The neural crest is a transient embryonic tissue that gives rise to a multitude of derivatives in an axially restricted manner. An in vitro counterpart to neural crest can be derived from human pluripotent stem cells (hPSCs) and can be used to study neural crest ontogeny and neurocristopathies, and to generate cells for therapeutic purposes. In order to successfully do this, it is critical to define the specific conditions required to generate neural crest of different axial identities, as regional restriction in differentiation potential is partly cell intrinsic. WNT and FGF signaling have been implicated as inducers of posterior fate, but the exact role that these signals play in trunk neural crest formation remains unclear. Here, we present a fully defined, xeno-free system for generating trunk neural crest from hPSCs and show that FGF signaling directs cells toward different axial identities within the trunk compartment while WNT signaling is the primary determinant of trunk versus cranial identity.

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WNT/β-catenin signaling is crucial for neural crest (NC) formation, yet the effects of the magnitude of the WNT signal remain ill-defined. Using a robust model of human NC formation based on human pluripotent stem cells (hPSCs), we expose that the WNT signal modulates the axial identity of NCs in a dose-dependent manner, with low WNT leading to anterior OTX + HOX − NC and high WNT leading to posterior OTX − HOX + NC. Differentiation tests of posterior NC confirm expected derivatives, including posterior-specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC exhibits a transient TBXT + /SOX2 + neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, which suggest a crucial role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected retinoic acid (RA) and FGF are able to modulate HOX expression in the posterior NC. Surprisingly, early RA supplementation prohibits NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.

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8The WNT/β-CATENIN pathway is critical for neural crest (NC) formation. However, the effects of the 1 9 magnitude of the signal remains poorly defined. Here we evaluate the consequences of WNT 2 0 magnitude variation in a robust model of human NC formation. This model is based on human 2 1 embryonic stem cells induced by WNT signaling through the small molecule CHIR9902. In addition 2 2 to its known effect on NC formation, we find that the WNT signal modulates the anterior-posterior 2 3 axial identity of NCCs in a dose dependent manner, with low WNT leading to anterior OTX+, HOX-2 4 NC, and high WNT leading to posterior OTX-, HOX+ NC. Differentiation tests of posterior NC confirm 2 5 expected derivatives including posterior specific adrenal derivatives, and display partial capacity to 2 6 generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC 2 7 transit through a TBXT+/SOX2+ neuromesodermal precursor-like intermediate. Finally, we analyze 2 8 the contributions of other signaling pathways in posterior NC formation, and suggest a critical role for 2 9 FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected RA and 3 0 FGF are able to modulate HOX expression in the posterior NC, but surprisingly, RA supplementation 3 1 prohibits anterior, but only reduces, posterior NC formation. This work reveals for the first time that 3 2 the amplitude of WNT signaling can modulate the axial identity of NC cells in humans. 3 3 3 4 KEY WORDS: neural crest, WNT dosage/magnitude, anterior-posterior axis, HOX genes, 3 5 human embryonic stem cells.