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Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGF beta pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGF beta family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGF beta pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGF beta/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGF beta/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

The EGF-CFC gene cripto governs anterior–posterior (A–P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII–activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal–ALK4–Smad2 signaling both in embryonic stem cells and cell-based assays. In criptoF78A/F78A mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, criptoF78A/F78A embryos establish an A–P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal–Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A–P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal–ALK4–Smad2 signaling pathway.

The advent of human induced pluripotent stem cell (hiPSC) reprogramming has revolutionized the field of developmental biology. To realize the full potential of this promising technology, it is imperative to understand mechanical and chemical signaling pathways that coordinate the process of reprogramming. Primary cilia have been shown to play a critical role in mechano-chemical signaling across a wide spectrum of cell types, with major implications in maintaining homeostasis. Their functions in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. This work focused on understanding how reprogramming affects the mechanical characteristics of primary cilia. Using immunofluorescence imaging assays and electron microscopy studies, we established for the first time the presence of primary cilia on fibroblasts derived hiPSCs. Using quantitative PCR assays, we identified changes of expression levels of pluripotency markers Nanog and Cripto as well as primary cilia signaling partners Hh, Ptch1, Gli1, Gli2, and Gli3. Furthermore, morphometric analysis revealed that reprogramming resulted in an increase in curvature of primary cilia from ∼0.16 μ−1 to ∼0.78 μ−1 (p