INTRODUCTION Neural crest cells (NCC) are embryonic multipotent stem cells that migrate along defined pathways to give rise to a wide variety of phenotypes including neuronal, mesenchymal, endocrine and pigment cells (Kalcheim and Le Douarin, 1999). Owing to their developmental properties, and the relative accessibility of the neural crest (NC) for experimental manipulation within embryos, NCC serve as a useful model for investigating the mechanisms involved in cell specification, migration, proliferation and differentiation, i.e. fundamental processes in the development of complex multicellular organisms. In common with the remainder of the autonomic nervous system, the neurons and glia that constitute the enteric nervous system (ENS) are entirely derived from the NC (Le Douarin and Teillet, 1973; Yntema and Hammond, 1954). The rhombencephalic (vagal) region of the NC, adjacent to somites 1-7, contributes the vast majority of ENS precursors along the entire length of the gut (Epstein et al., 1994; Le Douarin and Teillet, 1973). Upon leaving the NC, these vagal-derived precursors enter the foregut then migrate in a rostrocaudal direction, reaching the terminal hindgut of the chick at embryonic day (E) 8-8.5 (Burns and Le Douarin, 1998; Le Douarin and Teillet, 1973) and of the mouse at E14 (Kapur et al., 1992; Young et al., 1998). A second source of enteric precursors, the sacral NC, which is situated caudal to somite 28 in the chick and somite 24 in the mouse, contributes cells to the postumbilical gut only (Burns et al., 2000; Burns and Le Douarin, 1998; Le Douarin and Teillet, 1973; Pomeranz and 2785 Development 129, 2785-2796 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 DEV2890 The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH- diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal- to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/ signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia. Key words: Enteric nervous system, Quail, Chick, Neural crest cells, Gut, Neurons, Glia SUMMARY In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation Alan J. Burns 1, *, Jean-Marie M. Delalande 1 and Nicole M. Le Douarin 2 1 Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK 2 Institut d’Embryologie Cellulaire et Moleculaire, College de France et CNRS, Nogent-sur-Marne, 94736, France *Author for correspondence (e-mail: a.burns@ich.ucl.ac.uk) Accepted 2 April 2002