James A. Weston - US grants

Investigations are proposed to learn mechanisms that underlie the development of specific neurons and their connections in the CNS. The zebrafish, a vertebrate with marked advantages for developmental and genetic studies, will be utilized. The focus is on neurons that are segmentally arranged in the CNS, and that arise and begin to form their processes during the first day of embryogenesis. These neurons can be individually identified, in some cases before their processes grow, and examined in situ in the optically clear, small, and simply organized embryo. Specifically, (1) Lethal mutations will be obtained and the embryonic phenotypes the mutations produce will be studied, to identify genes that control CNS patterning and to characterize the roles of these genes during embryogenesis. (2) The relationships between development of brain segments and seginent-derived structures of the head periphery will be determined in normal and in experimentally perturbed embryos. These experiments may reveal whether brain segments are patterned as components of larger head metameres, and if head mesoderm is required for development of segmentation in the brain. (3) The relationships between brain segmentation and neuronal diversity in the brain will be examined by comparing the development of identified hindbrain reticulospinal neurons whose features vary according to the specific segments containing them. The origins of the dendrites of these neurons and of their early synaptic inputs will be determined. The studies will be carried out using antibodies to label the cells and newly forming synapses, and may also serve to identify molecules involved in synaptic target recognition.

Our major goal is to understand how genes control the structure, function, and development of the nervous system in a vertebrate. Initially, we plan to give the highest priority to the isolation and study of mutations which affect the structures of photoreceptor cells in the retina of the eye of the zebra fish (Brachydanio rerio.) A corollary goal of our work is to develop rapid and efficient procedures that will permit a variety of genetic analyses in a vertebrate at the level of the whole organism. Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. We have perfected methods for making homozygotes in zebra fish: Sperm whose genetic contribution has been eliminated by ultraviolet irradiation are used to activate eggs. The maternal haploid set of chromosomes is allowed to replicate once, and partitioning of the duplicated chromosomes into two cells is prevented by hydrostatic pressure or heat shock. A cell with two identical sets of chromosomes is thereby produced. We propose to mutagenize mother fish during their early development with ethylnitrosourea (ENU). Among the progeny, clones of mutants will be recognized by use of a vision-dependent feeding test. Mutants will be characterized through optomotor and optokinetic tests, flicker fusion frequency, electroretinogram shape, and retinal anatomy. In addition to the direct screening of mutagenized progeny, gamma-ray-induced mutations (possibly long deletions) with specific retinal lesions will be used to select ENU-induced point mutations. Through the study of these mutants we hope to learn what kinds of alterations are specific to retinal photoreceptor cells, what the developmental, structural, functional, and behavioral consequences of the mutations are, how mutation affects other cell types, and what the molecular nature of the mutations are.

Neural crest cells disperse during early vertebrate embryogenesis, localize precisely in various embryonic regions, and produce a variety of derivatives, including melanocytes, neurons and glia of the sensory, autonomic, and enteric nervous systems, neurosecretory cells, dental papillae, and other skeletal and connective tissues of the head and face. Genetic or developmental defects affecting crest cell behavior result in a variety of congenital malformations and diseases, such as craniofacial dysplasia, spina bifida, and phakomatoses. We wish to understand how localized environmental cues, encountered in interstitial spaces by dispersing crest cells, affect their developmental behavior. To this end, we propose to analyze the development of mouse embryos, homozygous for mutations (e.g. Patch, and piebald-lethal or lethal-spotting,) that adversely affect neural crest derivatives. Specifically, we will combine histochemical procedures and microscope-photometric measurements to compare the kinds, amounts and precise local distribution of extracellular matrix macromolecules in mutant mouse embryos and their normal littermates, at times and locations at which important crest cell morphogenetic events occur. By such comparisons, we hope to identify morphogenetically significant components of the interstitial matrix. To test the mode of action of identified matrix macromolecules, we will (i) isolate crest cell subpopulations with known developmental potentialities, using monoclonal antibodies against cell type-specific surface determinants, (ii) culture these cells on substrata containing matrix macromolecules with putative developmental signficance, and (iii) characterize the differentiative behavior of the cultured crest cells in response to the environmental cues that they encounter.

We propose a Program Project that addresses two crucial questions in vertebrate development: (1) how stable phenotypic differences arise in cell lineages of multicellular embryos, and (2) how migration and localization of different embryonic cell populations and neuronal processes is controlled. We propose to examine two cell populations--the neural crest and primary neurons--in a live vertebrate embryo, the zebrafish, Brachydanio rerio, where development can be observed and characterized directly. The neural crest gives rise to a variety of cellular phenotypes, many with useful markers of terminal differentiation. Since crest cells appear to disperse, localize and differentiate in response to developmental cues encountered very early in development, they are ideal for studying the role of embryonic environments in regulating cellular differentiation and morphogenetic behavior. Primary neurons underlie the earliest embryonic behaviors. They arise early in embryonic development, and are the first neurons to grow axons. The primary motor axons, which are the first axons to appear in the periphery, navigate through the same interstitial environment at the same time as crest cells. We will study these two systems using cellular, molecular and genetic approaches. First, to understand the relative roles of cell lineages and environment in specification of cell fate, we will determine the detailed lineages of individual crest cell precursors and we will analyze the migration and interactions of crest cells with their environment in living embryos. Second, we will use monoclonal antibodies to identify molecules that may determine neuronal specification. Using these antibodies as probes, we will isolate and ultimately characterize the genes that code for these developmentally important molecules. Finally, we propose to characterize genetic and developmental mechanisms that determine cell fates by generating and analyzing mutations that affect neural crest development.

We propose a Program Project that addresses two crucial questions in vertebrate development: (1) how stable phenotypic differences arise in cell lineages of multicellular embryos, and (2) how migration and localization of different embryonic cell populations and neuronal processes is controlled. We propose to examine two cell populations--the neural crest and primary neurons-- in a live vertebrate embryo, the zebrafish, Brachydanio rerio, where development can be observed and characterized directly. The neural crest gives rise to a variety of cellular phenotypes, many with useful markers of terminal differentiation. Since crest cells, appear to disperse, localize and differentiate in response to developmental cues encountered very early in development, they are ideal for studying the role of embryonic environments in regulating cellular differentiation and morphogenetic behavior. Primary neurons underlie the earliest embryonic behaviors. They arise early in early in embryonic development, and are the first neurons to grown axons. The primary motor axons, which are the first axons to appear in the periphery, navigate through the same interstitial environment at the same time as crest cells. We will study these two systems using cellular, molecular and genetic approaches. First, to understand the relative roles of cell lineages and environment in specification of cell fate, we will determine the detailed lineages of individual crest cell precursors and we will analyze the migration and interactions of crest cells with their environment in living embryos. Second, we will use monoclonal antibodies to identify molecules that may determine neuronal specification. Using these antibodies as probes, we will isolate and ultimately characterize the genes that code for these developmentally important molecules. Finally, we propose to characterize genetic and developmental mechanisms that determine cell fates by generating and analyzing mutations that affect neural crest development.

Dr. Weston will exploit the avian neural crest, which provides an experimentally accessible population of pluripotent embryonic cells that undergoes a characteristic sequence of restrictions of developmental potential. Phenotypically distinct crest-derived subpopulations can be detected at early stages of development using sensitive cell type-specific markers. Some of these subpopulations appear to arise from developmentally restricted, but functionally undifferentiated embryonic cells. Although the existence of developmentally restricted precursors has been inferred, they cannot now be identified directly. Therefore to characterize the initial appearance and ultimate fate of these cells, we propose: 1) to identify subpopulations of crest-derived cells that have distinct, limited developmental potentials using specifically identified monoclonal antibody markers; and 2) to characterize the normal developmental appearance of immunochemically identified cells, and the subsequent expression by these cells of other phenotypic traits characteristic of crest derivatives. To examine early restriction of developmental potential, we propose; 3) to analyze the response of identified precursors to environmental factors, including an assessment of a) the responses of a crest-derived neurogenic precursor subpopulation to specific growth and survival factors, and b) the normal responses of nascent neurons to environmental cues. %%% The developmental potential of embryonic cells becomes progressively more restricted during embryogenesis. The mechanisms by which this occurs remain to be elucidated. In this study Dr. Weston will address the important issue of how this diversity is generated, stabilized and propagated within cellular lineages during embryonic development.

In vertebrate embryos, neural crest cells disperse through interstitial spaces, encounter a variety of different environmental cues and subsequently express a remarkable diversity of cellular phenotypes including neurons, glia, gland cells, pigment cells and connective tissue. Failure of normal neural crest development in humans, results in numerous diseases or syndromes, including (a) hereditary dysplasias and neuropathies such as Familial Dysautonomia and Hirschprung's disease (aganglionic megacolon), (b) hereditary metaplasias, such as von Recklinghausen's disease (Neurofibromatosis); (c) numerous neural crest-derived neoplasias, such as melanomas, Schwannomas, neuroblastomas, and pheochromocytomas; and (d) congenital defects such as cleft lip/palate, and craniofacial defects associated with heart malformation, such as Pierre Robin syndrome. Clearly, a detailed understanding of the regulatory mechanisms of normal neural crest development in vertebrates embryos will help elucidate such disease processes in humans and animals. We propose to test the hypothesis that developmentally-restricted cells among early migrating crest populations encounter specific, localized environmental cues on their migration pathways. (1) We will use immunocytochemical procedures, in combination with selective removal of matrix components, to detect specific growth factor immunoreactivities localized on crest cell migratory pathways in situ. Then, (2) we will characterize the distribution of extracellular matrix and growth factor immunoreactivity surrounding premigratory neural crest cells in vitro and in vivo. (3) To establish the normal role of such growth factors, we will compare the distribution of growth factor activity in the interstitial spaces of normal and mutant mouse embryos in which neural crest development is perturbed. Finally, (4) we will characterize the differentiation of crest cells on normal and mutant extracellular matrix substrata in the presence and absence of exogenous growth factors whose distribution in interstitial spaces had been altered by mutation. The results of the proposed experiments will provide important insights about how neural crest cell diversification is regulated during early development. We anticipate that they will reveal: (1) what developmental signals are encountered by crest cells during their dispersal through embryonic interstitial spaces; (2) how relevant developmental cues are presented to responsive cells; and ultimately, (3) what molecular mechanisms mediate the responses of specific crest-derived subpopulations to these signals.

In vertebrate embryos, neural crest cells disperse through interstitial spaces, encounter a variety of different environmental cues and subsequently express a remarkable diversity of cellular phenotypes including neurons, glia, gland cells, pigment cells and connective tissue. Failure of normal neural crest development in humans, results in numerous diseases or syndromes, including (a) hereditary dysplasias such as Familial Dysautonomia, Hirschprung's disease (aganglionic megacolon), and hereditary neuropathies (e.g. ALS; Shy-Drager syndrome); (b) hereditary metaplasias, such an von Recklinghausen's disease (Neurofibromatosis) (c) numerous neural crest-derived neoplasias, such as gliomas, neuroblastomas, and pheochromocytomas; and (d) congenital defects such as cleft lip/palate, and craniofacial defects associated with heart malformation, such as Pierre Robin syndrome. Clearly, a detailed understanding of the regulatory mechanisms of normal neural crest development in vertebrates embryos will help elucidate such disease processes in humans and animals. We propose to test the hypothesis that developmentally-restricted cells segregate in a precise sequence from the neural crest lineage, and as a result of these early segregation events, subsets of crest-derived cells among early migrating crest populations respond differentially to localized environmental cues. Specifically, we will test our predictions that: (1) neurogenic precursors are present in early migrating crest cell populations In vitro; (2) survival of neurogenic precursors normally depends on their timely encounter with specific growth factor activities; (3) disappearance of the putative neurogenic precursor subpopulation is due to developmentally-regulated cell death; and (4) the lack of neurogenesis by crest-derived calls on the dorsolateral migration path, in vivo, is due to the absence of a neurogenic precursor subpopulation. The results of the proposed experiments will provide important insights about how neural crest cell diversification is regulated during early development. We anticipate that they will reveal: (1) the identity of developmentally-restricted populations that exist in the premigratory crest; (2) the specific responses of such crest-derived subpopulations to developmental cues; and (3) the identity of specific growth factors that mediate the developmental response of these subpopulations.

We will continue our Program Project to learn the mechanisms of cell signaling, specification and commitment during embryonic development. We propose three component projects, each addressing one of these three issues. We will carry out these studies in the zebrafish, Brachydanio rerio, because of the advantages it offers as an experimental system, including labeling and transplanting single cells, genetic analysis of developmental pathways, and production of transgenic animals. Specifically, we propose to examine induction of the nervous system and the development of two cell lineages -- primary neurons that form the earliest neural circuitry in the embryo and neural crest cells that form the peripheral nervous system and structures in the head and heart. Induction of these lineages involves cell signaling by inducing (growth/survival) factors, and their receptors, and depends on cell position in the embryo. Before overt differentiation, cells become specified. While these specifications may involve phenotypic changes, they do not represent irreversible commitments that restrict the cells to a single developmental course. We propose to examine whether these "conditional" specifications are temporally separable from commitment in neuronal and neural crest lineages. Our main long-term objectives are to identify the regulatory molecules and their genes that underlie signaling, specification and commitment during vertebrate development, and to discover their functional interactions. Disruption of such mechanisms during development of the human central and peripheral nervous systems results in numerous diseases or syndromes, including (a) hereditary dysplasias and neuropathies such as Familial Dysautonomia, Hirschprung's disease (aganglionic megacolon), and orthostatic hypotension; (b) hereditary metaplasias, such as von Recklinghausen's disease (Neurofibromatosis); (c) numerous neoplasias, such as gliomas, neuroblastomas, and pheochromocytomas; and (d) congenital defects such as spina bifida, cleft lip/palate, and craniofacial defects associated with heart malformation (e.g. Pierre Robin syndrome). A detailed understanding of the regulatory mechanisms of normal neural development in vertebrates embryos will help elucidate such disease processes.

Funds are requested to help support expenses for invited speakers for the 53rd annual symposium of the Society for Developmental Biology (SDB), which will be held June 15-18, 1994 at the University of Wisconsin at Madison. This year's symposium is organized by Charles B. Kimmel, current President of the SDB, and will be attended by researchers and teachers interested in the study of development, including graduate students, postdoctoral fellows. We expect that, as during the past 6 years, the meeting will be of medium-size (300-400 registrants), and we encourage junior level attendance by providing them some financial support. The meeting will include a broad range of topics, following the extremely successful organization of talks and posters that was newly established for last year's SDB annual meeting. Plant, animal and microbial systems will all be represented in sessions organized around-developmental problems and "themes" rather than particular systems or organisms. We plan 60 scientific talks, beginning with an opening evening keynote address (developmental genetics of yeast). Highlighted during the next 3 days will be a series of 6 plenary sessions that include 19 exciting invited speakers. Additionally, there will be 8 "minisymposia" arranged in 2 sessions (4 minisymposia running in parallel during each session). The minisymposia will be organized and chaired by experts in the areas, whereas most of the talks will given by junior people, thus providing them with an opportunity to speak at the national level. Posters, also providing opportunity for young people to present their work, will be available for viewing during the entire meeting, and 5 scheduled poster sessions will provide ample time for discussion. Reflecting the growing role of the SDB in the affairs and concerns of its membership, this year's meeting will, for the first time, include 2 issue-oriented workshops, on education and on the concerns of women and minorities.

We will continue our Program Project to learn the mechanisms of cell signaling, specification and commitment during embryonic development. We propose three component projects, each addressing one of these three issues. We will carry out these studies in the zebrafish, Brachydanio rerio, because of the advantages it offers as an experimental system, including labeling and transplanting single cells, genetic analysis of developmental pathways, and production of transgenic animals. Specifically, we propose to examine induction of the nervous system and the development of two cell lineages -- primary neurons that form the earliest neural circuitry in the embryo and neural crest cells that form the peripheral nervous system and structures in the head and heart. Induction of these lineages involves cell signaling by inducing (growth/survival) factors, and their receptors, and depends on cell position in the embryo. Before overt differentiation, cells become specified. While these specifications may involve phenotypic changes, they do not represent irreversible commitments that restrict the cells to a single developmental course. We propose to examine whether these "conditional" specifications are temporally separable from commitment in neuronal and neural crest lineages. Our main long-term objectives are to identify the regulatory molecules and their genes that underlie signaling, specification and commitment during vertebrate development, and to discover their functional interactions. Disruption of such mechanisms during development of the human central and peripheral nervous systems results in numerous diseases or syndromes, including (a) hereditary dysplasias and neuropathies such as Familial Dysautonomia, Hirschprung's disease (aganglionic megacolon), and orthostatic hypotension; (b) hereditary metaplasias, such as von Recklinghausen's disease (Neurofibromatosis); (c) numerous neoplasias, such as gliomas, neuroblastomas, and pheochromocytomas; and (d) congenital defects such as spina bifida, cleft lip/palate, and craniofacial defects associated with heart malformation (e.g. Pierre Robin syndrome). A detailed understanding of the regulatory mechanisms of normal neural development in vertebrates embryos will help elucidate such disease processes.

Hu protein is expressed in all embryonic and adult neurons, and is one of the earliest markers of neuronal differentiation. Hu is a member of a family of RNA-binding proteins that are homologous with the product of the elav gene in Drosophila. It has been known for more than a decade that mutations in elav result in specific loss of neurons, implying that ELAV is necessary for neuronal development. Recently, we have shown that misexpression of Hu in embryonic neural crest cells can promote the expression of neuronal traits. However, the mechanism by which Hu/ELAV protein elicits neuronal differentiation has remained elusive. Recently, ELAV was shown to be necessary for the neuron- specific splicing of neuroglian mRNA, suggesting that ELAV, and presumably the Hu proteins, mediate their effects by regulating the neuron-specific splicing of gene products required for neuronal differentiation. It is known that heterogeneous nuclear ribonucleoproteins (hnRNPs), abundant and ubiquitously expressed RNA- binding proteins, are involved in RNA splicing, but it is not clear how cell-type specific splicing is mediated. We have recently been able to show, however, that Hu proteins associate with hnRNPs in an RNA- dependent manner. Our preliminary results suggested a new hypothesis that binding of Hu proteins to hnRNPs confer neuron-specificity to the function of hnRNP complexes. We propose to confirm and extend our preliminary results, and thereby provide the foundation for future research proposals to establish the role of Hu proteins in the post- transcriptional regulation of neuronal differentiation.