During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. We have isolated a pair of oligodendrocyte lineage genes (Olg-1 and Olg-2) that encode bHLH proteins and are tightly associated with development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo.

We have identified a novel role of a signaling pathway comprised of PDGF, PI3K, and PKB in the control of morphogenetic cell movements during gastrulation. Furthermore, our findings provide insight into the relationship between cell polarization and directed cell migration at the onset of zebrafish gastrulation.

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a smallmolecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cγ1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.In the postgenomic era, assigning gene function and delineating signaling pathways require the combined effort of multiple disciplines and approaches. The use of chemical probes has immense potential in examining biological processes and developing specific therapeutic compounds. On the biological side, these goals can be achieved through the appropriate use of model systems. In vitro and cell-based assays have been widely used for drug discovery and chemical library screening 1-3 . Whole organism approaches are also possible using yeast, worms, flies or zebrafish embryos 4,5 . Of these, the zebrafish, as a vertebrate organism, has reasonable counterparts to many mammalian organs, tissues and cell types. As such, it affords an opportunity to investigate more complex biological processes 5 . The transparency of the zebrafish embryo has facilitated visual scoring of phenotypic defects. Thus, it has been used extensively for developmental biology and genetics, and in the last few years as a new model Correspondence should be addressed to J.C. (joanne.chan@childrens.harvard.edu).. 6 These authors contributed equally to this work. COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Chemical Biology website for details).Note: Supplementary information is available on the Nature Chemical Biology website. NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2006 August 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript for chemical biology 4,5 . However, tissue growth and differentiation are very different in an embryo versus in an adult animal. Embryonic development involves precise coordination of genetic programs that allow the building of a whole organism from a single cell. Therefore, chemical genetic analysis in embryos is dictated by the timing of developmental events. In an adult animal, organ maintenance and cellular needs are different, with turnover and repair being important. Another crucial consideration in developing therape...

Striking homology between signaling molecules in zebrafish and humans suggests that compounds known to inhibit human kinases may enable a chemical genetic approach to dissect signaling pathways in the zebrafish embryo. We tested this hypothesis using a vascular endothelial growth factor receptor inhibitor, PTK787/ZK222584. Zebrafish embryos treated with this compound lacked all major blood vessels. Overexpression of AKT/PKB, a putative effector of vascular endothelial growth factor signaling, allowed blood vessels to form in the presence of drug. Endothelial cell apoptosis induced by the drug is prevented by increasing AKT/PKB activity, thus establishing the physiological relevance of AKT/PKB in the angiogenic process. This approach allowed us to examine the effects of blood flow and the role of endothelial signals in organogenesis.

Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway. Growth factors acting through Ras primarily stimulate the Raf/MEK/ MAPK cascade of protein kinases. In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK-(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38 hog pathways. Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways. These studies suggest a high degree of cross-talk apparently not seen in vivo. We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways. We report preferential association between MAP kinases and their activators in eukaryotic cells. Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins. We show that SAPK(JNK) or p38 hog activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38 hog phosphorylation. Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members. The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.Genetic studies in yeast first revealed the existence of multiple distinct mitogen-activated protein kinase (MAPK) 1 related signal transduction pathways containing structurally similar protein kinase cascades mediating responses to mating factor, hyperosmolarity, and cell wall integrity (1). Pathways in mammals, which likely have different physiologic significance, have been discovered which utilize related protein kinase cascades (2-4). The archetypal MAPK pathway is activated in response to Ras-GTP loading as well as other processes such as phosphatidylinositol turnover (5-7). The pathway comprises a series of protein kinases such that activation of the Raf protooncogene causes phosphorylation and activation of MEK which, in turn, phosphorylates and activates the MAPKs, Erk1, and -2 (4, 8). The targets of these kinases include other protein kinases and transcription factors such as p62 tcf (9 -11). While mitogens and growth factors commonly stimulate this MAPK pathway, cells respond to cellular stress agents by induction of two structurally related but distinct pathways (2, 12-16). Stres...

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Vascular dysfunction has been reported in human cases of anthrax, in mammalian models of Bacillus anthracis, and in animals injected with anthrax toxin proteins. To examine anthrax lethal toxin effects on intact blood vessels, we developed a zebrafish model that permits in vivo imaging and evaluation of vasculature and cardiovascular function. Vascular defects monitored in hundreds of embryos enabled us to define four stages of phenotypic progression leading to circulatory dysfunction. We demonstrated increased endothelial permeability as an early consequence of toxin action by tracking the extravasation of fluorescent microspheres in toxin-injected embryos. Lethal toxin did not induce a significant amount of cell death in embryonic tissues or blood vessels, as shown by staining with acridine orange, and endothelial cells in lethal toxin-injected embryos continued to divide at the normal rate. Vascular permeability is strongly affected by the VEGF/ vascular permeability factor (VPF) signaling pathway, and we were able to attenuate anthrax lethal toxin effects with chemical inhibitors of VEGFR function. Our study demonstrates the importance of vascular permeability in anthrax lethal toxin action and the need for further investigation of the cardiovascular component of human anthrax disease.endothelial ͉ vascular permeability ͉ VEGF

Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/ RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.