Regulation of Mesendoderm Formation and Axial Elongation by Wnt Signaling in Mouse Embryonal Carcinoma Cells.

Abstract

Formation of the three germ layers from pluripotent embryonic cells is the key initial event for the development of the animal body. During gastrulation, the internal germ layers, i.e., mesoderm and endoderm (or mesendoderm), are generated and separated from the external layer, i.e., ectoderm. Mesodermal cells play critical roles in axial morphogenesis, particularly for the elongation of the embryo along the cranial-caudal body axis through their convergent extension movement. The molecular mechanisms that regulate mesendoderm formation and axial morphogenesis are not fully understood in mammalian embryos. To study these processes at the molecular and cellular levels, we use the P19 mouse embryonal carcinoma cell line as an in vitro model. When cultured in mono-layer, P19 cells maintain the undifferentiated state that is comparable to the epiblast, the precursor of all the three germ layers. However, when cultured as aggregates, P19 cells exhibit the pattern of gene expressions that are characteristic of nascent mesendoderm. Furthermore, P19 cell aggregates display elongation morphogenesis that is similar to the axial elongation at the caudal end of normal embryos. Here, by taking advantage of these properties of P19 cells, we investigated how mesendoderm formation and axial elongation in mouse embryonic cells are regulated by Wnt signaling. The Wnt signaling pathways are evolutionarily conserved signal transduction cascades that play critical roles in cell differentiation, growth, and patterning in a wide range of animal phyla. We first demonstrated that the activation of Wnt/beta-catenin signaling is both necessary and sufficient to induce the formation of mesendoderm in P19 cells. The up-regulation of mesendoderm-associated genes, such as Brachyury, Cdx2, and Snai1, was totally absent in cell aggregates, in which beta-catenin was knocked down by RNA interference (RNAi). Importantly, the up-regulation of Wnt3 upon cell aggregation was also dependent on beta-catenin, indicating the existence of a positive-feedback regulation of Wnt3 expression. The experimental activation of Wnt/beta-catenin signaling in mono-layered cells was sufficient to up-regulate these mesendoderm-associated genes. Furthermore, when the expression of Wnt3a was knocked down by RNAi, cell aggregates exhibited only partial up-regulation of Brachyury and other mesendoderm-associated genes. By contrast, the knockdown of Wnt5a expression by RNAi did not markedly impair the up-regulation of mesendoderm-associated genes in cell aggregates. However, the axial elongation morphogenesis was significantly diminished in Wnt5a-knocked-down aggregates. Because Wnt5a typically acts along the planar cell polarity (PCP) pathway, we investigated the involvement of PCP components, namely Rho-associated kinase (ROCK) and Jun N-terminal kinase (JNK), in the axial elongation of P19 cell aggregates. Pharmacological experiments indicated that the activation of ROCK, but not JNK, was essential for the elongation morphogenesis. Thus, Wnt signaling regulates mesendoderm formation and axial elongation at distinct levels through the expression of different ligands and downstream cascades.