Neural crest specification and Pax7 cis-regulation.

摘要

Neural crest cells are multipotent migratory stem cells unique to vertebrates that contribute to a diverse collection of tissues including facial bone and cartilage, the neurons and glia of the peripheral nervous system, and melanocytes. Neural crest defects can lead to a wide variety of human diseases, collectively known as neurocristopathies, including Waardenburg syndrome, Treacher Collins syndrome, and Hirschprung disease. Because of its significance in development and disease, neural crest development has been widely studied. Neural crest cells arise at the border of neural and non-neural ectoderm during gastrulation. They form at the dorsal tips of the neural folds and undergo an epithelial-to-mesenchymal transition to migrate from the dorsal neural tube to their final locations. The neural crest progenitors can first be identified by the expression of a combination of transcription factors that overlap at the neural plate border. In chicks, the transcription factor Pax7 is the earliest marker expressed exclusively at the neural plate border and later in neural crest.;It was thought that interactions between neural and non-neural tissue and/or mesoderm were required for the formation of neural crest, but recent evidence has shown that neural crest cells are specified during gastrulation, prior to the formation of these tissues. Understanding when and where cell types originate in the embryo is an important aspect of development, allowing researchers to then study the signals, both in terms of the identity and timing, responsible for the specification of that tissue. Both neural and epidermal tissues have been shown to be specified in the early chick blastula in the medial and lateral domains, respectively. Previous studies of the early avian blastula have not consistently found specified neural crest cells nor have fate maps been carefully examined with respect to the neural crest. In this study we show for the first time that presumptive neural crest cells are consistently specified in the early avian blastula. We found that explants from the border of presumptive neural and non-neural tissues in embryos expressed Pax7 after 25h in culture and Pax7 with HNK-1, a marker of migratory neural crest cells, after 45h. We also provide a preliminary fate map of the early blastula that is focused on the neural crest, demonstrating that a region of the epiblast in between presumptive neural and epidermal tissues will give rise to neural crest cells.;Pax7 is one of the earliest markers of the neural crest and is required for its formation, and is therefore a key factor in understanding the initial formation of neural crest cells. Several signaling pathways, including Wnts, BMPs, and FGFs have been shown to regulate Pax7, but it is unclear if these are direct or indirect relationships. In order to better understand how Pax7, and thus the neural crest, is regulated during early development, we searched for cis-regulatory elements upstream of the chicken Pax7 gene. We screened a ∼6kb region of genomic DNA and identified a novel enhancer that drives neural crest-specific expression of a reporter. We then characterized this enhancer further to identify the minimal element necessary and sufficient for enhancer activity. Through a combination of mutational analysis, overexpression, and in vitro EMSA we identified cMyb, a newly identified member of the neural crest gene regulatory network, as a novel regulator of Pax7 expression during gastrulation. We also examined the ability of this enhancer to label neural crest cells in human embryonic stem cell-derive neural crest precursors for use in prospectively isolating human neural crest cells that could then be used to study early events in human neural crest development, for which information is sorely lacking.