Slit-like 2, A Novel Zebrafish Slit Homologue That Might Involve In Zebrafish Central Neural And Vascular Morphogenesis And Dec1, Dec2 Differentially Regulate Zebrafish Cardiogenesis

(1)Nervous and vascular systems grow as parallel networks, indicating common cues in distal targets. We have identified a novel zebrafish gene Slit-like 2 (Slitl2) that might involve in zebrafish central neural and vascular morphogenesis. Whole-mount in situ hybridization of zebrafish embryo detected distinct signals of Slitl2 transcripts in zebrafish midline structure of central nervous system similar to that of Slits. Strong expression is also observed in zebrafish vasculature. Zebrafish Slitl2 shares amino acid sequence identity of 41% with Homo sapiens Slitl2 (vasorin) and Mus musculus Slitl2, and 35%, 33% with Danio rerio Slit3, Slit2. Analysis of zebrafish Slitl2 cripto growth factor domain, extracellular matrix protein Slit domain, and putative signal peptide confirms that as a secreted and cell-surface protein Slitl2 may be essential in axon guidance, vessel development, and axis patterning. Micro-injection of either Slitl2 siRNA vector or pcDNA3-Slitl2 cause lethal embryos, malformations in central neural midline and cardiovascular system, which implies that knock-down and over-expression of Slitl2 will disturb the normal formation of zebrafish central neural and cardiovascular system, as well as axial patterning. These results provide evidence that Slitl2 may play important roles in zebrafish central nervous system and vascular morphogenesis.(2)Dec1 and Dec2 have been investigated in cellular poliferation, differentiation and circadian regulation, however, the developmental roles of the two genes in embryogenesis are not clarified. We have cloned the zebrafish Dec2 gene that might involve in zebrafish heart morphogenesis, together with zebrafish Dec1. Sequence analysis shows that the two genes are evolutionally conserved and may have related functions. Whole-mount in situ hybridization reveals that Dec2 expresses dynamicly in central neural system, early somite and cardiovascular system, partially overlap, but distinct from that of zebrafish Dec1, and Dec2 expression is much weaker than that of Dec1 in early zebrafish embryogensis. The two genes have mutual exclusive intracellular localiztion with Dec1 in the nucleus and Dec2 presents predominantly in the cytoplasm. Down regulation of Dec1 results in zebrafish cardiac atrium expansion, while excess Dec1 causes disturbance in heart asymmetry patterning. Dec2 deficient embryos fail to undergo proper heart looping, and Dec2 over-expressed embryos show enlarged cardiac atrium. Quantitative PCR and whole mount in situ hybridization confirmed that Dec1 and Dec2 reciprocally cooperate to maintain the equilibrium in normal cardiogenesis, with Dec1 as a dominant factor. Our findings also provided an example of a pair of highly homologous genes regulate embryonic development in a reciprocal fashion.