Figure 11

ERK1 and ERK2 knockdown differently affect signals involved in patterning of the early embryo. (A-D): Schematic representation of the effects of ERK1 and ERK2 knockdown on the activities of Nodal, FGF, Wnt and BMP signaling pathways in late blastula embryos. (E): effect of ERK2 knockdown (ERK2MO) on early embryonic mesendoderm differentiation. The representation of predicted signaling activity in the wild type embryos is based on the potential range of signals, the expression patterns and range of antagonists, adopted from Schier and Talbot (2005). The combined signaling activities from these pathways are responsible for the differentiation and fate-map of the late blastula/early gastrula stage of the zebrafish embryo (E, late blastula stage, lateral view, dorsal to right, animal pole to top). In the zebrafish embryo, dorsal ventral patterning starts as early as the 128-cell stages by accumulation of β-catenenin at the nuclei of the dorsal cells, rapidly followed by the expression of goosecoid (A). Soon after mid-blastula transition, β-catenin also activates the expression of a number of zygotic genes, including chordin, bozozok and squint (A and D), and FGF signals (C). These genes act to inhibit the action of ventralizing factors or induce mesoderm and endoderm cell fates at the dorsal side. Subsequently, the expression of these genes quickly spreads over the complete margin (E). To establish a mesodermal zone, induction processes occur at the animal – vegetal axis. Complex signaling processes are used by the embryo to induce mesoderm. In a over-simplified manner, it can be said that Nodal (D) signaling is involved in initiation of mesoderm formation, FGFs (C) and Wnt (A) are involved in maintaining the mesoderm state and BMPs (B) are involved in further patterning of the mesoderm [33]. Knockdown of ERK1 (ERK1MO) resulted in an increased expression of the BMP-inhibiting protein smurf1/wwp1 and the ventrally expresses gene vent (A). In addition also the mesoderm marker tbx6 showed a reduced dorsal expansion of its expression domain (Fig. 7K). Combined, this indicates a reduction of ventral signaling, possibly leading to a mild dorsalization of ERK1 knockdown embryos. ERK2 knockdown (ERK2MO) promotes Nodal signaling by repressing the expression of Nodal inhibitors (vox, vent, ved and lft1) (D). Furthermore, it perturbs FGF signaling (repression of fgf8 and components of the RAS-ERK pathway and down regulation of its target genes) and Wnt signaling (repressed expression of frizzled receptors and key components of the Wnt pathway). In addition, perturbed BMP signaling results in incorrect patterning of the mesoderm (B). In summary, this shows that mesendoderm differentiation is still initiated by Nodal signaling (D), but mesendoderm maintenance by FGF and Wnt signaling is defected. This results in reduced expression of mesoderm (ntl, tbx6 and spt) and endoderm (gata5, sox32) marker genes (B, C and E), showing that ERK2 is essential for mesendoderm differentiation (E).