Papel de la cooperación FGF/BMP en las vías de señalización que regulan el proceso de cardiogénesis18

Abstract

Over the last few years, the challenge to understand those molecular determinants responsible for heart development has taken on special relevance. In the present Doctoral Thesis, we analyze the molecular determinants of cardiac specification of cells programmed to form the heart. With this perspective, we examine the prospective cardiogenic cells, from their initial location, at primitive streak level, and their migration to precardiac mesoderm and primitive cardiac tube formation, their molecular characteristics, specific gene expression patterns, and those inductive and repressive factors involved in cardiac specification. We use chick embryo in culture as a model to design groups of experiments based in gain- and loss-of-function. We perform gain-of-function experiments through electroporation and acrylic beads application on the prospective cardiogenic cells from the initial gastrulation by using Fgf8, Bmp2 and miR-130. Simultaneously, we perform loss-of-function experiments by means of Fgf8 siRNA; SU5402, blocker of FGF signal; noggin, which antagonizes Bmp activity; and anti-miR-130. Our results show that over-expression of Fgf8 lead to a decrease in the transcription factor Nkx- 2.5 expression, the earliest marker of cardiac differentiation, and Gata4. Moreover, Fgf8 over-expression decreases Bmp2. Our experiments based on Bmp2 over-expression resulted in an increased Nkx-2.5 and Gata4 expressions, as well as suppression of Fgf8 expression and reduction of Erk1/2 staining, at endocardial tube level. These data agree with the results we obtained by means of loss-of-function experiments, that show the opposite effects. Additionally, we analyze the role of miR-130, a microRNA that binds to specific sequences in the 3´UTR of target gene Erk1/2 (Mapk1). We observe that miR-130 expresses at precardiac mesoderm and underlying endoderm levels of the primary cardiac field. Our results show that over-expression of miR-130 suppresses Fgf8 expression at both primitive endocardial tube levels, and induces Nkx-2.5 and Gata4 expressions. Furthermore, it regulates miR-133 expression, specifically expressed in precradiac areas. The most relevant data, over-expression of miR-130 increase Bmp2 expression, a molecular factor which maintains a close relationship with other factors here analyzed, such as catecholamines pathways, which reveals an active role of these factors in the process of cardiac differentiation. All those data support the establishment of a negative feed-back between Bmp2 and Fgf8 mediated by miR-130 expression and Erk1/2 signal, constituting a crucial element in the precise patterning and early development of the heart.