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. The initial dorsal-ventral axis of the zebrafish embryo is established by transcriprional control exerted by the Wnt pathway effector molecule beta-catenin, which becomes localized in the nucleus of one side of the early embryo. This side will form the organizer at the early gastrula stage. One of the first consequences of beta-catenin function is activation of the homeobox gene encoding the transcriptional repressor Bozozok/Dharma/Nieuwkoid (hereafter called Boz). Boz functions by repressing ventral genes such as vent, vox, ved and bmp. We found that the maternally inherited E3 ubiquitin ligase LNX-Like can bind and ubiquitinate Boz, targeting it for proteasomal degradation. If LNX-L is depleted from the embryo, the embryo is dorsalized because of expansion of the domain in which Boz is active, consequently increasing the size of the organizer. The presence of LNX-L balances the organizer and ventral domains in the embryo, assuring appropriate axis formation and normal development. These studies point to the modulation of protein stability as a fundamental mechanism in the regulation of early embryogenesis in the zebrafish. 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. Several novel genes, not previously known to be differentially expressed in the pineal gland, were indentified in these studies. Detailed molecular-genetic analyses of selected genes in this group have been initiated, promising to provide insights into certain aspects of the differentiation of the pineal gland. 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 witth Alcian blue eexhibit 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. The establishment of left/right (L/R) asymmetry is a fundamental step in the development of bilaterian embryos. This topic has considerable applied interest as L/R asymmetry defects are comparatively common in humans, causing a variety of disease states including heart and vascular malformations and kidney defects. The molecular mechanisms of initial establishment of L/R asymmetry has been much studied, and the importance of the Nodal and Shh pathways has been emphasized. Figroblast Growth Factor (Fgf) signaling is also known to be involved in laterality establishment, but its role has not been analyzed in detail previously. In particular, is was not known what factors function downstream of Fgf signaling in this process. We have identified two previously known but uncharacterized proteins, Ier2 and Fibp1, as downstream effectors of Fgf signaling in the zebrafish embryo. The genes encoding both of these proteins are activated in response to Fgf signaling, and both Ier2 and Fibp1 are required for normal laterality establishment in the embryo. In embryos depleted of Fgf8, or of Ier2 and Fibp1, laterality is not establsihed normally. The proximal effect of a loss of Ier2 and Fibp1 are defects in the formation of Kupffer's vesicle, the laterality organ of the zebrafish. Specifically, cilia that normally line Kupffer's vesicle and have a role in laterality establishment, do not form in normal numbers and normal size in Ier2 plus Fibp1-depleted embryos. We suggest that an Fgf signal is mediated by the function of Ier2 and Fibp1 to promote ciliogenesis in Kupffer's vesicle during normal development in the zebrafish. The alpha2 macroglobulin gene of zebrafish was isolated during our gene discovery screens. This gene was shown to be expressed early in liver formation, and was required for liver differentiation. The alpha2 macroglobulin gene represents a useful marker for the developing liver in the zebrafish embryo. In a collaborative study with Brian Brooks of the National Eye Institute we studied the role of the related transcription factors Nlz1 and Nlz2 during closure of the optic fissure. Gene knockdown of Nlz1 and/or Nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. In a collaboration with David Thomas of Melbourne we used WIF1 minus mice to study the role of Wnt signaling in osteosarcoma. We had previously identified Wnt Inhibitory Factor 1 in zebrafish and Xenopus, and had generated knock-out mice for this gene. While these mice are viable and fertile, the group of David thomas could show that the absence of WIF1 leads to accelerated formation of osteosarcoma in mice.
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