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...

Genetic analyses in zebrafish identify a novel physical signaling mechanism that drives formation of invadopodia-like structures and promotes cell invasion in vivo.

Smooth muscle cells provide structural support for many tissues and control essential physiological processes, such as blood pressure and gastrointestinal motility. Relatively little is known about early stages of intestinal smooth muscle development and its relationship to the development of the enteric nervous system, which regulates intestinal motility. Here, we report an evolutionarily conserved 523 base pair regulatory element within the promoter of the zebrafish sm22α-b (transgelin1) gene that directs transgene expression in smooth muscle cells of the intestine and other tissues. Comparative genomic analysis identified a conserved motif within this element consisting of two Serum Response Factor binding sites that is also present in the promoters of many mammalian smooth muscle genes. We established a stable line expressing GFP in smooth muscle cell and used this line to describe lineage relationships among cells within different intestinal smooth muscle layers and their co-development with the enteric nervous system (ENS).

Adherens junction remodeling allows changes in cell shape and position. Zilberman et al. show through live imaging that CDC-42 is dispensable for epithelial cell polarization, but its RhoGAP-regulated activity is needed to control junctional actin organization during embryo elongation.

Background The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Methods Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Key Results Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. Conclusions & Inferences These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism.

Background The high molecular weight isoform of the actin-binding protein Caldesmon (h-CaD) regulates smooth muscle contractile function by modulating cross-bridge cycling of myosin heads. The normal inhibitory activity of h-CaD is regulated by the enteric nervous system; however, the role of h-CaD during intestinal peristalsis has never been studied. Methods We identified a zebrafish paralog of the human CALD1 gene that encodes an h-CaD isoform expressed in intestinal smooth muscle. We examined the role of h-CaD during intestinal peristalsis in zebrafish larvae by knocking down the h-CaD protein using an antisense morpholino oligonucleotide. We also developed transgenic zebrafish that express inhibitory peptides derived from the h-CaD myosin and actin-binding domains, and examined their effect on peristalsis in wild-type zebrafish larvae and sox10colourless mutant larvae that lack enteric nerves. Key Results Genomic analyses identified two zebrafish Caldesmon paralogs. The cald1a ortholog encoded a high molecular weight isoform generated by alternative splicing whose intestinal expression was restricted to smooth muscle. Propulsive intestinal peristalsis was increased in wild-type zebrafish larvae by h-CaD knockdown and by expression of transgenes encoding inhibitory myosin and actin-binding domain peptides. Peristalsis in the non-innervated intestine of sox10colourless larvae was partially restored by h-CaD knockdown and expression of the myosin-binding peptide. Conclusions & Inferences Disruption of the normal inhibitory function of h-CaD enhances intestinal peristalsis in both wild-type zebrafish larvae and mutant larvae that lack enteric nerves, thus confirming a physiologic role for regulation of smooth muscle contraction at the actin filament.

Lumen extension in intracellular tubes can occur when vesicles fuse with an invading apical membrane. Within the C. elegans excretory cell, which forms an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane and is required for its outgrowth, suggesting that exocyst-targeted vesicles extend the lumen. Here, we identify a pathway that promotes intracellular tube extension by enriching the exocyst at the lumenal membrane. We show that PAR-6 and PKC-3/aPKC concentrate at the lumenal membrane and promote lumen extension. Using acute protein depletion, we find that PAR-6 is required for exocyst membrane recruitment, whereas PAR-3, which can recruit the exocyst in mammals, appears dispensable for exocyst localization and lumen extension. Finally, we show that CDC-42 and RhoGEF EXC-5/FGD regulate lumen extension by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a pathway that connects CDC-42, PAR proteins, and the exocyst to extend intracellular tubes.

Lumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. Using acute protein depletion, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.