In situ hybridization of chemotactically bioactive molecules on whole mounted chick embryo

Abstract

In vitro assays showed that the morphogen, sonic hedgehog (Shh), the chemokine, stromal cell-derived factor-1 (SDF-1), and the growth factor, neurotrophin-3 (NT-3), behaved as chemotactic signals for neural crest cells (NCCs) and are associated with a topological distribution of chemotactic molecules on whole cultured chick embryo. In situ hybridization was conducted to show the expression of mRNA transcripts of Shh, SDF-1 and NT-3, while protein immunolocalization and immunolabeling of NCCs were performed on whole mount early chick embryo stages. Spatiotemporal expression of bioactive molecules was shown in the target fields of NCCs of early embryo cephalic sites, supporting the in vivo chemotactic behavior of this cell population. This evidence strongly suggests that directional migration of mesencephalic NCCs is modulated by guiding molecules segregated from the optic vesicle region, where NCCs colonize and differentiate into neurons and glia of the ciliary ganglia. The capacity to visualize the in situ expression of molecules involved in the chemotactic response of NCCs to concentration gradients established from target regions is intrinsically important for understanding the mechanism of oriented migration of embryonic cells, and also contributes to uncovering additional cell-guiding activities for chemokines, trophic factors and morphogens.