| The heart is one of the first organs to form during organogenesis in vertebrate.Heart development is complex and precise processes,which is controlled by a number of heart development related genes in spatial and temporary collaboratively.When the genes that control heart development are ectopic expression or dysfunctions,it can result in severe congenital heart development even death at embryos.Research on the functions of candidate genes that regulate heart development and theirs' molecular mechanisms,make us better understanding the processes of heart development and how the congenital heart disease happens at the molecular level.We used zebrafish as an animal model to research on functions of Pygol in the heart left-right asymmetry development,the maternal effects of Pygol and the role of gene fhlA in regulating of heart and skeletal muscle development.Homo sapiens Pygol gene localized in q21.1,reign of chromosome 15,the gene cDNA contains 1260 bp,consists of two introns and three exons.The protein Pygol contains a PHD zinc finger domain at its N-terminal,which belongs to the C6H5 zinc finger protein family.Pygol is highly conserved in evolution from invertebrates,vertebrates to Homo sapiens.Pygol has been identified as a new member involved in the Wnt signal pathway.In situ hybridization experiment was carried out to reveal the spatial and temporally expression pattern of Pygo1,digoxin labeled Pygo1 sense RNA was used as negative control.Zebrafish zygote expression begins in the period of blastosphere,at somatic stages 5-8,Pygo1 specifically expressed in Kupffer's Vesicle(KV);At somatic stages 15-18,Pygo1 specifically expressed on the heart progenitor cell,that is the lateral plate mesoderm;at somatic stage 22,it specifically expressed on the heart field;at somatic stage 25,Pygo1 exclusively expressed in the neural crest.Heart neural crest cells can migrate into the embryonic heart,involve in formation of aortopulmonary septal;endocardial cushion of outflow track and separation of outflow track;24hpf,Pygo1 specially expressed in the heart tube.The expression pattern of Pygo1 indicated that it might be involve in left-right asymmetry development as well as heart development.We used transgenic strain Tg:Nppa,which heart specifically expresses green fluorescence protein,and Tg(cmlc2:dsRed2-nuc),which heart specifically expresses red fluorescence protein in the nuclei,as materials.Injected with Pygo1 Morphoinos,zebrafish heart looping was disordered.Meanwhile,digoxin-label nppa anti-sense RNA was used as the probe.In situ hybridization experiments further proved that Pygo1 also regulates the wild type(AB strain)zebrafish left-right asymmetry development.Next,earlier development process was detected,in situ hybridization using myl7 and bmp4 probe showed that Pygol affect migration of heart progenitor cells;we further testing the development process with left-right asymmetric marker gene,such as:spaw,pitx2,lefty1 and lefty2,proving that Pygo1 may regulate the expression of left-right asymmetry development marks genes.Knockdown as well as over-expression of Pygo1 both resulted in randomized expression of left-right asymmetric mark genes,while co-injection capped Pygo1 mRNA and Pygo1 Morpholinos into zebrafish embryos,the phenotype turned to normal.The experiment further ascertained that Pygo1 regulates left-right asymmetric development;with the spinal marker gene shh as probe,in situ hybridization experiment showed that Pygol may control the spine development.The experiments demonstrate that Pygol regulates left-right asymmetry development through controlling the formation of the spine.Double color in situ hybridization experiments showed that Pygo1 and Charon,an antagonist of the Nodal signal pathway,co-expressed in Kupffer's vesicle.Morpholino knockdown of Charon and over expression of Pygol both result in left-right asymmetric developmental disorder.Furthermore,over expression of Pygol or knockdown of Charon both resulted in double KV like structure.The results suggest that Pygo1 may antagonist regulate left-right asymmetry development with Charon.At last,in vivo Co-IP experiments proved that Pygol may physically interact with Charon.Pygol regulate KV,s development by inhibiting Charon;our study showed that pygol may plays an important role in the "cross-talk"of Wnt signal pathway and Nodal signal pathway and act as a key"node" between them.Zebrafish fhlA is a homology gene of the fifth isoform of human FHL1,which is a member of four and a half LIM domain family,including four and a half LIM domain structure.Human FHL1 mRNA coding FHL1,SLIMMER and KyoT2 proteins through selective splicing,all the three proteins have related to muscle development.We cloned zebrafish fhlA ORF to pGEMT vector,in vitro synthed digoxin labeled anti-sense RNA,used sense digoxin labeled RNA as negative control,insitu hybridization experiment showed that fhlA expressed from 128 cells stage.From somatic stage 8-20,fhlA specifically expressed in the skeletal muscles;between 20-25 somatic stages,fhlA expressed both in the skeletal muscle and heart field,after somatic stage 25,fhlA exclusively expressed on the heart field,from 24 to 48 hpf,fhlA specifically expressed in the atrium-ventricle border and outflow track;at 72 hpf,the expression of fhlA almost disappeared.We used previous mentioned Tg:NPPA and Tg(cmlc2:dsRed2-nuc)embryos as material.Morpholinos knockdown technology showed that:52hpf,fhlA knocked down embryos showed expanded atrium and ventricle,over-expression of fhlA exhibited shrunked heart;In situ hybridization experiment further proved these results.At earlier development stages(22 somatic stage),in fhlA knocked down embryos,the expression area of cardiac differentiation genes was much larger than that of wide type,in fhlA over-expression embryos;the expression area of cardiac differentiation genes was much smaller than that of wide-type,showed fhlA inhibit heart early development stages.At 72 hpf,zebrafish embryonic heart dissection experiment showed that:knockdown of fhlA decreased the number of cardiac cells and over-expression of the gene increased the number of cardiac cells,indicating that the gene promotes the proliferation of cardiac cells during later developmental stages.At the same time,in situ hybridization experiment with MyoD probe and antibody staining with anti-body MF20 further verified that fhlA.promote skeletal muscle development.By comparing the development of both cardiac muscles and skeletal muscles simultaneously,we found that fhlA had an effect on cardiac muscle differentiation and skeletal muscle development.FhlA was found to regulate the switch of cells to skeletal muscle lineage by activating mef2ca from 12 somatic stages all the way through 24 hpf.Inhibition of fhlA expression was found to cause up-regulated expression of mef2cb and fate conversion of skeletal muscle into cardiac muscle in zebrafish at somatic stage 22.This is the first study to show that the same gene has antagonistic functions in both the regulation of cardiac morphogenesis and the development of skeletal muscle in vivo. |
PDF Full Text Request