The Mutant Phenotype Analysis Of The Left-right Asymmetry Gene Klhl31 In Zebrafish Heart Developmental

Nodal signalling is a key pathway to regulate the development of embryonic left-right asymmetry in vertebrates. Nomo(Nodal modulator) gene is an antagonist of Nodal signaling. The provius works in our lab revealed that human KLHL31 interacted with NOMO. Knock-down of klhl31 gene by klhl31 morpholino resulted into developmental block, slow heart rate and abnormal relative location between the atria and the ventricle in zebrafish embryos. In order to describe the functions of k1h131 in heart development, we used the transgenic zebrafish of Tg:Nppa and Tg(cmlc2::dsRed2-nuc) as models, and injected klhl31 morpholinos、klh131-mRNA and co-injected klhl31 morpholinos and klhl31-mRNA into the zibrafish embryos in the stage with 2-4 cells. Then, we observed the morphology of heart by a microscope and detected the atria marker genes nppa by in situ hybridization at 48hpf. The results showed that the embryos with klhl31 expression disturbed presented heart defects in asymmetric development. Moreover, the development of the dorsal tissues was abnormal in the embryos with knock-down of klhl31.By using Western-blot, the expression levels were detected of Lefty2、Pitx2 and Squint, the marker genes in the Nodal signaling pathway, in the groups of zebrafish which injected k1h131 morpholinos, injeced nomo morpholinos, co-injected k1h131 and nomo morpholinos and the group for control. The results showed that the interferences of klhl31 or/and nomo disturbed the expression of these marker genes of Nordal pathway. So, the conclusion was deduced that klh131 and nomo was the antagonist of each other in the Nodal signaling pathway. The results of in situ hybridization also showed that the expression of Lefty2, Pitx2 and spaw were disturbed.Our result of in situ hybridization showed that klhl31 expressed in the Kupffer’s Vesicle (KV), heart and skeletal muscle in normal zebrafish embryos. In the embryos which were interfered with k1h131 morpholinos, the size of KVs became bigger than that of normal embryos’ KVs, and double KVs or more were observed in some embryos interfered. The cilia in KV were detected by using tublin antibody. The changes were observed in the numbers and length of the cilias in the embryos interfered. The KV was the key organ to regulate the left-right asymmetric development in zebrafish, so klhl31 might be involved in heart left-right asymmetric development by regulating the conformation of KV in zebrafish. Our results in this paper, indicated that KLHL31 might play an important role in the heart development on the Left-right asymmetry.