Study In The Regulation Of Convergent Extension Movements By Ror2in Zebrafish

The receptor tyrosine kinase-like orphan receptor Ror2is a number of RTKsfamily and plays important function in human skeletal development and tumorgenesis.In mammalian like mouse, Ror2mutant mouse show respiratory dysfunction and leadto neonatal lethality. Xror2and its mutant also inhibit convergent extension. So Ror2may play important roles in early development stages, then we use zebrafish embryoas model to investigate it. The recent has shown that Ror2, as a non-Frizzled receptor,could interact with Wnt5a to mediate CE movement.Convergent extension is a major cell movement during gastrulation and playsimportant roles in the vertebrate early embryonic development. Specifically, multiplegenetic evidences underscore an important role in regulating CE movements duringzebrafish embryo gastrulation.Though Ror2could interact with Wnt5a to mediate CEmovement, zebrafish Wnt5a is expressed at low levels during gastrulation andknockdown did not result in CE defects. However, zebrafish wnt11/slb exhibitedcompromised CE movements. Wnt11could regulate cell movements in zebrafish byinteracting with Frizzled-7and Flamingo, then could Wnt11mediate CE movementvia Ror2? Hiroki Hikasa et al indicated that co-expression of XRor2with XWnt11,XFz7, or both, synergistically inhibited convergent extension in embryos; and theectodomain of Xror2interacted with Xwnt11in co-immunoprecipitation expreriment.This study provided some messages, but the interaction of Wnt11with Ror2in vivohas not been covered.So there are some questions. Could Ror2function duringzebrafish CE movement? Could Wnt11mediate CE movement via Ror2? CouldWnt11bind to Ror2? All of these questions need to be answered.We first cloned zebrafish Ror2, and showed high similarity with knownsequence from human, mouse and Xenopus Ror2. Analyzing embryos of developmentstages by RT-PCR displayed that Ror2is a maternal gene and highly expressed at6-9hpf, suggesting Ror2may be function during gastrulation. Forced expression ofRor2mRNA in zebrafish embryos inhibited the medio-lateral narrowing andanterior-posterior lengthening of the development axis, resulted in CE defects. Theresult suggested that Ror2could function in CE movement.This phenotype was similar to that observed in Wnt11mRNA injected embryos.Co-injection of low-dose Ror2and Wnt11mRNA induced severe CE defects, whereasthese low-dose mRNAs by themselves did not affect development. Threewell-characterized markers (hgg, dlx3b and ntl) were also performed to verify thisphenomenon. These data suggest that zebrafish Ror2and Wnt11could interact witheach other in CE movement. To further understand function of Ror2,Morpholinos-mediated Ror2knockdown was performed in zebrafish embryos, whichshowed that the phenotype were normal as wild type. This may be due to the presenceof maternal Ror2proteins as well as redundant functions of zygotic Ror2products. Then a dominant-negative form of Ror2, Ror2-TM, was used to inhibit the ability ofwild-type Ror2. Injection of dnRor2mRNA in zebrafish embryos caused CE defectsand incomplete separation of eyes at later developmental stages. This phenotype wassimilar to that observed in the slb/wnt11embryos. Co-expression low-dose of dnRor2and Wnt11MO acted in synergy to strengthen CE and eyes defects. Furthermore,co-expression of dnRor2and Wnt11mRNA inhibited the action of Wnt11mRNA inCE defects. These data suggest that Wnt11could interact with Ror2to modulate CEmovements in zebrafish embryos. To further test this hypothesis,co-immunoprecipitation analysis indicated that Wnt11was able to interact with Ror2.Further, Ror2Y647F and S863A mutants were co-injected with Wnt11mRNArespectively, but the synergy effect in CE movements were seriously impaired,suggesting that the two sites were crucial to mediate convergent extensionmovements.Taken together, in zebrafish, Wnt11-Ror2interaction regulates convergent andextension movements through the Y647and S863of Ror2. These results also providenovel insights into the convergent and extension movements in vertebrategastrulation.