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Analysis of Cranial Neural Crest Cells During Palatogenesis
Max Dougherty, George Kamel, MD, Graham Hickey, Michael Grimaldi, Renee Ethier, Eric C. Liao, MD, PhD.
Massachusetts General Hospital, Boston, MA, USA.
In all vertebrates, cranial neural crest cells (CNCC) follow stereotypic patterns of migration to form craniofacial structures. In particular, the anterior stream of migrating CNCC forms the palate (ethmoid plate in fish). We generated a transgenic animal (sox10:kaede) whose neural crest cells are labeled with kaede, a green fluorescent protein that can be selectively converted to red with targeted UV light. This model allows us to precisely analyze CNCC migration throughout embryogenesis. Study of CNCC migration perturbed by over-expression or knockdown of genes regulating craniofacial development, such as mirn140 and wnt9a, uncovers the mechanism of action of these genes on cellular morphogenesis.
The sox10:kaede transgenic animal was generated using Tol2 transposase mediated germline integration. Progeny of founder transgenic zebrafish were used for the photoconversion experiments. Specific regions of neural crest cells were labeled at 10 somites and at 23 hours post fertilization by targeted irradiation with 403 nm light using a confocal microscope. Embryos were photographed at the time of photoconversion and followed through embryogenesis to track cellular movements. wnt9a morpholino or mirn140 duplex RNA were injected at the one-cell stage and fate-mapping experiments were carried out.
Preliminary work on wild-type embryos has demonstrated the utility of this model in tracking cellular movements. We confirm that the kaede reporter protein is expressed in CNCC cells. wnt9a knockdown and mirn140 overexpression have unique effects on CNCC migration, where movement to populate pharyngeal arch and subsequent craniofacial structures are deranged.
The sox10:kaede transgenic line provides a reliable model for CNCC fate-mapping. We are able to utilize this system to uncover the morphogenetic mechanism of two genes that regulate CNCC migration, wnt9a and mirn140. These studies illustrate the advantage of the zebrafish model for application in the study of craniofacial development, where complex cellular movements are highly choreographed at molecular and spatiotemporal levels.
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