reduced melanoma metastasis. Separately, we have observed that melanoma cells exhibit angiotropism: expansion along the abluminal surfaces of blood vessels. From these results, we hypothesize that physical contact with macrophages induces actin dynamics in tumor cells to regulate intravasation in vivo. With the long-term goal of identifying potential markers for predicting metastatic risk as well as targets for therapies that block the early steps of melanoma metastasis, this proposal focuses on interactions between tumor associated macrophages and melanoma cells, and seeks to understand how this interaction impacts invadopodium formation during intravasation (Aim I) and angiotropism (Aim II), processes that are difficult to study in other in vivo systems. As melanoma cells adhere to the vasculature prior to penetrating matrix barriers for intravasation, we will further determine whether focal adhesion assembly and invadopodium formation are coupled events (Aim III) for efficient intravasation of melanoma cells. These experiments will reveal the mechanisms by which melanoma cells switch from adhering to matrix to degrading matrix during intravasation, a critical step in metastasis.

SUMMARY Topographic neural maps are ordered connections between the brain and the periphery in which spatial coordinates in the projecting field are represented in the target field. Topographic maps are a common motif in vertebrate nervous system organization and are critical for our ability to perceive the world and accurately respond to it, so their development is of fundamental interest to neurobiology. Examples of topographic maps are in the ordered projections of retinal neurons to visual centers in the brain and in the projections motor neurons in the spinal cord to specific target muscles in the limb. Cranial motor neurons in the vertebrate hindbrain exhibit a topo- graphic relationship with the pharyngeal arch-derived muscles in the head periphery that they innervate, whereby more anterior neurons innervate more anterior pharyngeal arches. Using the transparent zebrafish model for live imaging and transplantation of single motor neurons, we have found that a topographic map is detectable within the vagus (cranial nerve X) motor pro- jections to the posterior pharyngeal arches in the 3-day embryo, and have discovered two paral- lel strategies that govern its formation: a Hox-regulated molecular mechanism and a novel tem- poral mechanism in which timing of vagus axon initiation is regulated to match the sequential development of the pharyngeal arch targets. We call this a ?temporal matching? model as distin- guished from classical spatial matching (chemoaffinity) models of topographic mapping. The overall aim of this proposal is to discover how the Hox-regulated and temporal matching mech- anisms together regulate topographic mapping. We will identify the molecular mechanism by which timing of vagus axon initiation is spatially regulated in vivo and how it is matched to the timing of pharyngeal arch development in Aim 1. We will identify the guidance pathway that is regulated by hox5 genes in Aim 2. Finally, we will determine how the two mechanisms are coor- dinately regulated by spatial cues in Aim 3. Ultimately our goal is to elucidate novel mechanisms of topographic mapping, their regulation and integration during development.