The Role Of X-box Binding Protein 1 (XBP1)in Xenopus Laevis Heart Development

XBP1 is a member of basic leucine zipper(b ZIP)structure protein,it is also an important transcription factor during endoplasmic reticulum stress(ERS).When ERS happens,IRE1? endoribonuclease activity is activated,and splices the substrate X-box binding protein-1(XBP1),the XBP1 s splice variant then binds to ER stress response elements and the mammalian UPR element to regulate a variety of UPR-related genes.Previous studies showed that knockout of XBP1 mouse embryos die between embryonic days 10.5 and 14.5 which indicate that XBP1 play important roles in heart development.In our study,we investigated XBP1 function in heart development by using Xenopus laevis as a model system since the large size of the eggs,the large number of embryos,and easy to be treated.Specific antisense oligonucleotides morpholino(MO)were microinjected into four cells stage embryos of Xenopus laevis to knockdown XBP1.Embryos were collected at different stages and were detected via real time quantified PCR(q RT-PCR),western blot,whole mount in situ hybridization,immunohistochemistry,and Chromatin Immunoprecipitation(CHIP).We found that the XBP1 s MO-injected embryos grew with small heart.Expression of FHF(First Heart Field)mark gene Nkx2-5 and SHF(Second Heart Field)were down regulated in XBP1 s MO-injected embryos,as well as early cardiac marker genes Nkx2-3,GATA4,GATA6,FGF3 and FGF8.Myocardial differentiation related genes TNIC,desmin,?-actin,VMHC,MYH6 and NPPA were also down regulated.Overexpression of XBP1 s,the phenotype and gene expression were similar to that of XBP1 s MO-injected embryos.The rescue experiment of co-injected Nkx2-5 m RNA with XBP1 s MO shows that Nkx2-5 have a rescuing effect on knockdown of XBP1.In addition,XBP1 s binds the XBP1 binding site on the Nkx2-5 promoter in cardiac progenitor cells.The results indicated that XBP1 was involved in heart development of Xenopus laevis and maybe function by regulating Nkx2-5.