Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning.

In vertebrates, wnt8 has been implicated in the early patterning of the mesoderm. To determine directly the embryonic requirements for wnt8, we generated a chromosomal deficiency in zebrafish that removes the bicistronic wnt8 locus. We report that homozygous mutants exhibit pronounced defects in dorso-ventral mesoderm patterning and in the antero-posterior neural pattern. Despite differences in their signaling activities, either coding region of the bicistronic RNA can rescue the deficiency phenotype. Specific interference of wnt8 translation by morpholino antisense oligomers phenocopies the deficiency, and interference with wnt8 translation in ntl and spt mutants produces embryos lacking trunk and tail. These data demonstrate that the zebrafish wnt8 locus is required during gastrulation to pattern both the mesoderm and the neural ectoderm properly.

Reverse genetics in zebrafish.

The zebrafish has become a popular model system for the study of vertebrate developmental biology because of its numerous strengths as a molecular genetic and embryological system. To determine the requirement for specific genes during embryogenesis, it is necessary to generate organisms carrying loss-of-function mutations. This can be accomplished in zebrafish through a reverse genetic approach. This review discusses the current techniques for generating mutations in known genes in zebrafish. These techniques include the generation of chromosomal deletions and the subsequent identification of complementation groups within deletions through noncomplementation assays. In addition, this review will discuss methods currently being evaluated that may improve the methods for finding mutations in a known sequence, including screening for randomly induced small deletions within genes and screening for randomly induced point mutations within specific genes.

The role of cell adhesion molecules in Drosophila heart morphogenesis: faint sausage, shotgun/DE-cadherin, and laminin A are required for discrete stages in heart development.

Heart development in the Drosophila embryo starts with the specification of cardiac precursors from the dorsal edge of the mesoderm through signaling from the epidermis. Cardioblasts then become aligned in a single row of cells that migrate dorsally. After contacting their contralateral counterparts, cardioblasts undergo a cytoskeletal rearrangement and form a lumen. Its simple architecture and cellular composition makes the heart a good system to study mesodermal patterning, intergerm layer signaling, and the function of cell adhesion molecules (CAMs) during morphogenesis. In this paper we focus on three adhesion molecules, faint sausage (fas), shotgun/DE-cadherin (shg/DE-Cad), and laminin A (lam A), that are essential for heart development. fas encodes an Ig-like CAM and is required for the correct number of cardioblasts to become specified, as well as proper alignment of cardioblasts. shg/DE-Cad is expressed and required at a later stage than fas; in embryos lacking this gene, cardioblasts are specified normally and become aligned, but do not form a lumen. Additionally, cardioblasts of shg mutant embryos show a redistribution of phosphotyrosine as well as a loss of Armadillo from the membrane, indicating defects in cell polarity. The shg phenotype could be phenocopied by applying EGTA or cytochalasin D, supporting the view that Ca2+-dependent adhesion and the actin cytoskeleton are instrumental for heart lumen formation. As opposed to cell-cell adhesion, cell-substrate adhesion mechanisms are not required for heart morphogenesis, but only for maintenance of the differentiated heart. Embryos lacking the lam A gene initially developed a normal heart, but showed twists and breaks of cardioblasts at late embryonic stages. We discuss our findings in light of recent results that elucidate the function of different adhesion systems in vertebrate heart development.

Embryonic development of the Drosophila brain. II. Pattern of glial cells.

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion.

faint sausage encodes a novel extracellular protein of the immunoglobulin superfamily required for cell migration and the establishment of normal axonal pathways in the Drosophila nervous system.

We examined the structure of the nervous system in Drosophila embryos homozygous for a null mutation in the faint sausage (fas) gene. In the peripheral nervous system (PNS) of fas mutants, neurons fail to delaminate from the ectodermal epithelium; in the central nervous system (CNS), the positions of neuronal cell bodies and glial cells are abnormal and normal axonal pathways do not form. Sequence analysis of fas cDNAs revealed that the fas protein product has characteristics of an extracellular protein and that it is a novel member of the immunoglobulin (Ig) superfamily. In situ hybridization demonstrated that fas transcripts are expressed throughout the embryo but they are in relatively high concentrations in the lateral ectoderm, from which the peripheral nervous system delaminates and in the CNS. Antiserum directed against Fas protein was found to stain neurons but not glia in the CNS. We conclude that fas encodes a protein that, in the developing nervous system, is present on the surface of neurons and is essential for nerve cell migration and the establishment of axonal pathways.

Delamination and division in the Drosophila neurectoderm: spatiotemporal pattern, cytoskeletal dynamics, and common control by neurogenic and segment polarity genes.

Cytoskeletal changes occurring during the delamination of precursors of the peripheral (microchaete precursors in the pupal notum) and central nervous system (embryonic SI neuroblasts) were studied. The pattern of cell division in the ventral neurectoderm (VN) of wild-type embryos was analyzed using BrdU incorporation and correlated to the pattern of neuroblast delamination. Finally, defects in the pattern of proliferation of the VN and neuroblast delamination which occur in Notch and wingless mutant embryos were described. The results indicate that the patterns of delamination and mitosis are closely correlated: delamination occurs either immediately after a cell has divided (in case of microchaete precursors) or shortly before the division (in case of the neuroblasts). In addition, cytoskeletal changes similar to those occurring during mitosis can be seen in delaminating neuronal precursors. Thus, during both mitosis and delamination, the discrete apicobasally oriented microfilament-tubulin bundles break down. Microfilaments form a dense, diffuse cortical layer surrounding the entire cell body. Microtubules are concentrated at the apically located centrosome. The relationship between mitosis and delamination is supported by the finding that the neurogenic gene Notch and segment polarity gene wingless (wg) affect both proliferation and delamination in the ventral neurectoderm. Thus, in embryos expressing the trunkated cytoplasmic domain of the neurogenic gene Notch under heat-shock control (Struhl et al., 1993), all ventral neurectodermal cells go into mitosis prematurely, followed by the absence of neuroblast delamination. In wg loss-of-function mutants, mitosis in the VN is irregular and generally postponed, accompanied by irregularities in the timing of neuroblast delamination in general and the absence of a subset of neuroblasts.