Developmental Genetics in the Zebrafish Danio rerio

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

This laboratory has used forward genetic screens and gene expression analysis involving in situ hybridization and DNA microarray technology to identify genes that function in embryonic development. A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as the pharyngeal arches. The zebrafish Barx1 gene has been isolated and shown to be involved in the regulation of chondrogenesis during pharyngeal arch development. Barx1 is expressed in the cranial neural crest, the pharyngeal arches, the anterior aspect of the pectoral fin buds, and the gut wall. By 2.5 days post fertilization, embryos in which Barx1 expression in inhibited exhibit developmental delay exemplified by poor facial outgrowth and micrognathia. Histological analysis and labeling of cell membranes revealed reductions in differentiation and chondrocyte condensation within the arches. Affected larvae stained with Alcian blue exhibit small and dysmorphic arch cartilage elements, and expression of chondrogenic markers is perturbed. The expression of Barx1 is controlled by bone morphogenetic protein (BMP) as seen in bead implantation experiments. These results suggest a role for Barx1 at early stages of chondrogenesis within the pharyngeal arches during zebrafish development. It is possible that these observations will become relevant to the study of malformation in human craniofacial development. In a related project a relative of the Myc oncogene named Mych has been isolated from zebrafish and shown to be required for cell survival during neural crest formation. These studies extend other studies that have indicated a role for the Myc family of factors in neural crest formation. [unreadable] [unreadable] Protocadherin 18 is a member of a large family of cell surface proteins with multiple roles in determining cell behavior. This laboratory has characterized protocadherin 18 in zebrafish and shown its expression in several tissues during the development of the embryo. By cell transplantation it was shown that overexpression of protocadherin 18 causes diminished cell migration and reduced cell protrusions, resulting in a tendency of cells to stay more firmly aggregated, probably due to increased cell adhesion. These data suggest a role for protocadherin 18 in cell adhesion, migration and behavior but not cell specification during gastrula and segmentation stages of development.[unreadable] [unreadable] Brd4 is a member of the family of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. Brd4 has a role in cell cycle progression, making it indispensable in mouse embryos and cultured cells. Further, the N-terminal domain of Brd4 participates in a fusion oncogene. Brd4 associates with acetylated histones in chromatin, and this association persists during mitosis implicating Brd4 in epigenetic memory. This laboratory has shown that Brd4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition, indicating that the Brd4-chromosome association is a conserved property that is maintained even prior to zygotic transcription. The association of Brd4 with acetylated histones may also be conserved in early embryos as this laboratory found that histones H3 and H4 are already acetylated during pre-MBT stages.[unreadable] [unreadable] An extensive DNA microarray analysis was carried out to characterize the RNA populations in the zebrafish pineal gland and compare them to the RNA populations in the brain at different stages of development and adult life. The pineal gland has an important role in regulating circadian rhythm in the animal. The pineal transcriptome was analyzed at three embryonic and two adult stages of development, allowing developmental analysis of gene expression in addition to the identification of genes preferentially expressed in the pineal gland. The resulting experimental results allow evaluation of developmental changes in the pineal gland, of differences in gene expression in the pineal gland compared to the brain, and comparison of diurnal differences. The results indicate that a large set of genes is preferentially expressed in the pineal gland during development. The data support the known role of the fish pineal as a photoreceptor organ as genes involved in photoreception and transduction were prominent among the pineal-enriched genes. Frequently, different isoforms of certain proteins were expressed in the pineal gland as compared to other organs, e.g., the eye. Developmental changes visualized in these studied suggest that the embryonic pineal gland is enriched for regulatory factors such as transcription factors and signal transducers whereas adult pineal glands express genes involved in physiological functions such a enzymes, photo transduction factors, and structural components.