Study On Amphious Sox7-like Gene In Endoderm Development

Amphioxus, a cephalochordate, is regarded at the base of Chordata and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. Transcription factor Sox7and Sox17of vertebrates are expressed in endoderm, playing an important role in early embryonic development and endoderm formation. Lots of research of Sox7and Sox/7in zebrafish, clawed frogs and other creatures indicate that they can induce expression of the pan-endoderm marker Edd, showing that they are essential for endoderm formation. It is not clear whether there are any genes homologous to vertebrate Sox7or Sox17in Amphioxus, and whether it is related with endoderm formation and other regulatory activities. Here we report the sequence, phylogenetic analysis and expression pattern of AmphiSox7-like gene. Functional analysis of this gene in zebrafish embryos provided evidence for its role in endoderm signaling pathway. and the molecular basis and mechanism during embryonic development, and reveal the function evolution of AmphiSox7-like in the evolutionary process.The ORF of AmphiSox7-like is1824bp long, encoding a protein of607amino acids. Sequence analysis showed that AmphiSox7-like contained a HMG-box and a characteristic C-terminal Sox-C-TAD motif, the same as the Sox family members in other species, which are important for mature protein function. The AmphiSox7-like sequence conservation also implies the importance of its function in various species during evolution. Although only one AmphiSox7-like, which is homologous to both Sox7and Sox17, was found in amphioxus genome, phylogenetic analysis showed that AmphiSox7-like groups together with a vertebrate cluster of Sox7, which is independent from a cluster of Sox17.Real-Time PCR revealed that AmphiSox7-like transcripts exist in the zygote as a maternal mRNA. The maternal expression is reduced in3h early blastocyst. After that its zygotic expression increases by6h early gastrula stage. The highest expression level was detected in the late gastrula around9h. Then it declines until48h larval stage when very low level of expression remains. In situ hybridization confirmed the maternal expression of AmphiSox7-Iike transcripts. In addition, in situ hybridization revealed an asymmetry expression pattern of AmphiSox7-like in blastocyst. In vegetal hemisphere the AmphiSox7-/ike signal is higher than in the animal hemisphere. At gastrula stage the AmphiSox7-like is expressed in the inner layer predetermined mesendoderm cells.In order to study the function of the AmphiSox7-like, a pCS2+-AmphiSox7-like expression vector was constructed. AmphiSox7-like mRNA was transcribed in vitro and microinjected into zebrafish embryos at one cell stage. This resulted in abnormal phenotypes such as eyeless, small eyes, monolateral eyes, small heads, bent axis and cardiac edema. Real-Time PCR and in situ hybridization showed that the expression of endoderm marker Sox32was up-regulated, wherease mesodermal marker ntl and ectoderm marker otx2were down-regulated in AmphiSox7-like overexpressed zebrafish embryos. Furthermore, AmphiSox7-likt mRNA can rescue the phenotype of Sox7and Sox17morpholino knockdown zebrafish embryos in a dose dependent manner. The results indicated that AmphiSox7-like promotes the endoderm development and its function might overlap with DSox7and DSoxl7to some extent. This is the first gene identified in the regulatory cascade in amphioxus endoderm development, and the charatcrization of this gene implies the conservation of the mechanism of endoderm development in amphioxus and vertebrates.