The Pilot Study Of Cell Cycle Factor Geminin Regulating Zebrafish Heart Development

Heart is the first organ to form and has its function during embryogenesis. Its function is critical from early on for the viability of the mammalian , and it can be observed by the researcher easily. So,heart is also the first organ to be studied. Zebrafish becomes one of the best models to study heart development , because it fertilizes in vitro, and the embryos are transparent and develop very fast. There are many key processes during zebrafish heart development, and developmental abnormalities of the heart can cause many congenital heart malformations. So we study some genes, regulation on cardiogenesis can provide useful understanding and diagnosis of heart disease.Geminin is an important regulator during cell cycle. It directly interacts with Cdtl,a replication initiation factor, to block binding of the mini-chromosome maitenance(MCM) complex to origins of the replication again during cell cycle. There has been a research indicating that geminin overexpression or cdt1 knockdown decreased the number of mitotically active cells and this effect depended on direct interaction between Geminin and Cdt1 .This balanced interaction of Geminin and Cdt1 is critical for the cell-cycle regulation. But from now on there are few reports about its role on organgenesis. So it will be very significative to study its role in cardiogenesis. In this research ,we investigated what role geminin played in heart development.First of all ,we confirmed in which process geminin performs its fuchtion. We used whole mount in situ hybridization to check expression of heart marker gene nkx2.5. and we found if we knocked down geminin, the nkx2.5 expression area enlarged ,it indicated that geminin affected inducement and differentiation of heart progenitor cell, it inhibited extra inducement of nkx2.5 in non-heart area. And we also found there were abnormalities in heart phenotype at 48h, there were some problems about heart loop. For example, the ventricle and atrium in a line or the oposition and angle between ventricle and atrium was absolutely opposite to the wide type, about one in three embryos had this phenotype, this told us that the second phenotype after injecting GemMO is changing of heart left-right pattern.Then we wanted to know if increasing heart progenitor cell by GemMO injection had relationship with changing of heart left-right pattern.. We used a tool BMS453 which can enlarge nkx2.5 expression .After dealing with BMS453, about 22% embryos displayed loop reverse phenotype. This simulated geminin knockdown phenotype on certain extent. Otherwise, basing on that Geminin-Cdt1 balance affects cell cycle , Cdt1MO can rescue this phenotype if we injected GemMO and Cdt1MO together. On the other hand ,We also studied whether geminin affected left-right asymmetry pattern during development. We found that expression pattern of bmp4 which regulated left-right asymmetry, is changed after knock-down geminin. In wide type , bmp4 expression is uniform in the developing heart at the time of heart fusion(21-somite stage) just before jogging, the pattern of bmp4 expression becomes markedly asymmetric,with far more on the left than on the right side. This is called left-predominant asymmetry. But after injecting GemMO, bmp4 expression become right-asymmetry or symmetry. Also we found Cdt1MO could rescue this phenotype. This hinted that geminin also affected left-right pattern . we have known that if bmp4 expression is left-predominat followed by rightward bending of the ventricle and leftward bending of the atrium ,the heart tube will moves to the left. If bmp4 expression is symmetry followed by no bending of ventricle and atrium, then the heart tube will be in the middle. If bmp4 expression is right-predominant followed by left bending of the ventricle and rightward bending of the atrium ,the heart tube will moves to the right. So this is identical to the result we concluded before. So we think that geminin can regulate heart loop by changing the bmp4 expression pattern and changing the pathway and genes downstream bmp4. This function of geminin is associate with left-right asymmetry pattern. In order to confirmed this conclusion, I also checked expression of pitx2c , lefty2 and nt1, they are marker genes influencing left-right asymmetric pattern . I found that the asymmetric expression pattern of pitx2c and lefty2 were changed afer injecting GemMO, and the expression of nt1 is increased. This was consistent with the result above. So this was another pathway that geminin affected heart loop and its position. The two experiments above both have the same result, that is , geminin regulate left-right asymmetry pattern. But we can not conclude whether the increasing of heart progenitor cells lead to changing of left-right asymmetry pattern or changing of left-right asymmetry pattern lead to inreasing of heart progenitor cells.