Foregut Endoderm Requirement in Neural Patterning 311

Development 128, 309-320 (2001) 309 Printed in Great Britain © The Company of Biologists Limited 2001 DEV1634 Foregut endoderm is required at head process stages for anteriormost neural patterning in chick Sarah Withington1, Rosa Beddington2 and Jonathan Cooke1,* Division of Developmental Neurobiology, and Division of Mammalian Development, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK *Author for correspondence (e-mail: [email protected]) Accepted 8 November 2000; published on WWW 11 January 2001 SUMMARY Anterior definitive endoderm, the future pharynx and flexure and of heart migration. Important gene expressions foregut lining, emerges from the anterior primitive streak within axial mesoderm (chordin, Shh and BMP7) appear and Hensen’s node as a cell monolayer that replaces unaffected in all embryos, including those due to be hypoblast during chick gastrulation. At early head process pattern-deleted, during the hours following the operation stages (4+ to 6; Hamburger and Hamilton) it lies beneath, when anterior brain pattern is believed to be determined. lateral to and ahead of the ingressed axial mesoderm. A specific system of neural anterior patterning signals, Removal of the monolayer beneath and ahead of the node rather than an anterior sector of the initially neurally at stage 4 is followed by normal development, the removed induced area, is lost following operation. Heterotopic lower cells being replaced by further ingressing cells from the layer replacement operations strongly suggest that these node. However, similar removal during stages 4+ and 5 patterning signals are positionally specific to anteriormost results in a permanent window denuded of definitive presumptive foregut. The homeobox gene Hex and the endoderm, beneath prechordal mesoderm and a variable chick Frizbee homologue Crescent are both expressed sector of anterior notochord. The foregut tunnel then fails prominently within anterior definitive endoderm at the to form, heart development is confined to separated lateral time when removal of this tissue results in forebrain regions, and the neural tube undergoes no ventral flexures defects, and the possible implications of this are discussed. at the normal positions in brain structure. Reduction in The experiments also demonstrate how stomodeal forebrain pattern is evident by the 12-somite stage, with ectoderm, the tissue that will, much later, form Rathke’s most neuraxes lacking telencephalon and eyes, while pouch and the anterior pituitary, is independently specified forebrain expressions of the transcription factor genes by anteriormost lower layer signals at an early stage. GANF and BF1, and of FGF8, are absent or severely reduced. When the foregut endoderm removal is delayed until stage 6, later forebrain pattern appears once again Key words: Neural induction, Forebrain patterning, Gastrulation, complete, despite lack of foregut formation, of ventral Foregut endoderm, Stomodeal ectoderm, Chick INTRODUCTION Bird development involves pregastrulation movements, streak formation and then lower layer replacements in the Evidence has recently accumulated that in mammalian blastoderm that closely resemble those in the mouse egg development a separate site, distinct from the classical organiser cylinder. Definitive endoderm forms during gastrulation from before gastrulation, provides anteriormost axial patterning an expanding pool of anterior streak- and node-derived cells signals (mouse data reviewed by Beddington and Robertson, that insert themselves into the earlier lower layer or hypoblast, 1999, and see Knoetgen et al., 1999 for the evidence from displacing this to peripheral, extra-embryonic positions. rabbit). This site, the anterior visceral endoderm (AVE) is of Current work suggests that signals from that pregastrulation purely extra-embryonic fate, being initially the lower layer at the lower layer are not required for complete anterior development distal tip of the pregastrulation mouse egg cylinder. It moves to in chick, as they are in mouse (Knoetgen et al., 1999; define the future anterior side of the egg cylinder, opposite the S. Withington and J. Cooke, unpublished data). Studies in site where the streak will form, and is finally replaced as a layer Xenopus had led to a model whereby neurally induced tissue by definitive foregut endoderm as the head process forms (the is initially of anterior character, and is modulated only by phase of gastrulation equivalent to node regression in chick). Co- progressively posteriorising signals for production of the ordination of signals from the AVE with those from the node and complete neuraxial pattern in normal development (Niewkoop its derivatives is thought to be required, to achieve full anterior 1977: Nieuwkoop et al., 1985; Knecht et al., 1995; Holowacz neural pattern (see for example, Shawlot et al., 1999). and Sokol, 1999). However more recent results, especially 310 S. Withington, R. Beddington and J. Cooke those from other vertebrates, suggest that a default neural layer (endoderm) is accessible. Cultures were set up at stages 4-5 and specification may correspond only with a generalised forebrain brought to a temporary condition with the vitelline membranes (prosencephalic) state, with separate posteriorising and stretched on the rings but slightly convex from pressure of albumen anteriorising signal systems required to generate the territories medium from beneath, and the blastoderm just covered with a 1:1 of a complete neural rudiment. Thus the chick head process, mixture of Liebovitz air-buffered TCM (GIBCO/BRL): Hanks BSS anteriormost definitive embryonic mesoderm + endoderm that (buffered salt solution; made up with 1/10 the normal concentration, i.e. 0.1 mM, of Ca2+ and Mg2+). Such preparations were incubated emerges from the node or organiser during gastrulation, is (38°C) for a further 2 or more hours, whereupon development indeed important for completion of anterior neural pattern continues. Following addition of a little more TCM:BSS, these (Pera and Kessel, 1997; Foley et al., 1997; Dale et al., 1997). warmed embryos were in the condition most suited for selective In bird gastrulation, unlike in the mouse, axial mesoderm and removal of the lower layer from an anterior sector that includes definitive endoderm appear to form defined layers as they first Hensen’s node and extends to the periphery of the area opaca. In a emerge from the node (Sanders et al., 1978; Stern and Ireland, smaller sample of embryos, this layer and also the entire emerged 1981). This process of definitive endoderm emigration in bird axial and prechordal mesoderm were removed. Fig. 1A-C show gastrulation has been considered by most investigators to begin diagrammatically the versions of the lower layer operation performed, prior to the formation of axial mesoderm, such that in the region indicating the extents of emerged head process, while D-F show, in anterior to the prechordal mesoderm, definitive endoderm that sagittal section, the cellular anatomy of the head process stages. Following operations, the space above the membrane is drained and will eventually line the anterior foregut directly underlies the medium beneath replenished to give slight convexity for onward anterior neural and non-neural ectoderm. At these stages (4+ to culture. Too much convexity encourages incomplete dorsal closure, 5; Hamburger and Hamilton, 1951), we find that chick endoderm especially in neural tubes of reduced pattern, in embryos without is cleanly separable as an intact epithelium-like layer, without foregut tunnels. Following heterotopic lower layer replacements disturbing the integrity of the prechordal mesoderm. However, however, convexity and thorough draining of fluid was absolutely the endoderm is appreciably thickened just anterior to the limit required for good contact and healing of the flattened graft. of the prechordal mesoderm, so that after endoderm removal the exposed prechordal mesoderm appears narrower in plan view In situ hybridisation and histological procedures than the transilluminated intact structure. By stage 6, after about Operated and synchronous control embryos were inspected, and fixed only 2 hours of further development, this clean separation is either at later head process and headfold stages (up to 8 hours further culture), or at the 10- to 15-somite stages (18-24 hours further impossible to make without either disrupting the anteriormost culture). Fixation, after washing in BSS, was either in 4% prechordal mesoderm structure, or leaving in place cells that paraformaldehyde in PBS for whole-mount in situ hybridisation appear integrated with it, so that small gaps are created in the (Nieto et al., 1995), or in 75% ethanol: 23% formaldehyde (36%w/v): removed cell layer. Seifert et al. (Seifert et al., 1993) similarly 2% acetic acid, for embedding in Fibrowax, sectioning at 7 µm and describe the later chick ‘prechordal plate’, a region where staining with iron Haematoxylin/ Eosin. Gene expression signal was definitive endoderm and mesoderm layers cannot be resolved examined and photographed in whole mount, and after vibratome that lies just ahead of most prechordal mesoderm. DiI evidence sectioning at 50 µm. (not shown) indicates that a minority of cells from the lowest DiI labelling epithelium-like layer beneath Hensen’s node at stage 4 may become intercalated into the central part of the prechordal plate DiI (Molecular Probes) from a 0.5% stock ethanolic solution was diluted 1:10 with 0.3 M sucrose. With the embryo stretched

Load more