Eric Weinberg

genetic control of embryonic pattern, brain development, and organogenesis in the zebrafish embryo

Our laboratory works on developmental patterning, tissue differentiation, and organogenesis in the zebrafish embryo, focusing on development of the nervous system, inner ear, and heart. The zebrafish system is an excellent one for the study of vertebrate development since the organism has a rapid generation time, is easy to maintain, and is suitable for genetic analysis. A major advantage is that mutations can be screened easily in developing haploid or diploid embryos.

One group of projects centers on the early patterning of the forebrain and midbrain. We have been studying the zebrafish homeobox genes Otx1 and Otx2, which are expressed in the gastrula region that corresponds to the prospective forebrain and midbrain. The patterns of Otx expression during formation of the neural plate and neural tube are suggestive of a role in forebrain and midbrain specification and knockouts of the murine homologs show that Otx2 is necessary for formation of the anterior brain in the mouse. We have carried out RNA injection experiments to overexpress both Otx proteins in the zebrafish and found that one of the functions of these proteins may be to stimulate adhesion of the expressing cells. We have shown, using a novel lineage tracing method employing caged fluorescein, that cells in the prospective anterior brain maintain their cell-cell contacts through the period between gastrula and neural tube formation. The enhancement of cell adhesion in cells which ectopically express Otx proteins is thus consistent with the behavior of cells that normally express these proteins during embryogenesis.

Our work on early pattern formation concerns the maternal control of dorsoventral and anteroposterior axis formation. We have identified a recessive maternal effect mutation, ichabod, that results in embryos with a severe anterior and dorsal deficit. ichabod phenotype embryos completely lack heads or are partially deficient in anterior head structures, lack notochords, and often have an excess of posterior and ventral structures. Breeding experiments indicate that the mutation has its effect only in homozygous females. Phenotypes were further characterized by testing for activity of genes with characteristic anterior-posterior and dorsal-ventral expression patterns. Expression of several dorsal markers including goosecoid, Otx1, and Otx2, was absent or reduced and ventral markers such as gata1, eve1, and BMP-4 were expressed in expanded domains. These results indicate that the affected embryos have a respecified pattern of dorsoventral identity and that this altered pattern can be recognized even in the early gastrulating embryo. Although ichabod phenotype embryos can express the beta-catenin gene, no beta-catenin protein is detected in dorsal cell nuclei as in wild-type embryos. Injection of ichabod embryos with beta-catenin RNA can rescue the embryos and over half of such injected embryos survive at least two months. Injection of RNAs encoding proteins of the Wnt signaling pathway indicates that the mutation is in a gene upstream of beta-catenin, but is not in the beta-catenin gene itself. These results demonstrate that maternal components are required to properly utilize the beta-catenin signaling system to establish the organizer in zebrafish.

Our work on inner ear development is proceeding on several fronts. First, we are using lineage tracers such as uncaged fluorescein, to determine the gastrula fate map of the precursors of the otic vesicle placode. Second, we are performing transplantation experiments to discover the time of commitment of cells to become inner ear and to differentiate into particular inner ear structures. Third, we are studying a group of mutants affecting inner ear morphology, isolated in the Tübingen genetic screen. We also plan to obtain additional mutants by screening additional genomes.

With Alvin Chin, Associate Professor of Pediatrics with the Children's Hospital of Pennsylvania (CHOP), we are studying the formation of the heart in the zebrafish embryo. Dr. Chin has found that the BMP-4 gene is expressed in regions that are boundaries between developing chambers of the heart and vasculature. He has now used a series of mutant embryos – deficient in notochord and other structures – to demonstrate that initial heart laterality can be uncoupled from the direction of heart looping. This finding has led to a model of independent signals of laterality and looping.