Effect Of Pop3 On Cardiac Looping Of Zebrafish Analyzed By Whole Mount In Situ Hybridization

Heart looping is an important stage in heart development. Whether heart looping is normal or not is closely related to heart function, and heart looping is involved in exact expression of a series of heart genes in space-time. Preliminary studies in our laboratory have found that pop3 gives a high expression in heart and muscle of zebrafish, and pop3 might be involved in heart development analysed by using morpholino oligo-nucleotide knock-down techniques. By immunoprecipitation and other methods, the ATF4 protien was found to interact with POP3. This article is to study the effect of heart looping caused by expression change of pop3 and atf4 genes analyzed by the method of the whole mount in situ hybridization with the zebrafish as a model.In order to study the phenotype change of heart looping caused by pop3 gene, five cardiac marker genes nppa, cmlc2, vmhc, amhc and bmp4 were used as probes for whole mount in situ hybridization to observe the phenotype change of heart looping after knocking down the expression of pop3, overexpression of pop3, and rescuing the mutant phenotype of pop3. In wild-type embryos, nppa and cmlc2 were expressed specifically in the whole heart, vmhc was expressed in the cardiac ventricle, while amhc was expressed in the atrium and bmp4 was mainly expressed in heart valve. In embryos interferenced by pop3 morpholino, knocking down the expression of pop3 decreased the expression of nppa with appearance of heart linearization at 48 hpf. Knocking down the expression of pop3 also led to the reduction of the number of cardiac progenitors where cmlc2 was expressed, and resulted in heart linearization at 48 hpf. Simultaneously, knocking down the expression of pop3 led to the decreases of the vmhc expression in mRNA level and of the number of cardiac progenitors where vmhc was expressed at the period of 21 ss, with the smaller size of cardiac ventricule at 24 hpf and 48 hpf. The size of cardiac ventricle was decreased significantly at the period of 48 hpf, and the size of cardiac atrium was also reduced. When the expression of pop3 was knocked down, the expression of bmp4 was weakening, and the size of cardiac valve became smaller. When pop3was overexpressed, the cardiac mutant phenotype observed is opposite to those caused by knocking down the pop3 expression:the expression of nppa was upregulated and the cardiac chambers became larger, and the number of the cmlc2-expression cardiac progenitors was increased, with a larger cardiac chambers. When pop3 was overexpressed, the expression of both vmhc and amhc was upregulated, the number of the vmhc-expression cardiac progenitors was increased, with a larger cardiac ventricle at 24 hpf and 48 hpf, but with a slightly larger atrium. When pop3 was overexpressed, the expression of bmp4 was significantly enhanced and the size of cardiac valve was evidently increased. In embryos injected with pop3 morpholino and then rescued by pop3 mRNA, cardiac phenotype can be restored among most embryos, and there is no evident difference between them and wild-type embryos. At the same time the expression in mRNA level of the five marker genes mentioned above was returned to normal levels.In order to study the effect of heart looping caused by atf4 gene, five cardiac marker genes mentioned above were also used. The results showed that knocking down the expression of atf4 led to smaller cardiac chambers and heart valves, with hypoplasia of the ventricule and atrium, abnormal heart looping, and the expression of the five marker genes was down-regulated. The overexpression of atf4 mRNA resulted in a larger embryonic cardiac chambers and the expression of the five marker genes was upregulated. In embryos injected with atf4 morpholino and then rescued by atf4 mRNA, there was no obvious difference between the wild-type embryos and the embryos treated, and the expression in mRNA level of the five marker genes mentioned above was returned to normal levels.The results obtained by the whole mount in situ hybridization of the five cardiac marker genes showed that the expression change of pop3 and atf4 can cause abnormal heart looping of zebrafish, and pop3 and atf4 could be involved in a synergistical regulation of heart looping process of zebrafish. Besides, both pop3 and atf4 might be upstream regulatory genes of the genes nppa, cmlc2, vmhc, amhc and bmp4, and participate in the process of heart development by regulating the expression of them.