Signaling and transcriptional mechanisms of neural crest lineage specification in primary neural crest cultures
Neural crest cells are multipotent progenitors, giving rise to diverse cell types in the developing embryo. This raises the question of how neural crest cells commit to proper cell fate at the proper location and time during embryo development. cAMP signaling has been shown to be an important regulator for sympathoadrenal lineage and melanocytic lineage development, with the signaling and transcriptional mechanisms unknown. Employing quail primary neural crest cultures, I demonstrated that high-level activation of cAMP signaling mediates protein kinase A (PKA)-dependent Rap1-B-Raf-ERK1/2 activation. ERK1/2 activation leads to cytoplasmic accumulation of phospho-Smad1, thus terminating bone morphogenetic protein 2 (BMP2)-induced sympathoadrenal cell development. Concurrently, cAMP signaling promotes melanocytic lineage specification by inducing the expression of the melanocyte-determining transcription factor Mitf. Expression of a dominant negative CREB variant inhibits Mitf expression, supporting the notion that CREB activation is necessary for melanocyte specification. However, a constitutively active CREB variant is insufficient to mediate Mitf transcription, indicating PKA regulates additional aspects of Mitf transcription. To further investigate the regulation of Mitf transcription by PKA during melanocyte specification, the quail Mitf promoter was cloned and characterized. The Mitf promoter shows cAMP inducible and melanocyte specific activity. Gain-of-function and loss-of-function studies identified CtBP protein as a repressor for Mitf transcription. CtBP represses Mitf expression by sequestering β-catenin from binding to the Mitf promoter. Taken together, this study indicates that in primary NC cultures, the cAMP signaling network plays a dual role in NC cell lineage specification: attenuation of BMP2-induced sympathoadrenal lineage and induction of the melanocytic lineage. The pivotal regulatory effects of cAMP signaling observed in primary neural crest cultures support that the extracellular molecules that activate cAMP signaling regulate NC lineage specification in the developing embryo.
Andrisani, Purdue University.
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