br Studies in zebrafish vascular and neural development
Studies in zebrafish: vascular and neural development and the regulation of left–right asymmetry Zebrafish embryos represent an excellent model system to examine vertebrate vascular development. Major advantages of the zebrafish system include its genetic tractability, small size, external fertilization, rapid development and optical transparency, and studies on zebrafish have provided many novel insights into vascular morphogenesis . In early zebrafish embryos (at 30 hpf), ATX mRNA is detected in the head region, the neural tube and the vasculature, including the dorsal VU 0155069 mg . Knockdown of ATX by injecting morpholino oligonucleotides (MOs) against zATX into embryos resulted in severely reduced blood flow and aberrant vascular connections around the horizontal myoseptum. Additional LPA receptor knockdown studies suggested critical, synergistic roles for LPA1, LPA4 and LPA6 in regulating the zebrafish vasculature. LPA1 has also been implicated in lymphatic vessel development in zebrafish . The mechanism by which ATX–LPA signaling regulates zebrafish vessel formation is unknown, although circumstantial evidence points to a predominant role for ATX/LPA-mediated migration of endothelial cells, rather than their proliferation or differentiation . Another advantage of the zebrafish system is that embryos can develop without a fully functional vascular system up to 7days after fertilization. A recent gene silencing study has uncovered a role for ATX in promoting the differentiation of oligodendrocyte progenitor cells in the zebrafish hindbrain . This probably occurs via a paracrine signaling mechanism, with the cephalic floor plate being the most likely source of secreted ATX. It will be interesting to examine whether the ATX–LPA signaling axis may play a similar role in the maturation of mammalian oligodendrocytes. Conditional knockouts targeting implicated LPA receptor subtypes in the oligodendrocyte lineage should help to answer this question. Our own studies on ATX in zebrafish are largely in agreement with the above findings by Yukiura et al.  (A. Houben, unpublished results). Using the MO knockdown approach, we found that ATX-deficient embryos show vascular branching defects and a strongly reduced blood flow, most likely due to severe heart malformations. The major cardiac defects observed include weak contraction, edema and loss of cardiac jogging and looping resulting in a linear heart (Fig. 3A), suggesting a role for ATX in establishing left–right patterning. Direct evidence for ATX–LPA signaling playing an important role in the regulation of left–right (L–R) asymmetry comes from a recent study by Lai et al. . Through MO-based approaches, it was shown that the ATX–LPA3 receptor signaling axis is required for the formation of Kupffer's vesicle (KV), a central organ for establishing L–R asymmetry in zebrafish (equivalent to the mouse “node” around E8.0; ref. ), and subsequent induction of asymmetric genes, notably lefty1/2, pitx2 and spaw (Fig. 3B) . ATX/LPAR3-mediated KV formation was mediated by enhanced calcium signaling in the shield region and associated with Wnt signaling, as evidenced by the nuclear accumulation of β-catenin at the dorsal side of both atx and lpa3 morphants . Although KV formation in zebrafish may mechanistically differ from node formation in mice, future studies on ATX/LPA-dependent L–R patterning during mouse embryogenesis are warranted.
Concluding remarks Ennp2 knockout studies in mice have revealed a vital role for ATX in embryonic development, as ATX-deficient embryos die at midgestation with severely impaired vessel formation in yolk sac and embryo proper as well as other abnormalities. Furthermore, targeting studies in zebrafish confirm an essential role for ATX and multiple LPA receptors in regulating cardiovascular morphogenesis. ATX-mediated LPA production and subsequent LPA receptor signaling through the Gα12/Gα13-Rho-ROCK pathway, likely in cooperation with other G protein-effector systems, may contribute to vascular development by stimulating endothelial cell migration and invasion as well as by regulating adhesive interactions with the extracellular matrix. Consistent with this, the vascular defects observed in ATX-deficient mice resemble those in mice lacking genes involved in cell migration and adhesion such as fibronectin and focal adhesion kinase. Given the recent findings in zebrafish ,  it will be interesting to examine to what extent double and triple LPA receptor knockout mice recapitulate the Enpp2(−/−) phenotype and, furthermore, how the ATX–LPA receptor axis may govern left–right asymmetry. Further mechanistic insights into the importance of ATX in vertebrate development awaits the analysis of tissue-specific Enpp2 knockout mice, transgenic rescue studies, generation of mice deficient in multiple LPA receptors and further studies in zebrafish.