1. Proteomic Analysis Of Zebrafish Single Embryo During Different Stages Of Embryonic Development 2. The Effect Of Knock-down Vsx1 On Development Of Early Goldfish Embryos

Partâ… :Proteomic analysis of zebrafish single embryo during different stages of embryonic developmentZebrafish has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development.The zebrafish genome has been sequenced and annotated allowing for high-throughput proteomic analysis.In this work,we analyzed the proteome of zebrafish single embryo at 3 and 5 days post fertilization.We used a method to efficiently remove the yolk from zebrafish embryo at 3 days post fertilization.Then three methods of protein tryptic digestion, namely using buffer-assisted,1%SDC-assisted and mild performic acid oxidation(PAOT),were used to dissolve and analyze the proteomic of zebrafish single embryo at 3 days post fertilization and 5 days post fertilization.From the 3 days post fertilization embryo sample,a total of 366 proteins from SDC-solubilized sample,162 proteins from PAOT-solubilized sample and 363 proteins from buffer-assisted digestion,were identified by using trypsin digestion and liquid chromatography-electrospray ionization tandem mass spectrometer(LC-ESI MS/MS).Among the 493 identified proteins, 108(21.9%)were identified by three methods,86(17.4%)were identified exclusively by buffer-assisted digestion,and 92(18.7%)were identified exclusively by SDC-assisted digestion.We demonstrate that greater proteomic dataset can be achieved by combining the result from two methods of protein digestion:one in buffer,and the other in SDC.Using these methods,we identified a total number of 449 proteins from deyolked zebrafish embryo at 3 days post fertilization,260 proteins from zebrafish embryo at 3 days post fertilization and 212 proteins from embryo at 5 days post fertilization, respectively.About 43%of all identified proteins were detected at both embryonic stages.Identified proteins were functionally classified,and we discuss different expression and the functions of these proteins during the two different developmental stages of zebrafish embryo.Partâ…¡:The effect of knock-down vsx1 on development of early goldfish embryosVsx1 was proposed to be a regulatory gene related with spatial patterning of eyes and diploid-dependent regulatory mechanism of goldfish.In order to investigate the function of vsx1 in the development of goldfish embryos and the role in diploid-dependent regulatory mechanism,we performed Morpholino antisense Oligos knock-down experiment with microinjection,and found the change of proteome due to the silence of vsx1,then investigated the function and the regulatory pattern of vsx1.We used embryos injected with morpholino antisense oligos (ATG-MO)and with standard control(SCON-MO)as starting materials.By observed the development of embryos,we found that most of the embryos injected with ATG-MO could not continue to develop at the early stage of gastrulation.It indicated that ATG-MO leaded to the silence of vsx1 in gastrula stage.As is well know,the gastrulation is the crucial time for organogenesis and formation of endoderm,mesoderm and ectoderm. Therefore,we speculated that vsx1 is a multi-purpose gene, which participate in the specification and differentiation of germinal layer in the goldfish embryonic development process.Differential proteome expression analysis was performed by using 2-DE(two dimensional gel electrophoresis)and PDQUEST software,more than one hundred spots were visualized,and 84 spots were identified by Mass spectrometry.21 protein spots expressed in the embryos injected with ATG-MO only,and 6 protein spots expressed in the embryos injected with SCON-MO only.47 protein spots were up-regulated and 10 protein spots were down-regulated in the embryos injected with ATG-MO.By analyzing the function of the identified proteins,we found many proteins that involved in transcription regulation,cell cycle control,and cell differentiation,such as Prohibitin-2, Zinc finger transcription factor detour,BMP typeâ…¡a receptor, were all up-regulated or expressed only in the embryos injected with ATG-MO.It accorded with the previous report and suggested that vsx1 was a transcriptional repressor.It was suggested that vsx1 may regulate various functional genes through these genes indirectly.And we also find some differentially expressed proteins related with signal transduction,cell differentiation and nervous development,which are likely to regulated by vsx1 directly or indirectly.