The Role Of Inositol Requiring Enzyme 1α(IRE1α) In Xenopus Laevis Heart Development

Perturbation of endoplasmic reticulum (ER) associated functions (such as protein folding, lipid and sterol synthesis and Ca2+ homeostasis) results in ER stress via the activation of complex cytoplasmic and nuclear signaling pathways, collectively termed the unfolded protein response (UPR). Inositol requiring enzyme la (IREla) is a type I ER transmembrane protein that functions as a kinase and as an endoribonuclease. When ERS happens, IREla endoribonuclease activity is activated, and splices the substrate X-box binding protein-1 (XBP1), the XBP1s 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 IREla is involved in several pathological processes, and is essential for embryo development. In addition, previous study in our lab showed that knockdown of IREla slowdown the heartbeat which indicate that IRE1α may play a role in heart development. In this study, we investigated IRE1α function in heart development by using Xenopus laevis as a model system due to 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 IRE1α. Embryos were collected at different stages and were detected via real time quantified PCR(qRT-PCR), western blotting, whole mount in situ hybridization, tissue section, transmission electron microscope(TEM), immunohistochemistry, and gene chip. We found that the IRElaMO-injected embryos grew with small heart and cardiac structure and cell ultrastructure were destroyed. Expression of cardiac marker genes Nkx2-5, TnIc, Gata4, Gata6 and Tbx20 were down regulated in IRElaMO-injected embryos, but somite development was normal. Knockdown of XBP1s, the phenotype and gene expression were similar to that of IRElaMO-injected embryos. In addition, gene chip results indicated that Wnt signal pathway genes are up regulated in IRE1αMO-injected or XBP1sMO-injected embryos. The results indicated that IREla was involved in heart development of Xenopus laevis and maybe function by regulating Wnt signal pathway through XBP1 pathway...