br Materials and methods br Acknowledgments This work

Materials and methods
Acknowledgments This work was supported by a grant from the Novo Nordisk Foundation, the Lundbeck Foundation, and the Augustinus Foundation. N.O. is supported by the Lundbeck Foundation. I.K. is supported by grants from the Danish Natural Science Research Council and the Danish Medical Research Council.
Specifications table Value of the data Data Fgf8 constitutes a crucial factor involved in MAPK/ERK signaling pathway [8]. Also, FGF receptors have demonstrated to play a significant role during the early steps in the Fgf8/ERK signaling cascade [6,9]. Although there is no evidence about the role of FGF receptors during early cardiogenesis, there are previous studies where FGFR1 has been identified in the endoderm underlying the precardiac mesoderm [16,17]. However, its functional role has been preferentially involved in cell proliferation rather than in cell differentiation [19]. It has been reported that cardiomyocyte proliferation is suppressed after miR-133 overexpression [13], and that miR-133 is also involved in late stages of mouse cardiac development as well as in molecular mechanisms regulating adult cardiovascular diseases [1,2,7,12,18]. To further analyze what mechanism regulates these signaling pathways, we will explore herein miR-133 expression and function. Although previous studies have provided data about miR-133 expression in chick ginsenoside rg3 [5], it has only been analyzed in late stages of cardiac development. Thus, we have analyzed miR-133 expression from the primitive streak stages to the primitive cardiac tube formation (Fig. 1), expression which is already detectable at gastrula stages throughout the entire primitive streak, including the precardiac cells. Subsequently, its expression spreads laterally and concentrates into the first cardiac field (FCF) and the underlying endoderm, to finally express restrictively at the splachnic mesoderm of the primitive endocardial tube. This topographical location of miR-133 shows a similar distribution to that of Fgfr1[16,17]. Since FGFR1 has been proposed as a crucial factor involved in Fgf8/ERK signaling pathway in different experimental models [8,11], it is reasonable to consider a close relationship between miR-133 and Fgfr1 during cardiogenesis. To assess the potential involvement of miR-133 in Fgf8-Bmp2 cooperation during cardiogenesis, we analyzed the effects of miR-133 overexpression on the precardiac primitive streak cells, resulting in Fgf8 inhibition and Bmp2 increase. Subsequently, Nkx-2.5 and Gata4 increase as well (Fig. 2). Interestingly, our loss-of-function experiments through Fgf8 siRNA electroporation showed an increased miR-133 expression (Fig. 3). It is also noteworthy that our Bmp2 overexpression experiments induced miR-133 expression, whereas noggin soaked beads administration -specifically into FCF, showed a decrease of miR-133 expression in the ipsilateral endocardial tube (Fig. 4), suggesting that miR-133 is upstream-regulated by Bmp2. All the above results clearly indicate that a reciprocal repression between miR-133 and Fgf8 regulates cardiac induction through Bmp2 signaling (Fig. 5), thus constituting complementary data to those obtained after our miR-130 analysis [15]. However, although miR-133 may be identified as a putative microRNA that targets FGFR1 3′UTR -site broadly conserved among vertebrates, its connection for Fgf8/ERK pathway in this process remains to be further established.
Experimental design, materials and methods Fertilized eggs (Granja Santa Isabel, Córdoba, Spain) were incubated at 38°C in forced-draft humidified incubators. Embryos were staged (PS stages: [14]; HH stages: [10]) and subjected to early chick (EC) embryo culture [3]. Two groups of embryos were selected for experiments:
Acknowledgments We thank María Pérez for her invaluable technical support. This work has been partially financed with Grants to research groups CTS005 (to VGM) from the Junta de Extremadura, with FEDER co-financing, and CVI-6556 (to DF) from the Junta de Andalucía Regional Council.