Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity
Erik Willems
Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, BelgiumFax: +3226292759
Search for more papers by this authorCorresponding Author
Luc Leyns
Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, BelgiumFax: +3226292759
✉ E-mail: [email protected]Search for more papers by this authorErik Willems
Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, BelgiumFax: +3226292759
Search for more papers by this authorCorresponding Author
Luc Leyns
Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, BelgiumFax: +3226292759
✉ E-mail: [email protected]Search for more papers by this authorAbstract
Abstract Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.
Supporting Information
Fig. S1. Mesoderm patterning by graded ActivinA/Nodal signaling assessed by qRT-PCR. Legend indicates the used concentration of SB-431542 (SB) (yellow tints) and Nodal (blue tints) with the control in red (A). Different concentrations of nodal protein in SFM and of SB in SCM showing graded Activin A/Nodal signaling as revealed by the expression of the Nodal direct targets Lefty1 and Lefty2 (B). Effects of graded Nodal signaling on pan-mesodermal Brachyury (C), neuroectodermal Sox1 (C), posterior mesodermal Evx1 and Mesp1 (D), extraembryonic mesodermal Flk1 (D), mesendodermal Gsc, Lhx1 and Sox17 and definitive endodermal Cxcr4 (E) and axial mesodermal markers Shh, Foxa2 and Chrd are presented (F). Histograms with error bars represent the mean of three experiments and SEM, with dashed lines indicating the untreated control level. Asterisks indicate p?0.05 compared to untreated controls and dots indicated p?0.05 between the experimental samples as marked.
Fig. S2. Schematic representation of the major findings in mesoderm patterning. Circles represent different mesoderm subtypes that can be marked with specific genes. Arrows indicate the direction from a pan-mesoderm cell towards a more differentiated subtype. The effectors that induce mesoderm subtypes are indicated with a plus, the ones that repress are indicated with a minus. Patterning towards posterior and extraembryonic mesoderm (1), mesendoderm (2), endoderm (3), and axial mesoderm (4) are indicated.
Table. S1. Specific markers in the gastrulating mouse embryo used in this study.
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