At early gastrula stages of the chick embryo, cells with cardiogenic potential arise from a localized region of the primitive streak, just caudal to Hensen's node. These groups of cells migrate in an anterior-lateral direction to form the bilateral cardiogenic mesoderm, between the ectoderm and the endoderm, in the most anterior and lateral part of the embryo, constituting the primary cardiac field. At this moment, the precardiac mesoderm is surrounded by the adjacent endoderm, which comes from the more cranial part of the primitive streak, and which plays a crucial role during cardiac specification with the involvement of several molecular factors. Therefore, previous evidences suggest that Nkx2.5 expression is induced by Bmp2, emanating from the adjacent endoderm, which is also regulated by Fgf8.
To further analyze Bmp2-Fgf8 interaction during cardiac specification, we have analyzed the role of microRNA-130, which is expressed in the endoderm adjacent to precardiac mesoderm, in early cardiogenesis. For this purpose, we have electroporated Bmp2, Fgf8 and miR130 in precardiac cells as they pass the primitive streak. Our study has been complemented by experiments of ectopic administration of Bmp2, Fgf8 and SU5402 (an inhibitor of Fgfr1). We observe that Bmp2 induces the specific cardiac markers cNkx-2.5 and Gata4, and suppresses the endodermal endogenous expression of Fgf8. Moreover, Fgf8 suppresses Bmp2, cNkx-2.5 and Gata4 expression, while SU5402 administration suppresses the endogenous Fgf8 expression. Overexpression of miR130, a microRNA that binds to specific sequences in the 3′UTR of target gene Erk1/2 (Mapk1), induces the expression of Bmp2, cNkx-2.5 and Gata4, and suppresses the expression of Fgf8. The electroporation of antimiR130 shows the opposite results.
All our data supports the establishment of a negative feed-back between Bmp2 and Fgf8 through miR130, constituting a crucial element involved in the precise patterning and early development of the heart.
Funding: Spanish Ministry of Science and Innovation [BFU2007-66350], and the Junta de Extremadura with FEDER co-funding [CTS005].