foregut endoderm requirement in neural patterning - development

INTRODUCTION

Evidence has recently accumulated that in mammaliandevelopment a separate site, distinct from the classical organiserbefore gastrulation, provides anteriormost axial patterningsignals (mouse data reviewed by Beddington and Robertson,1999, and see Knoetgen et al., 1999 for the evidence fromrabbit). This site, the anterior visceral endoderm (AVE) is ofpurely extra-embryonic fate, being initially the lower layer at thedistal tip of the pregastrulation mouse egg cylinder. It moves todefine the future anterior side of the egg cylinder, opposite thesite where the streak will form, and is finally replaced as a layerby definitive foregut endoderm as the head process forms (thephase of gastrulation equivalent to node regression in chick). Co-ordination of signals from the AVE with those from the node andits derivatives is thought to be required, to achieve full anteriorneural pattern (see for example, Shawlot et al., 1999).

Bird development involves pregastrulation movements,streak formation and then lower layer replacements in theblastoderm that closely resemble those in the mouse eggcylinder. Definitive endoderm forms during gastrulation froman expanding pool of anterior streak- and node-derived cellsthat insert themselves into the earlier lower layer or hypoblast,displacing this to peripheral, extra-embryonic positions.Current work suggests that signals from that pregastrulationlower layer are not required for complete anterior developmentin chick, as they are in mouse (Knoetgen et al., 1999;S. Withington and J. Cooke, unpublished data). Studies inXenopushad led to a model whereby neurally induced tissueis initially of anterior character, and is modulated only byprogressively posteriorising signals for production of thecomplete neuraxial pattern in normal development (Niewkoop1977: Nieuwkoop et al., 1985; Knecht et al., 1995; Holowaczand Sokol, 1999). However more recent results, especially

309Development 128, 309-320 (2001)Printed in Great Britain © The Company of Biologists Limited 2001DEV1634

Anterior definitive endoderm, the future pharynx andforegut lining, emerges from the anterior primitive streakand Hensen’s node as a cell monolayer that replaceshypoblast during chick gastrulation. At early head processstages (4+ to 6; Hamburger and Hamilton) it lies beneath,lateral to and ahead of the ingressed axial mesoderm.Removal of the monolayer beneath and ahead of the nodeat stage 4 is followed by normal development, the removedcells being replaced by further ingressing cells from thenode. However, similar removal during stages 4+ and 5results in a permanent window denuded of definitiveendoderm, beneath prechordal mesoderm and a variablesector of anterior notochord. The foregut tunnel then failsto form, heart development is confined to separated lateralregions, and the neural tube undergoes no ventral flexuresat the normal positions in brain structure. Reduction inforebrain pattern is evident by the 12-somite stage, withmost neuraxes lacking telencephalon and eyes, whileforebrain expressions of the transcription factor genesGANF and BF1, and of FGF8, are absent or severelyreduced. When the foregut endoderm removal is delayeduntil stage 6, later forebrain pattern appears once againcomplete, despite lack of foregut formation, of ventral

flexure and of heart migration. Important gene expressionswithin axial mesoderm (chordin, Shhand BMP7) appearunaffected in all embryos, including those due to bepattern-deleted, during the hours following the operationwhen anterior brain pattern is believed to be determined.A specific system of neural anterior patterning signals,rather than an anterior sector of the initially neurallyinduced area, is lost following operation. Heterotopic lowerlayer replacement operations strongly suggest that thesepatterning signals are positionally specific to anteriormostpresumptive foregut. The homeobox gene Hexand thechick Frizbee homologue Crescent are both expressedprominently within anterior definitive endoderm at thetime when removal of this tissue results in forebraindefects, and the possible implications of this are discussed.The experiments also demonstrate how stomodealectoderm, the tissue that will, much later, form Rathke’spouch and the anterior pituitary, is independently specifiedby anteriormost lower layer signals at an early stage.

Key words: Neural induction, Forebrain patterning, Gastrulation,Foregut endoderm, Stomodeal ectoderm, Chick

SUMMARY

Foregut endoderm is required at head process stages for anteriormost neural

patterning in chick

Sarah Withington 1, Rosa Beddington 2 and Jonathan Cooke 1,*

Division of Developmental Neurobiology, and Division of Mammalian Development, National Institute for Medical Research, TheRidgeway, Mill Hill, London NW7 1AA, UK*Author for correspondence (e-mail: [email protected])

Accepted 8 November 2000; published on WWW 11 January 2001

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those from other vertebrates, suggest that a default neuralspecification may correspond only with a generalised forebrain(prosencephalic) state, with separate posteriorising andanteriorising signal systems required to generate the territoriesof a complete neural rudiment. Thus the chick head process,anteriormost definitive embryonic mesoderm + endoderm thatemerges from the node or organiser during gastrulation, isindeed important for completion of anterior neural pattern(Pera and Kessel, 1997; Foley et al., 1997; Dale et al., 1997).

In bird gastrulation, unlike in the mouse, axial mesoderm anddefinitive endoderm appear to form defined layers as they firstemerge from the node (Sanders et al., 1978; Stern and Ireland,1981). This process of definitive endoderm emigration in birdgastrulation has been considered by most investigators to beginprior to the formation of axial mesoderm, such that in the regionanterior to the prechordal mesoderm, definitive endoderm thatwill eventually line the anterior foregut directly underliesanterior neural and non-neural ectoderm. At these stages (4+ to5; Hamburger and Hamilton, 1951), we find that chick endodermis cleanly separable as an intact epithelium-like layer, withoutdisturbing the integrity of the prechordal mesoderm. However,the endoderm is appreciably thickened just anterior to the limitof the prechordal mesoderm, so that after endoderm removal theexposed prechordal mesoderm appears narrower in plan viewthan the transilluminated intact structure. By stage 6, after aboutonly 2 hours of further development, this clean separation isimpossible to make without either disrupting the anteriormostprechordal mesoderm structure, or leaving in place cells thatappear integrated with it, so that small gaps are created in theremoved cell layer. Seifert et al. (Seifert et al., 1993) similarlydescribe the later chick ‘prechordal plate’, a region wheredefinitive endoderm and mesoderm layers cannot be resolvedthat lies just ahead of most prechordal mesoderm. DiI evidence(not shown) indicates that a minority of cells from the lowestepithelium-like layer beneath Hensen’s node at stage 4 maybecome intercalated into the central part of the prechordal plateby stage 6. Thus the clearly epithelial, early lower layer issubstantially of definitive endodermal fate, but may includesome prospective (though not necessarily prespecified)prechordal mesodermal cells.

To investigate whether developmental information for neuralpatterning is carried in the chick anterior definitive endoderm,we have studied the results of removing it at stages between 4,the full length streak just before node regression begins, and 6,the head process just before headfold initiation.

During headfold stages 7 or 8, during which anterior neuralpatterning is believed to become stabilised, we have examinedexpression of various genes of known or putative signallingfunction, both in exposed axial mesoderm and in relation to theregion of lower layer that is removed.

We discuss the results in terms of current ideas about theroles of the genes mentioned, and about steps in vertebrateneural induction and anterior patterning.

MATERIALS AND METHODS

Embryo culture and operationsOperations were performed in modified New (New, 1955) ringculture, where blastoderms are developing inverted so that the lower

layer (endoderm) is accessible. Cultures were set up at stages 4-5 andbrought to a temporary condition with the vitelline membranesstretched on the rings but slightly convex from pressure of albumenmedium from beneath, and the blastoderm just covered with a 1:1mixture of Liebovitz air-buffered TCM (GIBCO/BRL): Hanks BSS(buffered salt solution; made up with 1/10 the normal concentration,i.e. 0.1 mM, of Ca2+ and Mg2+). Such preparations were incubated(38°C) for a further 2 or more hours, whereupon developmentcontinues. Following addition of a little more TCM:BSS, thesewarmed embryos were in the condition most suited for selectiveremoval of the lower layer from an anterior sector that includesHensen’s node and extends to the periphery of the area opaca. In asmaller sample of embryos, this layer and also the entire emergedaxial and prechordal mesoderm were removed. Fig. 1A-C showdiagrammatically the versions of the lower layer operation performed,indicating the extents of emerged head process, while D-F show, insagittal section, the cellular anatomy of the head process stages.Following operations, the space above the membrane is drained andthe medium beneath replenished to give slight convexity for onwardculture. Too much convexity encourages incomplete dorsal closure,especially in neural tubes of reduced pattern, in embryos withoutforegut tunnels. Following heterotopic lower layer replacementshowever, convexity and thorough draining of fluid was absolutelyrequired for good contact and healing of the flattened graft.

In situ hybridisation and histological proceduresOperated and synchronous control embryos were inspected, and fixedeither at later head process and headfold stages (up to 8 hours furtherculture), or at the 10- to 15-somite stages (18-24 hours furtherculture). Fixation, after washing in BSS, was either in 4%paraformaldehyde in PBS for whole-mount in situ hybridisation(Nieto et al., 1995), or in 75% ethanol: 23% formaldehyde (36%w/v):2% acetic acid, for embedding in Fibrowax, sectioning at 7 µm andstaining with iron Haematoxylin/ Eosin. Gene expression signal wasexamined and photographed in whole mount, and after vibratomesectioning at 50 µm.

DiI labellingDiI (Molecular Probes) from a 0.5% stock ethanolic solution wasdiluted 1:10 with 0.3 M sucrose. With the embryo stretched in culture(see Embryo Culture), small groups of surface or deeper-lying cellswere labelled by ejecting pulses of dye from a micropipette tip ofapprox. 10 µm diameter, onto the surface of the cells. A PicospritzerII (General Valve Corporation, USA) delivering a square pulse of airpressure was used, with empirical adjustment of the pressure head(approx. 10 psi) and pulse length (approx. 50 milliseconds) to givesuitable sized labelled cell groups. Following further development forvarious time periods, embryos were fixed in 4% paraformaldehydeand examined in a Zeiss Axiophot microscope under phase andepifluorescence optics.

RESULTS

Region of endoderm removedFig. 1 shows the lower layer region removed. This includes themost anterolateral definitive endoderm, which emits inductivesignals inducing cardiac development in mesoderm(Schultheiss et al., 1995). The bilateral precardiac mesodermsultimately migrate across this region, with both layersundergoing rostrocaudal sequence inversion and convergentextension, as the foregut and associated heart tube form. Wehave not mapped in detail any relative movements betweenendo- and mesodermal layers within head process duringits emergence, as has been done for axial mesoderm and

S. Withington, R. Beddington and J. Cooke

311Foregut endoderm requirement in neural patterning

neurectoderm (Dale et al., 1999). However, DiI fatemapping in normal development (not shown)indicates that endoderm may continue to slideforward and outward relative to other layers as thehead process advances (see also Bellairs, 1953a;Bellairs, 1953b). The removal operation is mostsafely carried out without puncturing ectoderm ifresection of the lower layer extends right to theanterior periphery of the area pellucida as shown. However,removals that are confined to the prospective area that will liearound and ahead of prechordal mesoderm at headfold stages,i.e. to the limit defined by dashed line in Fig. 1B (8 cases), giveequivalent results. This indicates that the forebrain patterningsignals we are studying reside there rather than moreperipherally, which maps to extra-embryonic definitiveendoderm + displaced hypoblast.

Anterior neural pattern defects depend upon stageof host at operationFollowing stage 4 operations, the window in lower layerquickly compresses anteriorly and closes up, so that all axialmesoderm emerging from the regressing node is covered bylower layer as normal (Fig. 2A-D). DiI labelling of the noderegion immediately after endoderm removal at stage 4, showsthat the window is filled in by additional cells ingressingthrough the node and moving anteriorly (Fig. 2H-K). This newlower layer forms a normal substratum for cardiac inductionand migration, and forms normal foregut. Thus, it either is, orhas regulatively acquired the properties of, the endoderm thatwould normally have underlain the head process. We have beenunable to ascertain to what extent this result occurs becausenode cells are normally still emigrating to form definitiveforegut endoderm at this stage, or because a regulatoryextension of this emigration takes place in response toendoderm removal.

Following later endoderm removals (stage 4+ onwards), thewindow of endoderm does not close up. Therefore all headprocess mesoderm that has left the node prior to the operationremains permanently uncovered (Fig. 2E-G). Middle layertissue denuded of the endodermal epithelium can sometimescollapse its intercellular spaces, superficially re-assuming anepithelial appearance. This can be seen in the hours followingoperation (Fig. 6E,F), and at more advanced stages (Fig. 3B,C),but is quite distinct from genuine re-covering by endodermallateral migration. Performance of the operation at head processstages 4+ or 5 leads to loss of forebrain pattern in the majorityof embryos (Fig. 1B,H). Since, despite Tables of NormalStages, development is continuous, the correlation betweenembryonic stage recorded at operation and the anatomicaloutcome is imperfect. However, this correlation is highlysignificant. When performed at stage 4, the operation usuallyleads to normal later development (Fig. 1A,G; 166/221essentially normal cases). From operations performed at stages4+ and 5, there were only 64/235 normal cases. In addition tothe forebrain patterning defect, the normally induced hearttissue is unable to migrate to the midline, and differentiates astwo separated lateral halves. Presumably due to lack of themechanical influence of foregut tunnel formation, the neuraxisremains stretched out in a line rather than ventrally flexed andraised from the general blastoderm.

Following similar endoderm removal operations attemptedat even later stages, from 5+ and especially at stage 6, the final

Fig. 1. Lower layer removal operations. (A-C) Schematicviews of the operation from the lower layer aspect asviewed in ring culture. Lower layer areas removed shown inred for operations at stage 4 (A), stages 4+ to 5 (B), andstage 6 (C). Dashed line in B indicates more restricted outerlimits of removal in a subset of cases producing the sameresult. (D-F) Sagittal sections of head process, anterior is tothe right and regressing node is to the left. (D,E) Twosuccessive stages when lower layer can be removed intactare seen. (F) A separate epithelial lower layer is no longerresolvable at stage 6. (G-I) Schematic views from ventralaspect, at around the 12-somite stage, following operationsof types A, B, and C respectively. Both the intermediate andthe older stage operations result in absence of foreguttunnel, paired lateral heart rudiments (red) and lack ofventral flexure in CNS structure (outlined in black), butonly operations performed at the intermediate stage givereduced anterior CNS pattern at high frequency. Shh-expressing axial mesoderm, dark blue; heart tissue, red;somite outlines, light blue. Typical windows of absentlower layer, including foregut endoderm, are indicated by adashed line. Stomodeal ectoderm (green) appears ventrallypositioned in the normally developing head, is absent fromthe pattern in H and appears as a ‘pipe’ stretching beneathforebrain from an anterior position in I. Note anterolateraleye vesicles and subdivision of forebrain in I. Scale bar,100 µm in D-F.

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forebrain pattern tends once againtowards being complete, althoughneuraxes are neverthelessunfamiliar in appearance in thecontinued absence of ventral flexure, foregut formation andheart migration (Fig. 1C,I). At the latter stage, it is not possible(either surgically or in light microscopy, see Fig. 1F) clearly toresolve an epithelial covering from prechordal mesoderm at itsanterior extreme. Thus, lower layer at these stages can neverbe removed entire, without gaps corresponding to cells thathave been left behind. (We do, however, make every effort toremove these remaining cells as well.) Results of these lateroperations are more variable, with the anteriormost geneexpressions variably restored as described below, and small ornormal sized optic vesicles appearing anterolaterally. Theperiod during which removal of endoderm alone can beperformed successfully, and cause defects in subsequentforebrain regionalisation, is therefore essentially restricted tostages 4+ and 5.

Fig. 3 shows sagittal and anterior transverse sectional views,at the 12- to 15-somite stages, of the structure of controlembryos (stage 4 operated, or with peripheral cutting but noresection of endoderm layer), in comparison with typicalpattern-reduced experimental ones. Examples are also shownof experimental embryos that nevertheless have normallyregionalised forebrains, following ‘late’ attempts at endodermremoval. Following the effectively timed endoderm removals,the dorsal forebrain territory remains as a simple-lookingvesicle, rather than becoming subdivided into anteriortelencephalon, and diencephalon associated with lateral optic

rudiments, However, this vesicle is bulbous, rather than thedistinctive, narrow proboscis-like shape (minimal growth) thatis associated with loss of Sonic hedgehog (Shh)gene functionin both mouse (Chiang et al., 1996) and chick (Towers et al.,1999). In addition, the early morphological appearance of theinfundibular region remains as a keel-shaped depression, in theventral midline of the neural cross-section associated with theanteriormost axial mesoderm (Fig. 3K,L). This neural ventralmidline morphology is suppressed following disruption of Shhfunction as such (Chiang et al., 1996; Towers et al., 1999).Interestingly, following late and only partially effectiveendoderm removals, the small eyecups that develop are situatedwide apart at lateral extremes of the expanded brain vesicle(Fig. 3F), whereas the small eyecups of threshhold Shhdeficiency are very ‘ventral’ and close together near themidline of the narrowed forebrain, giving a morphologicalseries from cyclopia to semi-synopthalmia (Incardona et al.,1998; Towers et al., 1999).

Loss of forebrain-specific gene expressionsfollowing endoderm removal Forebrain pattern was further assessed at the 12- to 15-somitestage by in situ hybridisation for expressions of anterior neuralmarker genes. Fig. 4 shows typical results. The telencephalicregion, although still small at the 12-somite stage, willultimately undergo the greatest growth of all brain parts. Its


Fig. 2. Behaviour of lower layer afteroperations. (A-D) Successivetimepoints in the hours following astage 4 operation. The window in lowerlayer shortens, then becomescompressed and is displacedperipherally, being occluded or scarcelydetected ahead of the normal headprocess by stage 5 (D). Embryosconforming to stage 4 by externalmorphology have a penumbra of diffusemesoderm around the denuded node(A), but no fan of organised prechordalhead process. (E-G) A similar seriesafter an operation at stage 5+, showinghow the window in lower layer narrows,but remains over midline. The entirehead process sector that had emerged atthe time of the operation, and an areaahead of it, thus remains denuded. (H,Iand J,K) Two pairs of images fromembryos in which the deep part of thenode was DiI marked just after a stage 4removal operation, and where lowerlayer recovery has occurred. Labelledcells are populating the recovered lowerlayer well ahead of the node, in an areamore extensive than that occupied bynewly emerging underlying mesoderm(seen as more solid red signal just aheadof node outline in J). Scale bar, 250 µmin A-G and 180µm in H-K.


absence in our experimental embryos was demonstrated by theuse of several marker genes. BF1 (Alvarez et al., 1998) awinged helix transcription factor gene with normal earlyexpression restricted to telencephalon and anteromedial eyecupregions (Fig. 4E), and essential for their development in mice(Dou et al., 1999), is not expressed (4F, 8/15 cases) or showssmall, asymmetrical remnants of expression (5/15). GANF, thechick homologue of the mouse homeobox gene Hesx1, is alsonormally expressed in the telencephalon at the 10- to 12-somitestage, but with a ventral extension beyond the BF1domain andback to prospective infundibular level (Fig. 4I-J). The pattern-

reduced brains usually show no neural GANFexpression (4K,16/25 cases), or only a small ventral patch remains. Forebrainexpression of FGF8is also specifically lost (12/16 cases),while all other aspects of its expression remain normal (Fig.4G,H). The retained FGF8expression marking the isthmus(midbain-hindbrain boundary) at the rear of two simplevesicles, together with the homogeneous dorsalPax-6expression throughout the more anterior of these vesicles (Fig.4D, 11 cases), strongly suggests a general prosencephalicidentity for the latter (Pera and Kessel, 1997). However,subregionalisation of this vesicle does not occur, since the

Fig. 3. Anterior CNS structure; definitiveendoderm removals and endodermal plusaxial mesoderm removals. (A-F) Sagittalsections. Asterisks mark futureinfundibular region of brain associatedwith prechordal mesoderm, normally(A,D) lying near tip of invaginated foregutand above stomodeal head ectoderm.White arrowheads mark the narrowing ofthe isthmic or midbrain-hindbrainjunctional region (see FGF8of Fig. 4),where this area is present in plane ofsection. (A) Normal axis at the 12-somitestage following sham-control operation.Note expanded forebrain anterodorsally,with morphological division betweentelencephalic and diencephalic rudiments(black arrowhead). (B,C) Specimens atsimilar stages to A, but following stage 5lower layer removal operations. Noteretention of early infundibular ventralmidline morphology despite non-flexure ofbrain and absence of foregut tunnel andstomodeal ectoderm, but relatively shortforebrain with undivided morphology.Anterior notochord shows better in Bwhere section is exactly sagittal.(D) Normal axis at the 15-somite stage.Note more progressed subdivisions indorsal brain structure. (E,F) 15-somiteembryos following stage 6 lower layerremovals. Forebrain in E appearsqualitatively complete though compressedby lack of ventral flexure, with expansionand telencephalic/diencephalic divisiondorsally. In lateral section (F) opticvesicles (ov) are seen directed anteriorly inline with forebrain. (These would bevisible only at extreme lateral section,ventrolateral to the downflexed forebrain, in a normal brain such as in D.) (G-N) Transverse sections, all at the 12- to 15-somite stages. (G)Sham-operated control, mid-optic vesicle level. Note lateral expansion. (H) Two sections, anteriormost above, through morphologicallytransformed complete axial pattern after stage 6 lower layer removal (anatomy similar to E,F). Tube of stomodeal-type ectoderm (diamond)passes ventrally beneath anterior preneural ectoderm, ending against neural wall between anterolateral optic vesicles. (I) Infundibular level innormal axis, sham-operated control as in A,D. Note ‘keeled’ ventral neural midline (dagger) associated with anteriormost midline mesoderm,lying just ahead of tip of foregut endoderm pocket. Note, head ectoderm at this level has been damaged in section. (J) Simple anterior brainvesicle, following stage 4+ to 5 lower layer removal. Dorsoventral compression, which may occur sometimes due to ring culture and/orsectioning, is quite distinct from optic vesicle formation (compare with G). (K,L) Slightly more posterior level, similar to that of I, in twospecimens following endoderm removal. Note considerable retention of ventral neural midline morphology (daggers), but with underlyingmesoderm abnormally closely apposed and compressed due to lack of foregut tunnel. (M) Anterior level grazing simple vesicle as in J, butfollowing removal of both lower layer and anterior head process mesoderm at stage 6. Note stomodeal ‘tube’ (diamond) as in H, despite absenceof forebrain regionalisation. Note, ectoderm has flexed ventrally to cover head and lift it off blastoderm despite removal of inner tissues. (N) levelas in K, L but in a specimen with simple forebrain vesicle (as in B,C) after stage 5 meso+endoderm removal. Little further deficit in early CNSventral midline morphology (dagger), or growth, follows removal of mesoderm in addition to endoderm. Scale bar, 200 µm throughout.

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normal down-regulation of Pax-6expression inthe dorsoanterior midline (Fig. 4C) is not seen.Pax-6 expression is not expanded ventrally,consistent with the presence of normalinhibitory signals from ventral midline.

Following endoderm removal at stage 5+ to6, where brain pattern is often complete but witheye vesicles extending anterolaterally due to thelack of foregut tunnel, brain-associated BF1 and GANFexpressions can be normal in extent. The transitional result isa brain with very small optic vesicles protruding anteriorlyfrom the lateral edges of the forebrain ‘vesicle’ (Fig. 3F). Suchbrains show small or partial expression patches of BF1corresponding to partial restoration of dorsal telencephalicterritory.

Early patterning of the anterior neural plate afterendoderm removalOtx2 is expressed throughout the forebrain and midbrain fromvery early stages (Bally-Cuif et al., 1995), and is essential fortheir development. In the experimental pattern-reduced brainsat the 12-somite stage, the rostrocaudal extent of tissueexpressing otx2 appears normal, although its architecture issimplified into two vesicles (Fig. 4A,B). Comparison ofexperimental FGF8and Pax-6expressions helps identify thesetwo vesicles as prosencephalon and midbrain, and confirms thelack of subdivision in the former. Such specimens do notsuggest a reduced initial allocation of ectoderm to neural plateanteriorly, or even a reduced allocation within the initial neuralplate to forebrain territory. We nevertheless investigated moredirectly whether, following the endoderm removals at stage 4+or 5, the overall extent of the neural plate might have beenreduced because of failure of an anterior part of it to retainstable neural fate. Use of the pan-neural marker Sox3at neuralplate stages, 6-8 hours after operations, confirmed that in theseexperimental embryos the extent of the early neural rudimentremains normal (not shown, 4 cases). Neural plate otx2

expression at the same stages was also of normal relative extent(4 cases). In addition, early expression of GANF in the anteriorneural plate, first seen at stages coinciding with those ofeffective endoderm removals, remains near-normal at headfoldstages following such operations (9/11 cases normal, 2/11reduced), even though it is later lost. Therefore a distinctiveanterior neural territory is initiated as normal, but latersubregionalisation within this territory is either not induced ornot maintained.

Telencephalic expression of BF1 is not initiated until later, the7- to 8-somite stage, in chick. In mouse, early FGF8 expressionin the anterior neural ridge (ANR) has been implicated ininduction and/or maintenance of BF1 (Shimamura andRubenstein, 1997). We therefore examined FGF8 expressionat the 5- to 6-somite stage, its first reliable ANR-associatedappearance in normal chick, both in sham operated controls andfollowing foregut endoderm removals expected to lead toanterior pattern loss. A striking deficit in initial activation of thisgene was indeed associated with the removal of foregutendoderm (13/22 cases without expression, 3/22 cases withreduced expression), suggesting that the effects on neuraxial(and stomodeal) pattern might result, at least in part, frominadequate early expression of FGF8signal in the ANR.

Autonomous specification of stomodeal ectodermFollowing endoderm removal too late to result in forebrainpattern reduction, a significant proportion of the axes displayan unusual pipe-like tube of ectodermal-type epitheliumstretching ahead of the brain. This tube extends in from the


Fig. 4. Forebrain gene expressions at the 12-somitestage after definitive endoderm removal. Successivegene expressions are shown in whole-mount pairsfrom the dorsal aspect, with the control appearanceon the left, and the pattern-reduced brain on the right(stage 4+ or 5 operation). For GANF,the normaldorsal, then ventral appearances are shown.(A,B) Otx2. In the experimental case, expressionoccurs in the anterior two simple brain vesicles.(C,D) Pax6. In experimental case, the expressionoccurs in the anteriormost simple brain vesicle, andin non-neural ectoderm that would in the normalcase be applied to optic vesicles. (E,F) BF1.Experimental case lacks normal anterior neuralexpression.(G,H) FGF8. Experimental case lacksthe normal forebrain-associated expression, butretains isthmic (mid-hindbrain junctional)expression. Comparison of FGF (H) with Otx2(B)and Pax6 (D) helps diagnose the nature of the twosimple brain vesicles as prosencephalon andmidbrain, even though the former lacks subdivision.(I-K) GANF. In the experimental case, normalexpression associated with forebrain folds and withmore ventral facial (stomodeal) region is missingScale bar, 250 µm.


undersurface of ectoderm some way ahead of the forebrain, toend blindly at or near the infundibular region and its associateddispersed prechordal mesoderm (Figs 3H and 5). Suchembryos sectioned at around the 8-somite stage reveal that thistube is already developed. GANF and Shhare both expressedin this tube, as they are in normal stomodeal ectoderm(compare Fig. 5A,E with B,F), while the ectodermal pattern ofFGF8 expression near its base also strongly suggests itsidentity as stomodeum (Fig. 5C,D). It therefore appears thatthe stomodeum originates from its proper fate-map location inanterior ectoderm, but is unable to achieve its normal ventralposition due to lack of foregut tunnel and proper faceformation. This ectodermal region, once specified asstomodeum, apparently actively seeks cellular association withthe other components of the infundibular brain region,necessary for subsequent development of its normal derivatives(Rathke’s pouch and anterior pituitary). In these embryos thatlack ventral flexure and face formation, because anteriormostforegut endoderm was removed after having induced thestomodeal region, this association behaviour leads to theformation of the abnormal tube.

A further point of interest is that after later (stage 6)removals of prechordal and anterior chordamesoderm aswell as the endoderm, embryos that lack dorsal forebrainregionalisation similar to that after stage 4+-5 endodermremovals (see later subsection) may nevertheless displaythis anterior ‘pipe’ of stomodeal ectoderm leading to theinfundibular region (Fig. 3M compared with H). Therefore,although after endoderm removal, stomodeum is only specifiedwhen forebrain regionalisation has also occurred due to thelateness of that removal, specification of stomodeum does notrequire intact anterior forebrain pattern itself.

Midline signalling gene expressions in axialmesoderm are unaffected by endoderm removalShh and chordin are intercellular signal genes, expressedthroughout much of the axial mesoderm and the overlying

neural midline in the chick head process. We examined bothShhand chordin expressions in axial head process mesodermof appropriate endoderm-excised embryos, at both headfoldstages 7-8 HH (2-4 somites; 9 cases each gene) and the 12somites stage (Shh, 10 cases). There were no detectable deficitsin expression of either gene (Fig. 6A-D). BMP7expression instage 7-8 chick prechordal mesendoderm has been implicatedexperimentally in conferring anterior character to ventralneural midline (Dale et al., 1997; Dale et al., 1999; Vesqueet al., 2000). We therefore similarly examined this aspectof BMP7 expression in experimental embryos. Again, noappreciable deficit in prechordal mesodermal expression wasapparent (Fig. 6E,F; 10/11 cases).

Significance of normal gene expressions in theremoved endoderm regionThe homeobox gene Hex has expression in pregastrulationmouse AVE, the extra-embryonic region that is necessary forcomplete anteriormost axial patterning (see Introduction).However, it has recently been demonstrated that the essentialrequirement for Hex in forebrain patterning resides in itslater site of expression, the definitive embryonic endoderm(Martinez-Barbera et al., 2000). In chick, as well asexpression in the extra-embryonic lower layer, there is also aprominent patch of Hex expression in anterior midline foregutendoderm, centrally within the removed region in ourexperiments (Fig. 6G and Yatskievych et al., 1999). Webelieve that normal chick Hexexpression extends to cells ofthe prechordal mesoderm by later headfold stages, althoughthis has been difficult to detect decisively on sagittal sections(see also Yatskievych et al., 1999). Conditions of lower layerremoval that lead to anterior neural pattern reduction, bydefinition, result in absence of the prominent endodermal Hexexpression underlying the prechordal mesoderm and anteriorneural ridge around stage 7 (Fig. 6H; 10 cases). Furthermore,the complete absence of prechordal mesodermal expressionin 8/10 of these whole-mount specimens suggests that any

Fig. 5. Specification of stomodeal ectoderm withoutforegut formation. The abnormal pipe or tube ofstomodeal ectoderm (see Fig. 3H, M), tends to contractand bend beneath the brain during the whole-mount insitu hybridisation process. Pairs of specimens are shownat the 12- to 14-somite stage; normal above, stage 5+ to 6operated, below. (A,B)GANF. Expression in the normalstomodeum is seen ventrally, in the face, beneath theinfundibular brain region, and also marks the tube thathas been pulled back beneath the fully patternedforebrain in the foregut-less experimental case(asterisks). (C,D) FGF8. Two transverse expressionpatches flank the stomodeal region in the normal ventralview (C, compare with A). In the experimental case, alsoin ventral view, these patches (asterisks) instead flank theposition of an abnormal tube beneath anterior ectoderm,now stretching at right angles to the plane of paper.Curved edge of tube is in focal plane. Other (forebrainand isthmic) FGF8 expression sites are also visiblebeneath focal plane, because foregut tunnel is absent.(E,F) Shh. Sections of specimens stained as wholemounts. Expression is specific to the normal stomodealectoderm area (asterisk), which becomes closely appliedto anteriormost foregut that also expresses Shh. In experimental case, the expressing region corresponding to stomodeum has formed a tube(asterisk), that will make contact with anteriormost Shh-expressing area in neural midline (presumptive rostral diencephalon/ hypothalamus).

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normal middle layer Hexexpression, whether imported bycell intercalation or induced due to presence of underlyingendoderm, has also been prevented.

Chick Crescentencodes a putative secreted protein of theFrizbee group (Pfeffer et al., 1997), having the structure ofthe Frizzled receptor proteins for the Wnt signal pathwaybut lacking their intracellular signal transducing domain.Crescent is expressed in prestreak extra-embryonic lowerlayer, the hypoblast, and then strongly in the axial part of theearly head process endoderm. We find a broad, intenseexpression domain in anterior definitive endoderm, centredon, but considerably more widespread than, the site of Hexexpression. Seen in plan view, the area of Crescentexpressionat headfold stage corresponds rather closely with the lowerlayer area whose removal during stages 4+ or 5 distinctivelyreduces subsequent neural pattern (Fig. 6I). We find indeedthat when removals are carried out at stage 4, normalendodermal Crescent expression is already recovered byheadfold stages, as the gap in the endoderm layer is filled inand shrinks anteriorly. Following the stage 4+ and 5operations where the window underlies the axial mesoderm,the massive lower layer expression is absent at this positionas would be expected, and only the localised axialmesodermal expression remains (Fig. 6J; 8 cases).

Foregut endoderm provides instructive signals andnot just a maintenance environment, to headprocessWe performed experiments to address the issue of positionaldistinctiveness in signals from anterior definitive endoderm. Inone approach (Fig. 7A), embryos with this layer freshlyremoved in the stage 4+ or 5 operation were grafted with largeheterotopic pieces of correctly orientated lower layer, eitherfrom the prenodal sector of stage 4 donors (i.e. with hypoblastand presumptive extra-embryonic endoderm that would bedisplaced ahead of head process endoderm; 8 cases), or fromthe perinodal and posterior head process level of stage 6 or6+ donors (i.e. with more posterior presumptive foregutendoderm; 6 cases). Pieces were flattened to overlie an areacentred on the exposed prechordal mesoderm of hosts. Thedifficulties of this operation meant that the exposed ‘window’of the hosts was never entirely re-covered by lower layer, andmany cases were rejected from analysis because the layercontact achieved did not sufficiently resemble that in normaldevelopment. Nevertheless, in no case did the graft appear tohave rescued the anterior brain patterning deficiency as seenat the 12-somite stage. There was also little evidence forrestoration of the ability of precardiac tissue to migrate acrossthe re-covered anterior headfold area, or of foregut tunnelformation. In a second approach (Fig. 7B), endoderm fromover and just ahead of the stage 5 prechordal plate (i.e.centrally positioned within the normal removed region), wasexcised and re-inserted heterotopically, between endoderm andpresumptive mid- or hindbrain neural plate at one side of theregressing node of the same embryo. At headfold stagesfollowing a further 6-8 hours in culture, significant asymmetryof GANF expression was observed, biased to the grafted side(compare Fig. 7C with F; 8/15 cases). Four cases had separateareas of altered neural fold appearance with ectopic GANFexpression in contact with the graft, well posterior to the gene’snormal expression site (Fig. 7D,E). Similar lower layer piecesfrom more lateral and peripheral positions caused nodisturbance of either morphology or GANF expression (6cases).


Fig. 6. Gene expressionsin axial mesoderm andendoderm afteroperations. Normalwhole-mountappearances from ventralaspect (left) arecontrasted with those inspecimens operated onduring stage 4+or 5, thusexpected to loseforebrain patterning(right). A,B. Shh.At the12-somite stage shown,superimposednotochord/floorplateexpressions are often lessintense posterior to brainlevels, and are obscuredby the presence of heartand foregut tunnel in A.Lateral zones in theanterior fan of Shhsignalrepresent ventral neuralexpression.(C-J) Headfold stages,4-6 hours after time ofoperations.(C,D) Chordin.(E,F)BMP7.In somenormal specimens(though not in E), thereis a diffuse anteriorcrescent of expression inthe junctional areabetween foregut andextra-embryonicendoderm. In F, the sharpinner edges of such anarea reveal the windowin endoderm but theprechordal mesodermexpression remains.(G,H) Hex. Completeloss of central Hexexpression in theexperimental case. Theremaining crescenticlower layer expression inH shows that the removaloperation was of themore restricted typeindicated by dashed linein Fig. 1B, whichnevertheless producesthe patterning loss.(I,J) Crescent. Veryintense region of lowerlayer expression in I extends well lateral and anterior to subjacentprechordal mesoderm. Distribution corresponds well with theendoderm region whose removal deletes forebrain patterning. Scalebar, 200 µm.


The relative roles of endoderm andof axial mesodermIn our hands, extirpation during stage 5 ofthe entire emerged head process mesoderm,along with the definitive endoderm layeralready described, results in embryos whose anterior CNSstructure appears closely to resemble that resulting from lowerlayer removal alone. An indistinguishable result also followslater (stage 6) removals that include just this anteriorchordamesoderm along with the endoderm (compare Fig.3M,N with J-L). We have noted incidentally that these defectsfollowing mesoderm + endoderm removals, like thosefollowing the early removal of endoderm alone, differmorphologically from the defect resulting from Sonichedgehog (Shh) gene disruption as such. Despite their loss ofdorsal-anterior brain regionalisation, the embryos presentedhere retain a relatively expanded cross-section and pronouncedpre-infundibular midline morphology (see earlier subsection).We thus see little to suggest a greater loss of early CNS midlinespecification or growth patterning, both believed to be due toShhsignal, when the mesoderm extirpation is added to that ofendoderm (though see Pera and Kessel, 1997). By stage 6, asalready described, complete selective removal of endodermalone is not possible from over the prechordal region, and/oris ineffective in surrounding regions, since completelyregionalised neural patterns develop after attempts at thisoperation (sagittal sections of 3E,F and anterior transverse of3H).

DISCUSSION

We present evidence for a role of the chick definitive foregutendoderm in regionalisation of the forebrain. Followingremoval of this tissue as a complete epithelium-like layer atstages 4+ and 5, a simple-shaped but expanded prosencephalicvesicle forms, which lacks expression of telencephalic markersBF1, GANF and FGF8. This pattern deficit is not due to anoverall reduction in the neurally induced area, which isbelieved to be already defined by stage 4 or 4+ in normal

development (reviewed by Smith and Schoenwolf, 1998).Indeed, anterior neural GANFexpression is already detectablein normal embryos at the time of these operations, and is stillseen at headfold stages following stage 4+ to 5 endodermremoval. The deficit is rather in the failure to maintain or refinesubregionalisation within the prosencephalic territory, and thiscould be at least partly due to failure of early FGF8expressionin the ANR region. We have shown that the definitiveendoderm emits specific signals, since it can induce ectopicGANF expression in presumptive hindbrain territory whentransplanted. We also propose that the foregut endoderm is abledirectly to induce stomodeum in ectoderm at an anteriormostposition, before the ventral flexure of head formation is due tobring the latter into contact with ventral diencephalon and theforegut tunnel.

Separation of dorsal-anterior and neural ventralmidline patterningAt least following successful endoderm removal alone, the‘information’ that is deleted in our experimental embryos is notthat for neural ventral midline specification itself, viamaintenance of Shh orchordin expressions. We observeconsiderable retention of early midline morphology, and Pax6expression does not encroach abnormally upon the ventralmidline, as occurs in midline-deficient embryos. Even thedistinctive anteriorisation of midline character to give RostralDiencephalic Ventral Midline (RDVM; Dale et al., 1997; Daleet al., 1999) may be unaffected, since prechordal mesodermalBMP7 expression at headfold stages appears unaffected byendoderm removal at stage 4+ to 5. Instead, there is a lack ofone or more other signals that operate in combination withmidline patterning, to achieve normal regionalisation of moredorsal forebrain territories, so that territories that wouldrespond to midline-derived (Shhand other) signals by thedistinctive growth programmes e.g. of optic vesicle formation,

Fig. 7. Positional specificity in lower layer.(A) Operations to replace anterior definitiveforegut endoderm during stage 5 with stage 4(largely extraembryonic) lower layer (left) orwith more posterior definitive endoderm fromstage 6+ (right). (B) Operation ectopicallyrepositioning anteriormost definitive foregutendoderm, to underlie the mid-hindbrain regionin normal orientation. Operations are shownfrom lower layer aspect. (C) Normal GANFexpression at headfold stage 6-8 hours aftertime of operations. (D,E) Whole-mount andsectioned views of specimen at headfold stagefollowing the operation B, showing ectopicregion of GANF-expressing neural fold withaltered morphology at mid-hindbrain level overgraft (asterisks). (F) Another specimen afteroperation B, with ectopic backward extensionof GANF expression on grafted side. Whole-mount specimens are shown from dorsal aspect.Scale bar, 500 µm for C,D,F; 300 µm for E.

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have not been specified. More than Shh mutants, the presentexperimental embryos resemble other mouse mutants (Shawlotand Behringer, 1997; Andreazzoli et al., 1999; Dattani et al.,1998; Martinez-Barbera et al., 2000).

Transfer of information between embryonic layers;the role of HexMost recent work has assumed that neural-inducing signals perse in vertebrates, as distinct from signals for regionalisation,come from the classical gastrular organiser, the node-equivalent or its precursors, principally by planar but perhapsby some later ‘vertical’ transfer. By head process stages,anterior neural regionalising signals in vivo would be expectedto act only by locally modulating this already established stateof general neural induction. While such signalling could inprinciple occur by further planar transfer along the early neuralplate, underlying mesoendoderm is now believed to exert majorinfluences. Correspondingly, ‘deep’ (thus mesendodermal)tissue, emerged from the newly regressing chick node, isreported to be a poor neural inducer, while being an effectiveneural anterioriser (Foley et al., 1997).

In chick, endoderm and axial mesoderm layers of the headprocess are clearly distinct both as tissue leaves the node andin the head process through stage 5. In mouse, axial mesodermand definitive endoderm emerge from the node as a commonhead process population, only later separating, so that detailsof information transfer in the head process may differ.Complete anterior development in mouse has been shownto require a pregastrulation, extra-embryonic signallingcomponent (Rhinn et al., 1998; reviewed by Beddington andRobertson, 1999). So far, the evidence is that this necessarycomponent is not included in chick development (Knoetgenet al., 1999; S. Withington and J. Cooke, unpublished data).However, there appears also to be a subsequent necessarytransfer of anteriorising information to the CNS from mousedefinitive endoderm (Martinez-Barbera et al., 2000) andmesoderm (Ang et al., 1994; Shawlot et al., 1999) in the headprocess stage. We have shown here that the equivalentendoderm tissue in chick plays a similar anterior regionalisingrole. A further question concerns whether patterninginformation from endoderm acts directly on the ectoderm layer,or by modulating or stabilising the subsequent signallingproperties of head process mesoderm (see also Vesque et al.,2000). By stage 6, there may have been incorporation of cellsfrom the definitive endoderm into mesoderm at the prechordalregion, where these components become indistinct (Seifert etal., 1993).Any crucial information transfer from lower layerinto mesoderm could thus, in principle, have occurred throughsuch cell intercalation as well as by influence of secretedmolecules. During stage 6, dorsal-anterior forebrain patterningappears to have become stable in the presence of mesodermalone, though not yet stable if anterior head process mesodermis also removed.

Our results overall are consistent with either of two models.In a successive transfer model, ‘information’ influencing eventhe dorsal-lateral forebrain pattern that originates in definitiveendoderm, would be first transmitted as an influence on (oreven cellular incorporation into) head process mesoderm. Themesodermal inductive properties thus supported would thentake some further time, to complete their own influence onectoderm.

An alternative model is that the information our experimentsreveal is transferred directly from endoderm to ectoderm, inparallel with any information from mesoderm that itself mayproceed via midline patterning or independently of it. Thepresumptive anterior and lateral neural ridges at early stages,and the presumptive stomodeal ectodermal area ahead of them,lie outside the extent of head process mesoderm and aredirectly underlain by that region of foregut endoderm thatdisplays, for instance, strong Crescentexpression (see below).FGF8 expression in this ectodermal area is known to beimportant in organising its normal derivatives (Shimamura andRubenstein, 1997; see also Martinez-Barbera et al., 2000). Inthis model, although there is a quite clear endodermal epithelialstructure across the early head process midline, its removalthere would be irrelevant to our results. Only in presumptivelateral-dorsal regions of neural plate would it be providingdistinctive and direct information. We have been unable tomake sufficiently controlled subregional removals to test this.

Of relevance to an ultimate discrimination between thesetwo models, is the observation that the chick Hex expressionis very circumscribed (Fig. 6G), underlying prechordal plate inmidline endoderm and perhaps later invading it by an inductiveprocess or by cell transfer. Our endoderm removals clearlyablate this Hexexpression, and the dorsal anterior patterningdeficits we see closely resemble the phenotype in mousefollowing ablation of this gene’s head process endodermalexpression (Martinez-Barbera et al., 2000). Hex is thus likelyto be in the pathway that supports the inter-layer signals, fromendoderm to overlying structures, revealed by our experiments.

The developmental signals revealed in the presentexperiments would appear to be separate from those controllingCNS cross-sectional pattern in relation to the midline. We stressthat our incidental morphological observations, that in theanterior region at least, the early signs of CNS midlinepatterning and growth appear immune to the removal of axialmesoderm, should be expanded in a separate study by use ofsuitable gene markers. Following our mesoderm extirpations,the rostral diencephalic midline or pre-infundibular region mayalready have been exposed to significant extrinsic midline Shhand chordinsignals (see also Gunhaga et al., 2000). Moreover,intrinsic neural midline expression of these genes parallels oreven precedes their mesodermal expressions in this region, andremains normal for hours following the mesoderm extirpation(S. Withington and J. Cooke, unpublished observations; see alsoLe Douarin and Halpern, 2000). However Pera and Kessel(1997), following presumed removals of both mesoderm andendoderm layers in prechordal region, observed synopthalmiaand loss of ventral neural gene expressions, and suggested thatmidline patterning remains immediately dependent upon axialmesoderm signals at these stages and positions, just as for later-developing more posterior ones (Placzek et al., 2000). If this isthe case, then despite their similar appearance, our forebrainpattern losses following stage 6 mesoderm removals may notreally be the equivalents of those following earlier loss ofendoderm only. This would make harder the elucidation of anyrole for mesoderm in transfer of endodermal information forforebrain pattern (model 1 above), that is separate from its ownmidline patterning role.

Positional specificity within the endodermThe expressions of several genes such as Shh, chordinand



anteriorly, BMP7 are left intact in axial mesoderm for manyhours following definitive endoderm removals, despite theabnormally exposed development. This suggests that anteriorendoderm provides distinctive signals or conditions, and notjust a more general, protective or maintenance environment forhead process mesoderm. Positionally distinctive geneexpressions in the definitive endoderm, such as Hex andCrescent, reinforce this view. We have also shown positionalspecificity within the endoderm; endoderm from just anteriorto the prechordal mesoderm can induce ectopic GANFexpression in presumptive hindbrain territory, while moreperipheral and lateral endoderm cannot. The majority ofoperations removed the entire lower layer sector to the anteriorlimit of the area pellucida, but the similar effects of some morerestricted removals from around and just ahead of prechordalmesoderm (see Fig. 1) again suggest that the patterning signalswe are studying lie there rather than more peripherally. It hasbeen difficult to find suitable other lower layer regions, fromsynchronous donor embryos, and to graft these successfully inplace of anterior definitive endoderm in order to demonstratethe spatial specificity of its role. Most peripheral regions,known to have strong expression of ventralising BMP genesfor instance, may superimpose their own actively distortingsignals rather than revealing the effect of a ‘neutral’ epithelialcovering for head process mesoderm. However, we have failedto observe any capacity of lower layer from ahead of the nodeat stage 4, or of stage 6 perinodal/posterior head processendoderm, to substitute for anterior foregut endoderm itself inrescuing either forebrain pattern or heart migration.

Crescent expression in foregut endodermCrescent, a chick homologue of the Frizbeegroup of genes, isstrongly and distinctively expressed in the removed area ofanterior foregut endoderm (Pfeffer et al., 1997). Frizbeeproteins are candidates for secreted signals that may act bymodulating or antagonising Wnt signal pathways, throughcompetitive sequestration of the ligands. Anteriorly expressedgenes of this group, which might locally antagonise Wntsignals in establishing the conditions for head determination,have been found in Xenopusas well as mouse, and have theappropriate properties on experimental ectopic expression inXenopus. It is suggested that antagonism of a Wnt role, inspatial conjunction with antagonism of certain BMP andperhaps of Nodal signalling roles, provides the conditions fordetermination of anteriormost territories, and additional genesencoding proteins that exhibit more than one of theseantagonist functions have indeed been characterised fromvarious vertebrates (Leyns et al., 1997; Wang et al., 1997;Shawlot et al., 1998; Glinka et al., 1998; Piccolo et al., 1999;Pearce et al., 1999; Hoang et al., 1998). Genetic analysis is atan early stage (e.g. Simpson et al., 1999; Belo et al., 2000) butsuch genes of Frizbeeand other families are quite numerous,and co-expressed, so may act redundantly in supporting thisnormal developmental step. After lower layer removals sotimed as to result in later pattern truncation, the domain ofCrescent expression is not restored, even though earlierremovals followed by re-emigration of lower layer do restoreit. Thus, while we do not claim that Crescentitself provides aunique endoderm-derived anteriorising signal that ourembryological results reveal, we suggest that Crescent proteinand others functionally parallel with it are candidates for such

signals. A little-noted but relevant observation is that followingearly lithium treatment in Xenopusembryos, now thought todistort axial body pattern by global and sustained over-activation of the canonical Wnt pathway, a small but distinctiveanteriormost sector of neural pattern is deleted as well as thelarger and more widely recognised trunk-tail sector (Cooke andSmith, 1988).

Induction of stomodeal ectoderm by foregutendodermIt is of interest that a distinct region, lying at the conventionallydefined anterior limit of neural induction, becomespredetermined as stomodeum. Acquisition of this stomodealcharacter is revealed by the relationships of GANF, Shhand FGF8 expressions (Fig. 5), and by the capacity of thisepithelium actively to seek interaction with its normallyassociated structures at the base of the brain. In the absence offace formation this produces a deep tube or ‘pipe’, extendingback from beneath the anterior ectoderm to the ventrallyexposed hypophyseal region (Fig. 3H,M). This stomodealstructure is specified if anterior endoderm has been in placelong enough to support normal patterning of the forebrainitself, i.e. after lower layer removals delayed until stage 6.However, it is also produced after stage 6 axial mesoderm-plus-endoderm removals, which results in forebrains lackingtelencephalic structure. This suggests that, rather than byplanar ectodermal signalling, which depends on prior completeforebrain specification, stomodeum is specified by directsignals from an anteriormost position in definitive endoderm,where this layer directly contacts ectoderm.

We thank the following for gene probes; Anthony Graham forchordin, Ivor Mason and Anthony Graham for FGF8, Henk Roelinkfor Shh, Paul Thomas for cHex, Juan-Carlos Izpisua-Belmonte forCrescent, Philippa Francis-West for BMP7, Angela Nieto for BF1,Michael Kessel for GANF, Massimo Gulisano for Otx2, FabiennePituello for Pax6. We thank Juan Pedro Martinez-Barbera forinstructive discussion and access to unpublished work, and Photo-Graphics section, NIMR, for invaluable help with figures.

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foregut endoderm requirement in neural patterning - development

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