Regulation of extraembryonic endoderm stem cell differentiation by Nodal and EGF-CFC signaling

The signaling pathway for Nodal, a ligand of the transforming growth factor-beta (TGF-beta) superfamily, plays a central role in regulating the maintenance and/or differentiation of stem cell types that can be derived from the peri-implantation mouse embryo. Extraembryonic endoderm stem (XEN) cells are derived from the primitive endoderm of the blastocyst, which normally gives rise to the parietal and the visceral endoderm in vivo, but XEN cells do not contribute efficiently to the visceral endoderm in chimeric embryos. We have found that treatment of XEN cells with Nodal and/or Cripto, an EGF-CFC co-receptor for Nodal, results in up-regulation of markers for visceral endoderm as well as anterior visceral endoderm (AVE). Re-introduction of treated XEN cells into chimeric embryos by blastocyst injection or morula aggregation results in contribution to visceral endoderm and AVE. In culture, XEN cells do not express Cripto, but do express the related EGF-CFC co-receptor Cryptic and require Cryptic for Nodal signaling. Notably, the response to Nodal can be blocked by treatment with the ALK4/ALK5/ALK7 inhibitor SB431542, but Cripto treatment is unaffected, suggesting that its activity is independent of type I activin receptors. Gene set enrichment analysis of genome-wide expression signatures generated from XEN cells under these treatment conditions confirms the differing responses of Nodal- and Cripto-treated XEN cells to SB431542. Our findings define distinct pathways for Nodal and Cripto in the differentiation of visceral endoderm and AVE from XEN cells, and provide new insights into the specification of these cell types in vivo.

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Kruithof-de Julio M, Alvarez MJ, Galli A, Chu J, Price SM, Califano A, Shen MM