All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chick<sup>GFP</sup> embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. Chick<sup>GFP</sup>-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Carbocyanine dye, Chimera, Developmental biology, Enteric nervous system, Intraspecies grafting, Issue 99, Neural crest cells, Neural tube, Transgenic GFP chicken, Vascular network, Vessel painting,
Journal of Visualized Experiments
Department of Clinical Genetics

Delalande, J.-M, Thapar, N, & Burns, A.J. (2015). Dual labeling of neural crest cells and blood vessels within chicken embryos using chickGFP neural tube grafting and carbocyanine dye Dil injection. Journal of Visualized Experiments, 2015(99). doi:10.3791/52514