RET/GDNF signalling in vagal neural crest-derived neurons of the chick embryo cloaca

Introduction  The growth factor, ‘Glial cell line-Derived Neurotrophic Factor’ (GDNF), is involved in the development of enteric ganglia, using the tyrosine kinase receptor ‘REarranged during Transfection’ (RET) to stimulate the proliferation and differentiation of neural crest-derived precursor cells. To date, the presence of these signalling molecules have not been studied in the developing cloaca, thus the aim of this study was to investigate the distribution of RET and GDNF, and analyse their co-localisation in vagal-derived neurons of the cloaca using quail-chick chimera embryos. Materials and methods  Chicken embryos were incubated until the 10–12 somite stage. The vagal neural tube was microsurgically ablated in ovo and replaced with the vagal neural tube from age-matched quail embryos. Quail-chick chimera embryos were harvested at E12, fixed and embedded in paraffin wax, and serially sectioned. Immunohistochemistry was performed using human natural killer-1 (HNK-1), quail-cell-specific perinuclear (QCPN), GDNF and RET antibodies. Results  HNK-1 labelled all ganglia in the myenteric and submucosal plexuses of the cloaca, while the quail-specific QCPN antibody labelled all ganglia derived from the transplanted quail vagal neural tube (Fig. 1, a, b). RET and GDNF were found both co-localised and expressed in separate ganglia in the cloaca (Fig. 1, c, d). The majority of QCPN-labelled vagal-derived neurons also expressed RET and GDNF. Fig. 1  HNK-1 (a), QCPN (b), GDNF (c) and RET (d) immunoreactivity in the chick cloaca at E12. Arrows show ganglia displaying co-immunoreactivity for all four antibodies

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Conclusion  Results show that GDNF and RET signalling play a major role in ENS development in the chick embryo cloaca. We have shown, for the first time, that the majority of vagal neural crest-derived neurons co-express RET and GDNF, thus highlighting the importance of these signalling factors in cloacal development.