FGF8 and Shh promote the survival and maintenance of multipotent neural crest progenitors.

The developmental mechanisms that control the building of the complex head of vertebrates and particularly, facial skeletogenesis, remain poorly known. Progenitor cells derived from the embryonic neural crest (NC) are the major constituents and players of facial tissue development. Deciphering the cellular and molecular machinery that controls NC cell (NCC) differentiation into bone, cartilage, fat and other mesenchymal tissues, is thus a main issue for understanding vertebrate facial variations. In this work, we investigated the effects of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (Shh), two signaling molecules essential for craniofacial development, on the in vitro differentiation and multipotentiality of mesencephalic NCCs (MNCCs) isolated from the quail embryo. Comparison of distinct temporal treatments with FGF8 and/or Shh showed that both promoted chondrogenesis of MNCCs by increasing the amount and size of cartilage nodules. Higher rates of chondrogenesis were observed when MNCCs were treated with FGF8 during the migration phase, thus mimicking the in vivo exposure of migrating NCCs to FGF8 secreted by the isthmic brain signaling center. An in vitro cell cloning assay revealed that, after concomitant treatment with FGF8 and Shh, about 80% of NC progenitors displayed chondrogenic potential, while in untreated cultures, only 18% exhibited this potential. In addition, colony analysis showed for the first time the existence of a highly multipotent progenitor able to clonally give rise to adipocytes in addition to other cephalic NC phenotypes (i.e. glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes) (GNMFCA progenitor). This progenitor was observed only when clonal cultures were treated with both FGF8 and Shh. Several other types of multipotent cells, which generated four, five or six distinct phenotypes, accounted for 55% of the progenitors in FGF8 and Shh treated cultures, versus 13,5% in the untreated ones. Together, these data reveal an essential role for both FGF8 and Shh together in maintenance of MNCC multipotentiality by favoring the development of NC progenitors endowed with a broad array of mesectodermal potentials.
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