Studies On The Amphioxus Development Correlated Gene: Smads, Tmp21 And Comparative Researches On The Homologous Rab23 Gene In Amphioxus And Zebrafish

Cephalochordate amphioxus, widely considered as the closest living invertebrate relative to vertebrates, is a valuable model for investigating the animal embryonic development and evolution. Here, we report the studies on the gene of AmphiSmad1, AmphiSmad4, AmphiTmp21, and comparative researches on AmphiRah23 and ZebraRab23. Among these genes, AmphiSmad1 and AmphiSmad4 encode two key signaling transducers functioned in TGF-β pathway; AmphiTmp21 encodes a kind of protein correlated with the membrane trafficking process; Rab23 encodes a negative regulator which is essential in sonic hedgehog signaling pathway.TGF-β(transforming growth factor-β) superfamily members, which are expressed in most cell types, regulate cell proliferation, differentiation, immigration, apoptosis and play important roles in the development of whole body and the process of tissue morphogenesis. Most signal tranducers in this superfamily orchestrate the induction of mesoendoderm and the formation of dorsal-ventral body axis. The Smads protein family are the only established intracellular effecttors of TGF-β signaling, which transfer the TGF-β signal from cell surface to the nucleus, then promote the transcription of target gene. Besides, Smads can regulate the transcription of correlated genes involved in VEGF(Vascular endothelial growth factor) and BMP(Bone morphogenetic factor) pathways. So studies on the Smads genes make great sense in biological science. Collaborated with Sun Yat-Sen(Zhongshan) Universtiy, we cloned the complete sequence of Smad1 and Smad4 in amphioxus. Then we independently analyzed these two genes with systematic evolutionary methods. Also, the spatial and temporal expression patterns of AmphiSmad1 and AmphiSmad4 were investigated by whole mount in situ hybridization followed by sectioning. The results showed that AmphiSmad1 and AmphiSmad4 are highly conservative in their functional domains; they may have close relationships with the differentiation of amphioxus mesoendoderm; AmphiSmad1 may play roles in the development of amphioxus myotomes.Tmp21 is a kind of transmembrane tranficking protein, also called p23, i.e. p24deltal. It belongs to the p24 protein family, which usually exist in form of complex and are enriched in COPI-coated vesicles, these vesicles are involved in membrane traffic between the ER and Golgi complex. Through knock-out experiment, the generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. In addiotion, through immunohistochemistry examination in a patient with cancer-associated retinopathy syndrome, besides correlated lung cell carcinoma cell lines, the only other tissue in which immunoreactivity against p23 could be found was the optic nerve. Analyzing the subtract cDNA library of Branchiostoma belcheri tsingtauense online, we screened the EST of AmphiTmp21. Through online Blast tool on the genome website of Branchiostoma floridae, we got the complete cDNA sequence of AmphiTmp21. Then systematic and evolutionary analyses were performed. The spatial and temporal expression patterns in amphioxus enbryo and adults were investigated by whole mount in situ hybridization followed by sectioning. The results suggested that AmphiTmp21 may be closely related with the process of pigment diposit in the development of amphioxus sensory system.Sonic Hedgehog(Shh) pathway plays important roles in the process of embryonic development, whether its signaling is regular or not is straight related to the formation of dorsal-ventral axis in neuron tube and the development of the somites. In adult tissues, irregular activation of Shh pathway will lead to carcinogenesis. Rab23 is an essential negative regulator of Shh pathway. Based on the homologous characteristic of this gene in various species, we cloned the complete cDNA sequence of AmphiRab23, investigated the spatial and temporal expression pattern in amphioxus. In order to explore the embryonic function of AmphiRab23, the expression vector containing the whole AmphiRab23 ORF was build up, then we transcripted its mRNA in vitro and injected the mRNA into the zebrafish embryos. In zebrafish, we found the complete cDNAs of Rab23 in the library of Genebank. As same as in amphioxus, the expression patterns in zebrafish embryos were investigated and the expression vector containing the whole ZebraRab23 ORF was build up. Compared to the overexpression of ZebraRab23, we build up a RNAi vector for gene silence so as to further evaluate its developmental funtion. The elementary studies showed that Rab23 may play important role in the later development of the neron tube both in amphioxus and zebrafish. In addition, Rab23 is highly conservative from invertebrate to vertebrate and there is no functional difference between AmphiRab23 and ZebraRab23.