Functional roles of Wnt and Snail2 Target Genes in the Neural Crest
The Neural Crest
The Taneyhill lab studies the vertebrate neural crest, a transient population of migratory cells that ultimately differentiate to become a wide range of structures, including the peripheral nervous system, pigment cells (melanocytes), and the bones and cartilage of the face and neck. Consequently, many human congenital and hereditary malformations (craniofacial abnormalities, heart defects), diseases (Hirschprung) and cancers result from aberrant neural crest development. Neural crest cells detach from the neural tube and migrate away into the periphery by undergoing an epithelial-to-mesenchymal transition (EMT) characterized by the loss of various epithelial properties (for example, cell-cell adhesion) and the subsequent acquisition of a mesenchymal (motile) phenotype, an event molecularly similar to EMTs observed in metastatic or invasive cancers when cells leave the primary tumor site.
Our lab is studying neural crest formation in the avian (chicken) embryo to better understand overall animal growth and development. The chicken provides an excellent model system for understanding neural crest development for a number of reasons: 1) The embryo is large and develops externally, making it amenable to both in ovo (in the egg) and in vitro manipulations; 2) the availability of the genomic sequence allows us to do molecular biology and biochemistry-based assays; and 3) the wealth of descriptive literature detailing neural crest development gives a rich history upon which we can draw for our studies. Thus, the chicken is the ideal organism in which to study the neural crest and elucidate how abnormalities in neural crest development give rise to various diseases, disorders and cancers. To address these effects on health and proper development, we must first ask some general questions about neural crest formation. How do these cells arise in the dorsal neural tube, and what distinguishes them from the rest of the neural tube? What molecular cues transform these non-motile precursors (the premigratory neural crest) to migratory neural crest cells? Collectively, we aim to understand the functional role of Wnt signaling, and genes regulated by Wnt, during this process.
In ovo electroporation of morpholino antisense-oligonucleotides (MOs) into the neural tube targets a specific mRNA in the premigratory neural crest of the chick midbrain. MOs are employed in order to knock-down/inhibit translation of the mRNA of interest. MOs are labeled with a fluorophore (here lissamine, red) to allow for visualization with a fluorescence microscope.
Identification of Wnt target genes in the neural crest
The secreted Wnt protein family is known to function in cell fate specification, including neural crest development. But what genes does Wnt signaling regulate in the neural crest? What is/are the molecular mechanism(s) underlying this regulation? Taking advantage of a Wnt-induced gene expression profile in the neural crest obtained from a microarray screen, our lab is exploring how these genes function during avian neural crest development. Current experiments are aimed at determining the functional role of these genes in the induction, migration and differentiation of the neural crest, as well as elucidating the molecular mechanism(s) by which Wnt signaling regulates gene expression. We use a combination of molecular biology (quantitative PCR - QPCR, generation of expression constructs) and embryological (electroporation of DNA or oligonucleotides, in situ hybridization, cell injections, placement of protein-coated beads) approaches to address these questions.
Overexpression of a putative Wnt target gene enhances neural crest cell migration. Whole-mount in situ hybridization was performed for the premigratory and migratory neural crest cell marker FoxD3, followed by transverse sectioning. Note the presence of more FoxD3-positive cells (arrow in section), as indicated by the more intense purple staining observed on the side of the embryo in which the gene is overexpressed (right), compared to the contralateral control side (left).
The Wnt target Snail2 and neural crest EMT/emigration
Because of the known role of Snail transcriptional repressors in both neural crest development and tumor cell EMT, our lab is examining the function of Snail2 (formally called Slug) during neural crest EMT and emigration of cells from the dorsal neural tube. What genes does Snail2 target and repress during neural crest EMT? Are there distinct molecular similarities (or differences) between neural crest EMT and tumor cell EMT? One example of this is the direct transcriptional repression of the cell adhesion molecule cadherin6B (Cad6B) by Snail2 during neural crest EMT, mediated by the binding of Snail2 to E boxes (Snail2 binding sites) in the Cad6B regulatory region. Interestingly, the molecular mechanism of this regulation is analogous to that seen when Snail1 represses transcription of another cadherin, E-cadherin, during tumor cell EMT. Using molecular biology (QPCR), embryological (electroporation, in situ hybridization), and biochemical (chromatin immunoprecipitation - ChIP, gel shifts - EMSAs, luciferase assays) techniques, we are investigating the function of Cad6B, and other genes regulated by Snail2, in neural crest EMT.
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