Cloning, Developmental Expression, And Functional Analysis Of The Zebrafish Roof Plate-specific Spondin3Gene

Wnt signaling is one of the key signaling pathways controlling cell proliferation,differentiation, and morphogenesis during embryogenesis and in adults. TheR-spondins (Rspos) are a recently described family of4secreted proteins (Rspo1-4).Reported activities for Rspo proteins include essential roles in vertebrate development.Their ligand-type activities overlap substantially with those of the canonical Wntligands in that both Rspo and canonical Wnt signaling result in the activation ofβ-catenin. However, their structures and functions in teleost fish are poorlyunderstood. In this study, we report the identification and characterization of thezebrafish Rspo3gene. Structure analysis reveals that the zebrafish Rspo3is unique inthat it has3furin-like domains in contrast to the presence of2furin-like domains inall known mammalian Rspo3genes. To determine whether this unique structure iscommon among teleost Rspo3, we determined the structure of medaka, fugu, andstickleback Rspo3and found that they all contained3furin-like domains. Rspo3mRNA was expressed in all embryonic and larval stages in zebrafish. The Rspo3mRNA level increased gradually from0to12hpf (hours post fertilization) andmaintained at relatively high levels thereafter. Rspo3mRNA was detected in multipletissues with the highest levels in neural and skeletal tissues. Forced expression ofRspo3mRNA in zebrafish embryos resulted in severe morphological abnormality,including enlarged head, shortened tank, and coiled tail. These phenotypes ofanteriorization of the neuroectoderm, anomalies of the dorsal nervous system, andloss of the posterior body structure were similar to those observed in the Wnt3a-and/or Wnt8knocked down embryos, also similar to those observed in dkk1or Frzbinjected embryos. We next co-injected Rspo3mRNA with Wnt3a or Wnt8.1mRNA.Co-expression of Rspo3markedly reduced the A/P elongation phenotype induced byWnt3a or Wnt8.1expression, suggesting that zebrafish Rspo3acts as antagonist in the canonical Wnt signaling pathway. Since it has been reported that mammalian Rsposcan be modulated by dkk1activity, which is a negative modulator of theWnt/β-catenin pathway, we next co-injected zebrafish embryos with Rspo3mRNAand dkk1mRNA. Co-expression of Rspo3and dkk1enhanced dkk1-caused dorsalphenotypes (enlarged head structure and shortened anterior-posterior axis). Theseresults suggest that fish Rspo3has unique structural features and may play a complexrole in modulating the Wnt/β-catenin signaling pathway.