James A. Lister - US grants

The neural crest of vertebrates gives rise to a diverse array of cell types, including neurons and glia of the peripheral nervous system, head and neck cartilage, and pigment cells. In humans, the neural crest is the target of a number of developmental abnormalities as well as cancers, collectively referred to as neurocristopathies. To better understand the mechanisms of neural crest cell fate specification and differentiation, we have undertaken studies of this system in the zebrafish. We have identified a zebrafish gene required for development of neural crest-derived melanin-producing pigment cells, known as melanophores. Zebrafish homozygous for mutations at the nacre locus lack all melanophores from embryonic through adult stages, but have normal eye pigmentation and development of other neural crest derivatives. In this proposal I describe the following experiments to clarify the role of nacre in melanophore development: the nacre defect will be characterized at the cellular level by cell labeling and transplant studies. To identify potential candidate genes nacre will be placed on the genetic linkage map. The role of the zebrafish homologue of the microphthalmia-associated transcription factor (Mitf), a known pigment cell regulator in mice, in melanophore development will be investigated by misexpression in wild-type and nacre embryos. The proposed research will extend our understanding of the specification and differentiation of pigment cells in vertebrates and shed light on human conditions in which these processes are disrupted, such as piebaldism and melanoma.

DESCRIPTION (Provided by Applicant): Heterozygous mutation or deletion of the retinoic acid induced 1 (RAI1) gene results in Smith-Magenis syndrome while duplication of the genomic region containing RAI1 results in the dup(17)(p11.2) syndrome. SMS is a complex syndrome that encompasses developmental, physical, and behavioral abnormalities. Persons with SMS have variable developmental delays, mild craniofacial anomalies, obesity, and significant abnormalities in sleep, circadian rhythm, attention, and self-control. Individuals with RAI1 mutations exhibit overgrowth, with all reported cases at >90th percentile for height and weight. Interestingly, individuals with dup(17)(p11.2) display developmental delays, behavioral problems, and autism spectrum disorder, in addition to significant pre- and post-natal growth delays. Further supporting the human data, mice overexpressing Rai1 are growth delayed, while those with a heterozygous targeted knockout of Rai1 are obese, and both mouse models have neurological deficits. Taken together, these findings strongly suggest that gene dosage of RAI1 is critical for normal development, behavior, and growth. The overall goal of this application is to determine the biological role of RAI1 in the cell. The studies proposed seek to develop zebrafish as model for studying gene dosage requirements for rai1. The following studies are proposed: 1) Determine spatial and temporal expression of rai1 in zebrafish embryos by RNA in situ hybridization and assess the effect of retinoic acid (presence or absence) on this expression. 2) Develop zebrafish models to assess rai1 dosage threshold requirements and the primary developmental defects associated with reduced, absent, or increased expression of rai1. 3) Evaluate the functional consequences of rai1 knockdown or overexpression on the expression of putative RAI1-regulated genes. Thus, the studies proposed here will: 1) investigate the hypothesis that RAI1 is regulated by retinoic acid;2) create an rai1 antisense morpholino model to evaluate the effects of rai1 knockdown and an rai1 overexpression transgenic model to assess dosage effects;and 3) investigate putative RAI1-interacting genes utilizing the rai1 morpholino and transgenic models. Understanding the developmental and behavioral effects of rai1 gene dosage is critical. Disorders involving gene dosage, such as Smith-Magenis and dup(17)(p11.2) syndromes, together function as an ideal model in which to study a gene that has significant broader implications for common human phenotypes, including disorders involving sleep, mental illness, behavior disorders, and obesity. Thus, the investigators'studies will show that RAI1 regulates genes involved in development, obesity, sleep, and behavior, and that retinoic acid is a tissue-specific regulator for normal RAI1 function. PROJECT NARRATIVE: The proposed studies focus on a gene called RAI1 that plays a role in normal development. The project is focused toward developing a zebrafish model in which to study this gene.