Zebrafish Heart Development Related Gene ¦Ä-sg And The Foxp1 The Cloning, Expression And Function Studies,

(1)δ-SG,one member of the sarcoglycan complex,is a very conservative muscle specific protein exclusively expressed in the skeletal and cardiac muscle of vertebrates.Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy(LGMD)and dilated cardiomyopathy(DCM)in humans.Here we report molecular cloning,characterization and developing expression of a novel zebrafishδ-SG gene.And we adopte the gene-specific morpholino modified antisense oligo to carry out gene knock-down assay and analysis its influence to zebrafish embryogenesis.The full length of zebrafishδ-SG cDNA is 1938 bp and it encodes a polypeptide of 290 amino acids which shares 72%,73%,73% and 69% overall identity with human,mouse,hamster and chickenδ-SG,respectively.Characterization of zebrafishδ-SG genomic sequence reveals that it spans about 200kb and contains eight exons and seven introns.The expression pattern ofδ-SG in zebrafish embryonic development is studied by whole-mount in situ hybridization.The onset of zygotic transcription is at early segmentation period.Strong expression ofδ-SG was observed in various muscles including those of the segment,heart,eye,jaw,pectoral fin, branchial arches and swim bladder in zebrafish embryo.Expression could also be found in midbrain and retina after 72hpf.Furthermore,we carry out gene knock-down by morpholino modified antisense oligonucleotide.The MO-injected embryos showed severe abnormality in both the cardiac and skeletal muscle.However,the embryos injected with the same dose control mopholino oligonucleotide showed no difference from the uninjected wildtype embryos.Tthe evidences provided by MO-GFP and rescue experiment are valid enough to confirm that MO effectively represses the translation ofδ-SG protein.Some severe ones had serious morphological abnormality such as hypoplastic head,linear heart,very weak heartbeats and runtish trunk,all dead within 5 dpf.Whole-mount in situ hybridization analysis showed that inδ-SG knockdown embryos adaxial cells and muscle pioneers were affected.In addition,absence ofδ-SG protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle,and the cardiac left-right asymmetry was dramatically changed in morpholino treated embryos.Immunoblot analysis shows downregulation of the sarcoglycans.These data together suggest thatδ-SG plays an important role in early heart and muscle development.(2)The winged-helix/forkhead transcription factor gene family is a large group of proteins that share a conserved 110-amino acid DNA-binding domain termed as winged-helix or forkhead domain.Members of this family have been shown to play important roles in the development of various organs,including brain,cardiovascular system,lung,gut and kidney,with their diverse functions including pro-liferation, cellular differentiation,chromatin remodeling,mitotic program,and neoplastic transformation.In this report,we described the isolation,characterization and expression of Foxp1 in zebrafish.The full-length zebrafish Foxp1 cDNA was 2440bp encoding a 659-amino-acid protein which shares 72%,68%,68%,70%,65%,and 62% overall identity with human,mouse,rat,chicken,avian,and frog Foxp1,respectively.The product of Foxp1 gene contains a zinc-finger domain,a leueine zipper domain and a forkhead domain.5'RACE and RT-PCR have confirmed that zebrafish Foxp1 transcripts contain at least four different splicing variants named Foxp1A-D.The results of whole-mount in situ hybridization and RT-PCR showed Foxp1 have very complex and dynamic expression pattern during early embryonic development.The spatial and temporal expression patterns of four splice isoforms are different.Remarkable signals of Foxpl were detected in many domains of the developing central nervous system, especially in mid-hind brain boundary,hindbrain,and spinal cord.Strong and broad expression was also observed in retina,ear,branchial arches,hatching gland,heart, pronephric duct,gut,proctodeum,pectoral fin and swim bladder.Knock-down had been used in studying the gene function in zebrafish development.Almost all the Foxp1-MO-injected show highly consistant phynotype including hypoplastic head,small eyes,pericardial edema and weak heartbeats.The embryos injected with the same dose control mopholino oligonucleotide showed no difference from the uninjeeted wildtype embryos.These results provide evidence that Foxp1 might be very important in the development of the central nervous system, heart and many other organs.