2477Development 124, 2477-2488 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV3669

Mechanisms of dorsal-ventral patterning in noggin-induced neural tissue

Anne K. Knecht and Richard M. Harland*

401 Barker Hall, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3204, USA

*Author for correspondence

We have investigated mechanisms of dorsal-ventral pat-terning of neural tissue, using Xenopus ectoderm neural-ized by noggin protein. This tissue appears to be patterneddorsoventrally; cpl-1, a gene expressed in the dorsal brain,and etr-1, a gene largely excluded from the dorsal brain,are expressed in separate territories in noggin-treatedexplants (Knecht, A. K., Good, P. J., Dawid, I. B. andHarland, R. M. (1995) Development 121, 1927-1936). Herewe show further evidence that this pattern represents apartial dorsal-ventral organization. Additionally, we testtwo mechanisms that could account for this pattern: a dose-dependent response to a gradient of noggin protein withinthe explant, and regulative cell-cell interactions. We showthat noggin exhibits concentration-dependent effects,

inducing cpl-1 at low doses but repressing it at high doses.Since noggin acts by antagonizing Bone MorphogeneticProtein (BMP) signaling, this result suggests that BMPsalso may act in a dose-dependent manner in vivo. However,in the absence of a noggin gradient, regulative cell-cellinteractions can also pattern the tissue. Such regulation isfacilitated by increased motility of noggin-treated cells.Finally, the response of cells to both of these patterningmechanisms is ultimately controlled by a third process, thechanging competence of the responding tissue.

Key words: noggin, dorsal/ventral patterning, neural tissue,forebrain, morphogen, pattern regulation, competence, Xenopus

SUMMARY

INTRODUCTION

Over the last decade, significant advances have been made inidentifying tissue interactions and molecules that pattern theneural tube dorsoventrally. Ventrally, the neural tube ispatterned by signals from the notochord and floorplate (vonStraaten et al., 1988; Smith and Schoenwolf, 1989; Yamada etal., 1991), with sonic hedgehog being a strong candidate forthe molecule mediating these effects (Echelard et al., 1993;Krauss et al., 1993; Roelink et al., 1995; Chiang et al., 1996).Induction of dorsal fates, particularly neural crest, involves aninteraction between neural tissue and prospective epidermis(Moury and Jacobson, 1989; Dickinson et al., 1995; Selleckand Bronner-Fraser, 1995). Liem et al. (1995) suggested amechanism for this induction: they found that Bone Morpho-genetic Proteins (BMPs) 4 and 7 are expressed in the epidermisand are capable of dorsalizing neural tissue. How BMPsmediate this patterning is unknown. However, a BMP homologin Drosophila, decapentaplegic (dpp), has been shown to actas a morphogen, inducing different fates at different concen-trations (Ferguson and Anderson, 1992, Nellen et al., 1996).Thus vertebrate BMPs may also act as morphogens, withhighest concentrations inducing epidermis (Wilson andHemmati-Brivanlou, 1995), slightly lower concentrationsinducing dorsal neural fates, and still lower concentrationsproducing more ventral neural fates.

A convenient way to study neural patterning is to use neuraltissue induced by noggin. When ectodermal explants, or‘animal caps,’ are dissected from Xenopus blastulae and treated

with noggin protein, the tissue is directly converted intoanterior neural tissue (Lamb et al., 1993). Since these explantscontain little epidermis and no mesoderm, and thereby lackthese sources of patterning signals, one would expect theexplants to be unpatterned dorsoventrally. Surprisingly, weobserved gene expression patterns that suggested dorsal-ventral organization; cpl-1, a gene expressed in the dorsalbrain, and etr-1, a neural marker largely excluded from thedorsal brain, are expressed in separate regions in noggin-treated explants (Fig. 1A,B; Knecht et al., 1995). Here, usingadditional markers, we provide further evidence that thisexpression pattern represents partial dorsal-ventral patterning.

We proposed three mechanisms that could be responsible forthis pattern. First, explants might exploit a cryptic prepatternin blastula ectoderm. There is a dorsal bias in neural com-petence (Sharpe et al., 1987; Otte et al., 1991; London et al.,1988), which may be established by signaling from dorsalmesoderm (Savage and Phillips, 1989; Otte and Moon, 1992).However, using ventralized embryos which lack dorsalmesoderm (reviewed by Gerhart et al., 1989), we found thatdorsal-ventral patterning of explants does not require prepat-terning by dorsal mesoderm (Knecht et al., 1995).

Secondly, patterning of explants may result from unequalexposure to noggin. Cells in different locations within theexplant are likely to have differential access to noggin protein.Animal caps contain several cell layers, and the outerepidermal layer is thought to be impermeable to growth factors(Cooke et al., 1987); thus noggin can only diffuse into thetissue through the inner surface. Also, noggin binds tightly to

2478 A. K. Knecht and R. M. Harland

heparin (L. Zimmerman and R. M. H., unpublished data), so itmay not diffuse well through some extracellular matrices.

Unequal exposure to noggin in explants could generatedorsal/ventral pattern in two ways. First, if noggin does notpenetrate the explant completely, some cells might remainepidermal, while others would be induced to form neuraltissue. The resulting interaction between neural tissue andepidermis would dorsalize neural tissue at the boundary, as isthought to occur normally at the boundary of the neural plate.Secondly, noggin may play a more direct role in dorsal-ventralpatterning: noggin may act as a morphogen, inducing differentfates at different locations in the explant, depending upon thelocal concentration of noggin protein. Furthermore, noggin’smorphogenetic effects could be explained through its inhibi-tion of BMP-4, which is normally present in animal caps (Daleet al., 1992), and which may also act as a morphogen, asdescribed above. Zimmerman et al. (1996) have shown bio-chemically that noggin binds to BMP-4 and prevents it frombinding to its receptor; they therefore suggest that noggin actsby antagonizing BMP signaling. Since BMP-4 can dorsalizeneural tissue, one would predict that high doses of nogginwould block the formation of dorsal neural fates.

Lastly, neural tissue may organize itself, by the classicalprocess of regulation. Although poorly understood at amolecular level, regulative cell-cell interactions are a wellknown property of neural fields (Jacobson and Sater, 1988). Innoggin-induced neural tissue, such interactions may involvethe downregulation of dorsal potential except in one region ofeach explant, in a mechanism similar to lateral inhibition(reviewed by Muskavitch, 1994).

Here we show that cells respond to noggin in a concentra-tion-dependent manner, but regulation also occurs, involving adramatic change in cell motility. Therefore we suggest thatboth mechanisms participate in the dorsal-ventral organizationof noggin-treated explants, and are likely to be important invivo. However, an additional process, changing competence,determines exactly how cells respond to these two mecha-nisms. The response of Xenopus ectoderm to different signalschanges continuously and autonomously, with neural compe-tence declining at the mid-gastrula stage (Kintner and Dodd,1991; Servetnick and Grainger, 1991). Interestingly, we havefound that neural competence increases dramatically in disso-ciated cells between stages 8 and 10. Furthermore, during thatsame period, neuralized explants lose their ability to regulatepattern. These experiments strongly suggest an important rolefor changing competence, as well as concentration-dependentresponses to noggin and BMP, and regulative cell-cell interac-tions, in dorsal-ventral patterning of neural tissue.

MATERIALS AND METHODS

Preparation of embryos and noggin-treated explantsPigmented or albino Xenopus eggs were obtained and fertilized invitro, as described previously (Condie and Harland, 1987). Embryoswere staged according to the normal table of Nieuwkoop and Faber(1967).

Animal cap ectoderm was dissected at stages 8, 9 and 10, as describedby Lamb et al. (1993). Explants were dissected in 75% NAM solution(Slack and Forman, 1980), then transferred into very low calcium andmagnesium Ringer’s solution (vLCMR; 65.5 mM NaCl, 0.925 mMKCl, 0.185 mM CaCl2, 0.095 mM MgCl2, 1.2 mM NaHCO3, 2.5 mMHepes, pH 7.2, and 50 mg/ml gentamycin; Lamb et al., 1993) to prevent

explants from healing closed prior to treatment. Explants were treatedin low calcium and magnesium Ringer’s (LCMR; Hemmati-Brivanlouet al., 1990) + 0.5% protease-free BSA (Sigma #A-3294), with orwithout 1 µg/ml purified human noggin (a gift from Regeneron Phar-maceuticals). After 30 minutes of treatment, explants were washedextensively and cultured in 75% NAM until stage 22-23.

Cell dissociation and reaggregationCells were dissociated following the methods of Green and Smith(1990), with the following variations. Throughout the procedure, weused petri dishes and pipettes coated with 50 mg/ml poly HEMA (Poly-sciences, Inc.) dissolved in 95% ethanol. After all explants weredissected as described above, vLCMR was replaced with calcium- andmagnesium-free medium (CMFM; Sargent et al., 1986). Cells weredispersed by gentle swirling and intact outer layers were discarded.Cells were divided into equal pools and transferred into CMFM + 0.5%BSA containing varying concentrations of purified noggin protein.After 30 minutes, cells were washed in 75% NAM, swirled together,and allowed to reaggregate. For in situ hybridizations, the initial reag-gregated cell mass was subsequently split into ten smaller reaggregates.

For noggin and BMP experiments, cells were dissociated andtreated with varying concentrations of purified noggin protein, asabove. After 15 minutes of treatment, cells were washed extensivelywith CMFM, and half of the cells in each treatment were transferredinto CMFM + 0.5% BSA containing 1 ng/ml purified human BMP-2(a gift from Genetics Institute). After 15 minutes, all samples werewashed extensively in 75% NAM and allowed to reaggregate.

Continuously dissociated cells were washed with CMFM afternoggin treatment. One-third of the cells were transferred into 75%NAM, where they were washed and reaggregated as described above.The remaining two-thirds were frequently dispersed and washed inCMFM. Just prior to harvesting, dissociated cells were pelleted bycentrifugation for 1 minute at 110 g.

Five-explant recombinantsExplants were dissected as described above. To hold explants openuntil recombination, explants were treated in vLCMR + 0.5% BSA,with or without 1 µg/ml noggin protein. Recombinants were made byplacing five explants together in a small well in an agarose-coatedPetri dish. For aging experiments, explants were cultured in vLCMRuntil sibling embryos reached the appropriate stage.

β-galactosidase lineage tracing followed the method of Smith andHarland (1991). One-cell stage embryos were microinjected in theanimal pole with 0.5 ng of synthetic mRNA encoding nuclear-localized β-galactosidase. Animal caps were dissected from injectedor uninjected embryos, treated as described above, and combined, oneinjected cap with four uninjected caps. β-galactosidase activity wasvisualized by magenta-gal (Biosynth AG) staining.

Fixation and whole-mount in situ hybridizationAll samples were fixed for 1 hour and then processed according to themethod of Harland (1991), using modifications and the double in situhybridization protocol described by Knecht et al. (1995).Magenta/light blue double stains were performed using magenta-phos(Biosynth AG) as the first staining substrate, and BCIP as the second.Dark blue/brown double stains used Boehringer Mannheim purpleAP-substrate first, then alkaline phosphatase substrate kit II (VectorLaboratories). Samples were photographed in methanol (Fig. 2) orcleared in BBBA (2:1 benzyl benzoate/benzyl alcohol) (Figs 7-10).

Reverse transcription-PCR analysisRNA was harvested as described (Condie and Harland, 1987). RT-PCR was carried out following the protocol of Wilson and Melton(1994). Primer sets were described for NCAM by Hemmati-Brivanlouand Melton (1994); for cardiac actin, EF-1α, and epidermal keratinby Wilson and Melton (1994); and for XAG-1 by Blitz and Cho(1995). We designed primers for cpl-1: upstream, GTCTTAG-

2479Neural D/V patterning by noggin

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situ hybridizations showing expression of dorsal and ventral forebrainryos and noggin-treated explants. (A) Triple staining of cpl-1 (blue),1 (a light blue ring marking the cement gland), in the head. Expressionin overlaps some etr-1 expression, though etr-1 is mainly expressed) Double staining of cpl-1 (blue) and etr-1 (purple) in separateted explants. cpl-1 is expressed in several protrusions (arrowheads).BF-1 (blue) and etr-1 (brown) in the head. XBF-1 expression is

orebrain. (D) Double staining of XBF-1, in blue, and etr-1, in purple, in The pattern of expression of XBF-1 is identical to that of cpl-1, in B.ion of XeNK-2 in the ventral neural tube. (F) XeNK-2 is not expressedts. Scale bars in A, B, and C represent 0.5 mm. D-F are the same

GCAAGTGGTAC (211-218); downstream, ATCATCAGCGAGTC-CTTG (601-618). (Numbers correspond to sequence in the GenBankdatabase, accession number X84414.)

RESULTS

Partial dorsal-ventral pattern in noggin-treatedexplantsWe wondered whether the gene expression patterns observedin noggin-treated explants indicate dorsal-ventral patterning.Previously we showed that the neural markers cpl-1 and etr-1are expressed in separate territories in noggin-treated explants,with cpl-1 often being expressed in small pits or protrusions(arrowheads, Fig. 1B; Knecht et al., 1995). cpl-1 is normallyexpressed exclusively in the dorsal brain, while etr-1 isexpressed broadly throughout the nervous system, but excludedfrom regions of the dorsal brain (Fig. 1A; Knecht et al., 1995).Therefore we suggested that theseparate regions of expression innoggin-treated explants might representdorsal and ventral domains. Here, weexplore this interpretation using addi-tional dorsal or ventral forebrainmarkers.

To see whether expression of cpl-1, alipocalin, indeed indicates dorsal fates,we analyzed explants for expression ofXBF-1, a transcription factor expressedin the dorsal forebrain (Fig. 1C;Papalopulu and Kintner, 1996). XBF-1shows the same expression pattern ascpl-1 in noggin-treated explants: bothgenes are expressed separately frometr-1, and both frequently coincide withpits or protrusions (Fig. 1D). Thus thesestructures express two very differenttypes of genes which both mark thedorsal forebrain.

We also analyzed explants for thepresence of ventral forebrain using thegene XeNK-2, which is expressedstrictly in the ventral neural tube (Fig.1E; Saha et al., 1993). Unlike the moregeneral marker etr-1, XeNK-2 is notexpressed in noggin-treated explants(Fig. 1F). Therefore we conclude thatnoggin-treated explants contain apartial dorsal-ventral pattern: the mostventral fates, marked by XeNK-2, areabsent; however, dorsal forebrain islocalized to one region, separate fromthe remaining tissue which is presum-ably intermediate between dorsal andventral.

Overlapping expression ofnoggin and cpl-1 in the anteriorneural foldsWe subsequently investigated themechanisms responsible for restrictingthe dorsal cpl-1-expressing cells to one

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Fig. 1. Whole-mount in markers in stage 23 embetr-1 (purple) and XAG-of cpl-1 in the dorsal braventrally in the brain. (Bterritories in noggin-trea(C) Double staining of Xrestricted to the dorsal fnoggin-treated explants.(E) Side view of expressin noggin-treated explanmagnification as B.

patch. First we explored the possibility that noggin is involvedin patterning as well as neuralizing. If noggin is involved indorsal-ventral patterning of neural tissue in vivo, it should bepresent in the right places at the right times to affect thisprocess. We therefore re-examined the expression of noggin.Throughout neurulation noggin is expressed in the notochordand prechordal plate, as previously reported (Fig. 2A; Smithand Harland, 1992). However, at early neurula stages (13-17),noggin is also expressed at the anterior boundary of the neuralplate (Fig. 2A), as is cpl-1 (Fig. 2B; Knecht et al., 1995). Byperforming double in situ hybridization, we found that theseexpression patterns overlap (Fig. 2C,D). Thus noggin maydirectly induce dorsal brain fates. Furthermore, the expressionof noggin, in both the notochord and the neural/non-neuralboundary, supports the idea that noggin may play a role inneural dorsal-ventral patterning, since both regions are thoughtto provide important patterning signals.

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Fig. 3. cpl-1 is induced by low doses and repressed by high doses ofnoggin. RT-PCR analysis of dissociated cells treated with a range ofconcentrations of noggin protein, as indicated, or untreated(‘control’). For comparison, non-dissociated ‘intact caps’ weretreated in parallel, with medium alone (‘cont.’), or medium + 1 µg/mlnoggin (‘nog’). All samples were harvested at stage 22-23. As apositive control, varying amounts of whole embryo control cDNAswere used in PCR reactions, as indicated; for a negative control,reverse transcriptase was omitted (‘−RT’). All samples wereanalyzed using primers specific for NCAM, cpl-1, XAG-1, epidermalkeratin, EF-1α (a loading control; Krieg et al., 1989), and cardiacactin (a muscle marker; Gurdon et al., 1985).

cpl-1 is induced by noggin in a dose-dependentmannerNoggin’s patterning of explants may be mediated by unequalexposure to noggin protein within the intact tissue, withdifferent effective concentrations of noggin inducing differentfates. We tested dose-dependent effects of noggin, using a dis-sociated cell assay (Green and Smith, 1990) to ensure equalnoggin exposure. Cells were treated with different concentra-tions of noggin protein, then washed and reaggregated.Expression of marker genes was analyzed by RT-PCR.

Dorsal fates, indicated by expression of cpl-1, are inducedby noggin in a dose-dependent manner, with weakly neuraliz-ing doses of noggin inducing cpl-1 most strongly. Neuraliza-tion was assessed by analyzing expression of the general neuralmarker NCAM (Kintner and Melton, 1987). Dissociated cellsexpress some NCAM at 1 ng/ml noggin; at 10 ng/ml nogginNCAM levels peak, and the epidermal marker epidermalkeratin (Jonas et al., 1989) is completely turned off, as cellsare converted from prospective epidermis into neural tissue(Fig. 3). cpl-1 is most strongly induced at this epidermal/neuralthreshold (1-10 ng/ml noggin); its expression declines withhigher noggin concentrations (Fig. 3; see also Fig. 7). Sincecells treated with high noggin doses are still neural, as shownby NCAM expression, declining expression of the dorsalmarker cpl-1 suggests that cells are being converted into a more

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Fig. 2. Dorsoanterior views of the overlapping expression of nogginand cpl-1 in stage 13 embryos. All embryos were processed togetherfor double in situ hybridization; however, the brown-stainingsubstrate was omitted from the embryos in A, and the blue substratewas omitted from the embryos in B. (A) noggin expression (blue), atthe anterior edge of the neural plate, and in the prechordal plate andnotochord. (B) cpl-1 expression (brown), also in the anterior neuralfolds. (C) An embryo double stained for noggin and cpl-1, showingoverlapping expression in the anterior neural folds. (The two stainstogether appear bluish-brown; compare with the blue noggin-expressing notochord). (D) Higher magnification view of C. Scalebars in A and D represent 0.5 mm. B and C are the samemagnification as A.

ventral type of neural tissue. Noggin also has a dose-dependenteffect on the induction of cement gland (Fig. 3), an ectodermalstructure marked by XAG-1 (Sive et al., 1989). XAG-1 isinduced by dissociation alone, but is strongly upregulated bythe lowest neuralizing dose of noggin, 1 ng/ml. At highernoggin concentrations, XAG-1 is turned off, because cells arecompletely neuralized. Thus noggin exhibits concentration-dependent effects on induction of both dorsal forebrain andcement gland.

Neural competence and cpl-1 induction vary withstageIn dissociated cells, the concentration of noggin that inducescpl-1 most strongly varies considerably, depending upon whenanimal caps were dissected. This variation is due to a dramaticincrease in neural competence between stages 8 (early blastula)and 10 (early gastrula). Cells from explants dissected and dis-sociated at stage 8 are induced to express NCAM by only a highdose of noggin (1 µg/ml; Fig. 4). However, stage 9 cells expressNCAM weakly when treated with only 1 ng/ml noggin, andstage 10 cells are neuralized by dissociation alone (Fig. 4). Thislast result is consistent with previous findings that dissociationcan neuralize animal cap cells (Godsave and Slack, 1989;Grunz and Tacke, 1989; Wilson and Hemmati-Brivanlou,1995), though this effect is quite weak in our experiments (seecontrol lane, Fig. 3). Dissociation does seem to sensitize cellsto noggin, since dissociated cells are neuralized by much lowerdoses of noggin (1 ng/ml noggin; Fig. 3) than is required forintact explants (1 µg/ml; Lamb et al., 1993).

Since cpl-1 is induced at the epidermal/neural threshold, itsinduction also changes over time. At stage 8, cpl-1 is inducedweakly by 1 µg/ml noggin (Fig. 4). At stage 9 it is inducedstrongly by 1 ng/ml noggin and repressed by 1 µg/ml noggin,

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Fig. 4. cpl-1 induction shifts with increasing neural competence. RT-PCR analysis of noggin dose-response, using explants dissected anddissociated at stage 8, stage 9, or stage 10. For each stage, cells wereuntreated (‘control’), treated with 1 ng/ml noggin protein, or 1 µg/mlnoggin (N, noggin protein). All samples were cultured until stage 22-23. Controls and primers are the same as described for Fig. 3.

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se-response profiles are shifted in the presence of BMP-2. RT-PCRissociated at late stage 9/early stage 10 and treated with a range ofnoggin protein, as indicated. Cells were then divided into two pools;eated, and the other was additionally treated with 1 ng/ml purifiedles were harvested at stage 22. Controls and primers are the same as 3.

as in Fig. 3. Finally, at stage 10 it is induced by the dissocia-tion, and turned off progressively by increasing noggin con-centrations. Thus cpl-1 is consistently induced by a weak neu-ralizing signal and then repressed by stronger neuralization;however, the degree of neuralization depends critically on thestage of the responding tissue.

Addition of BMP antagonizes the effects of nogginZimmerman et al. (1996) showed that noggin can block BMP-4 signaling. If noggin mediates dorsal-ventral patterning in ourassays by inhibiting the dorsalizing effects of BMPs, thenapplication of BMP protein should antago-nize the effects of noggin, causing a shift inthe dose-response profile toward higherdoses of noggin. To test this idea within areasonable range of noggin concentrations,we used cells dissociated at late stage 9/earlystage 10, which possess maximal neuralcompetence (see Fig. 4). We also used a verylow dose of purified BMP-2 (1 ng/ml), sincehigher BMP doses have been shown to com-pletely block neuralization (Wilson andHemmati-Brivanlou, 1995). (AlthoughBMP-2 is not expressed during gastrulationand early neurulation (Clement et al., 1995),it is used in these studies to substitute forBMP-4, a more likely candidate for theneural dorsalizer, since BMP-2 and -4 haveidentical biological activities in earlyXenopus development (Clement et al., 1995;Hemmati-Brivanlou and Thomsen, 1995).)

In the presence of added BMP-2, higherdoses of noggin are required to elicit thesame effects observed with noggin alone. Inthe absence of BMP-2, cpl-1 is inducedmaximally by dissociation alone, 1 ng/ml

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noggin, and 10 ng/ml noggin; however, when BMP-2 is added,cpl-1 is induced maximally by 10 and 100 ng/ml noggin (Fig.5). Consistent with the idea that cpl-1 is induced by weak neu-ralization, this shift in dose-response correlates with a shift inthe epidermal/neural threshold. Without BMP-2, the epidermalmarkers (XAG-1 and epidermal keratin) are turned off, andNCAM is turned on, at 10 ng/ml noggin. However, in thepresence of BMP-2, 100 ng/ml noggin is required to suppressepidermal markers and induce NCAM. Thus BMP-2 is able toantagonize all the effects of noggin in this assay, in support ofthe idea that noggin acts by inhibition of BMP signaling.

cpl-1 is upregulated in continuously dissociatedcellsOur results suggest that noggin, via its inhibition of BMPs, canhave a direct effect on patterning. However, the results couldalso be explained by the model of neural patterning via inter-actions between neuralized and non-neuralized cells. If disso-ciated cells are heterogeneous in their neural competence, amixture of neural and epidermal cells could result at athreshold concentration of noggin. Subsequent reaggregationwould allow epidermal cells to contact and dorsalize neuralcells, thereby inducing a high level of cpl-1.

We tested the requirement for cell-cell contact by comparingreaggregated cells with cells that were continuously dissoci-ated. Long term dissociation exacerbates the weak neuralizingeffect of dissociation, such that untreated cells express NCAMweakly and express reduced levels of epidermal markersepidermal keratin and XAG-1(Fig. 6). cpl-1 is not significantlyinduced in any of the reaggregated cells, presumably becausethe critical dose of noggin was not tested. (Since cpl-1induction varies considerably with stage of dissection (Fig. 4),it was not possible to predict exactly which doses of nogginwould induce cpl-1.) Surprisingly, cpl-1 is greatly upregulatedin all continuously dissociated treatments. These results showthat cell-cell contact is not required for induction of cpl-1,

2482 A. K. Knecht and R. M. Harland

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Fig. 6. Continously dissociated cells express high levels of cpl-1. RT-PCR analysis of noggin dose-response in dissociated cells, treated asin Fig. 4 (N, noggin protein). After treatment, one-third of the cellswere reaggregated (‘reagg.’), and the rest were kept dissociated(‘dissoc.’). All samples were cultured until stage 20. Controls andprimers are the same as described for Fig. 3.

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d cpl-1 expression in stage 22 cell reaggregates. Dissociated cells were with the following noggin protein concentrations: 0.1 ng/ml (B), 1er magnification in D), 10 ng/ml (E, shown at higher magnification inµg/ml (H). Darkly stained protrusions are observed at 1 ng/ml noggin at higher noggin concentrations (E-H, see arrowheads in F and H).

epresent 0.5 mm. F is the same magnification as D; all others are the.

thereby discounting the neural/non-neural interaction hypoth-esis. Moreover, the upregulation of cpl-1 in the absence of cell-cell contact suggests that communication between cellsnormally acts to repress cpl-1 expression, perhaps by a processsimilar to lateral inhibition. We have further investigated theability of cells to self-organize in the following experiments.

Regulation of cpl-1 expression in reaggregated cellsWe hypothesized that the localized expression of cpl-1 in intactexplants occurs because of unequalexposure to noggin, with cpl-1 beingexpressed by only the cells seeing a par-ticular dose of noggin. If this mechanismis solely responsible for cpl-1 localiza-tion, then one would predict that disso-ciated cells, with equal exposure tonoggin, should express cpl-1 uniformly.We tested this prediction by performingin situ hybridizations for cpl-1 on disso-ciated cell reaggregates.

The patterns of cpl-1 expressiondemonstrate the ability of these cells toself-organize. Overall expression levelsverify our RT-PCR results (Fig. 3 forexample), showing declining cpl-1expression at higher noggin doses (Fig.7). The weak expression of cpl-1 at highnoggin doses (100 ng/ml and 1 µg/ml;Fig. 7G,H) is observed as diffusestaining throughout the explants.However, the strong expression at lowdoses (1 and 10 ng/ml; Fig. 7C-F) isextremely non-uniform, with littlestaining in some regions within theaggregates, and concentrated staining inother regions. Strikingly, at 1 ng/mlnoggin these concentrations ofexpression are often associated with pro-

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Fig. 7. Partially localizeuntreated (A), or treatedng/ml (C, shown at highF), 100 ng/ml (G), or 1 (C,D). Pits are observedScale bars in A and D rsame magnification as A

trusions (Fig. 7C,D), reminiscent of the cpl-1-expressing pro-trusions in intact explants. These protrusions are rarely seenwith higher noggin concentrations (Fig. 7E-H); instead, theseaggregates exhibit large pits (arrowhead, Fig. 7F), producing acup-like shape (arrowhead, Fig. 7H). These morphologicalchanges and the partial localization of cpl-1 both indicate thatthese cells are capable of self-organization.

We also tested the effects of changing competence on cpl-1 induction and localization (Fig. 8). The dissection ofexplants at progressively later stages has a dramatic effect onoverall levels of cpl-1, which is induced by lower doses ofnoggin as neural competence increases (as shown in Fig. 4).However, reaggregates that express cpl-1 strongly aregenerally equally capable of localizing cpl-1 expression,regardless of stage (Fig. 8E,G,H). cpl-1 expression in stage 8cells is diffuse (Fig. 8C), but that may be due to the weaknessof the signal. Protrusions were not observed in this experi-ment, but since only two concentrations of noggin weretested, we may have missed the critical dose for protrusionformation. Many neuralized aggregates do exhibit large pits(arrowhead, Fig. 8H). Thus, regardless of stage of dissection,cells are consistently able to localize cpl-1 expression andself-organize.

Regulation occurs in five-explant recombinants, butvaries with stageTo further explore this process of regulation, we devised anassay in which five animal caps were treated with noggin andthen allowed to heal together into one recombinant (Fig. 9A).If regulation occurs, recombinants should express cpl-1 in onepatch. However, if each explant retains its own patterning infor-mation, five cpl-1 patches should result.

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sion in reaggregated cells, dissociated at stage 8 (A-C), stage 9 (D-F), (A,D,G), treated with 1 ng/ml noggin protein (B,E,H), or 1 µg/mle 20. Neuralized reaggregates often appear cup-shaped (arrowhead in

We found that regulation occurs but is very stage-dependent.Explants dissected at stage 8 regulate, expressing cpl-1 in onelarge patch which is almost always associated with a pit(arrowhead, Fig. 9Ba). However, early stage 9 explants occa-sionally show additional patches of cpl-1 (Fig. 9Bb), and latestage 9 explants exhibit such extensive cpl-1 staining thatdiscrete patches cannot be identified (Fig. 9Bc). Therefore,over time, explants lose their ability to regulate the cpl-1pattern.

One explanation for this observation is that later explants,while still in the embryo at stage 9, receive additional pattern-ing information which prevents subsequent regulation. In thiscase, the stage of dissection is critical. Alternatively, the stageat which explants are combined may be important, since itestablishes when cell-cell signaling can begin. Lastly, the stageof noggin treatment may be crucial, as we have established thatcells exhibit changing competence to respond to noggin duringthis period.

To test whether time of dissection is critical, we dissectedexplants at stage 8, early stage 9, and late stage 9, but agedearlier explants in culture to late stage 9 before treatment andcombination. This aging causes early explants to lose theirability to regulate, such that all recombinants express multiplepatches of cpl-1 (Fig. 9Bd,e), as do late stage 9 explants thatwere not aged (Fig. 9Bc,f). This experiment shows that timeof dissection is unimportant, and embryonic signaling is notrequired for loss of ability to regulate, since early explants canlose this ability in vitro.

Secondly, to distinguish between time of treatment and timeof combination, wedissected explants at thesame three stages andtreated with noggin immedi-ately after dissection. Aftertreatment, however, earlierexplants were aged until latestage 9, when all recombi-nants were made. Thistreatment rescues the abilityof stage 8 explants toregulate cpl-1 expression toone patch (Fig. 9Bg). Thustiming of noggin treatmentis critical in determining theproperties of the resultingtissue, with only earlytreated tissue being able toregulate.

Early, regulatingrecombinants exhibitgreater cell movementWe wished to investigate themechanism by which thispattern regulation occurs.One possibility is that dorsallocalization occurs vialocalized specification ofdorsal fates within theneural field. A second possi-bility is that dorsal cell fates

A

D

G

st. 8

st. 9

st. 10

contr ol

Fig. 8. Partially localized cpl-1 expresor stage 10 (G-I). Cells were untreatednoggin (C,F,I), then cultured until stagH). Scale bar in A represents 0.5 mm.

are specified at multiple locations, but dorsal cells later cometogether by cell movement. To test the latter possibility, weused a lineage tracing technique diagrammed in Fig. 10A.Animal caps were dissected, at stage 8 or late stage 9, fromembryos injected with RNA encoding β-galactosidase (β-gal),and from uninjected embryos. All explants were immediatelytreated with noggin; then one β-gal-injected explant wascombined with four uninjected explants. After culturing, theposition of cells from the injected explant was determined byβ-gal staining with magenta-gal. Additionally, by performingin situ hybridizations for cpl-1 (stained blue), we could relatethe location of β-gal-stained cells to the location of the dorsalpatch.

Noggin-treated stage 8 recombinants, which regulate toexpress one patch of cpl-1, show remarkably extensive cellmovement (Fig. 10B,C). Furthermore, this movement onlyoccurs in recombinants undergoing regulation, since weobserved no evidence of cell movement in non-regulating latestage 9 recombinants (Fig. 10E), or untreated stage 8 recom-binants (Fig. 10D). The movement observed in regulatingrecombinants appears to include expansion of the entire popu-lation of labeled cells, often into large arcs (Fig. 10C), and notjust migration of a dorsally specified subpopulation. Thus itseems unlikely that cell movement can solely account for cpl-1 localization, if dorsally specified cells are initially widelydispersed. Nonetheless, these explants are clearly undergoingconsiderable reorganization, and the strong correlation withregulation suggests that cell movement may contribute to thelocalization of dorsal fates.

2484 A. K. Knecht and R. M. Harland

DISCUSSION

We have found that noggin-treated explants possess a partialdorsal-ventral organization. Furthermore, we have presentedevidence for two mechanisms that could mediate this organiz-ation: (1) unequal exposure to noggin, which can inducedorsal-ventral fates in a concentration-dependent manner; and(2) regulation by the responding tissue. However, both mech-anisms are subject to a third mechanism, changing competence,

s

treated and combinedimmediatel y

treated and combinedat late st. 9

treated immediatel y, combined at late st. 9

A

B

a

d

g

+

Fig. 9. Regulation in five-explantrecombinants, cultured until stage22-23. (A) Recombinants were madeby allowing five noggin-treatedexplants to heal together. Ifregulation occurs, one large patch ofcpl-1 expression should result;otherwise, each explant shouldexpress cpl-1 independently, suchthat five separate patches result.(B) cpl-1 expression in five-explantrecombinants, made from explantsdissected at stage 8 (a,d,g), earlystage 9 (b,e,h), or late stage 9 (c,f,i).(a-c) Explants were dissected at thestages indicated, then treated andcombined immediately. Stage 8recombinants regulate to form onepatch of cpl-1 expression (a), whichoften contains a large pit (arrowheadin a); later recombinants (b,c) exhibitmultiple, fused patches of cpl-1. (d-f)Explants were dissected at the stagesindicated, but aged until late stage 9before treatment and combination.All recombinants exhibit multiplecpl-1 patches (d-f). (g-i) Explantswere treated immediately after theirdissection, but all were aged to latestage 9 before combination. Stage 8recombinants regulate to form onecpl-1 patch (g), as in a. Scale bar in arepresents 0.5 mm.

which ultimately determines how cells respond to patterningsignals.

Partial dorsal-ventral pattern in noggin-treatedexplantsOur results show that noggin-treated explants are partiallypatterned dorsoventrally. Two genes expressed in the dorsalforebrain, cpl-1 and XBF-1, are both expressed in explants inthe same small region, which often contains a protrusion or pit

tage 8 early sta ge 9 late sta ge 9

b c

e f

h i

= regulation

no regulation

in situfor cpl-1

noggin =

2485Neural D/V patterning by noggin

(Fig. 1). The identity of these structures is unknown. However,the anterior neural folds, where cpl-1 is initially expressed (Fig.2B), give rise in part to the telencephalon (Eagleson and Harris,1990); thus one can speculate that the protrusion representstelencephalon outgrowth.

While dorsal fates are restricted to one patch in noggin-treated explants, the remaining tissue is not ventral, since itdoes not express XeNK-2. Ericson et al. (1995) have shown thata homolog of this gene, Nkx-2.1, can be induced in chickforebrain treated with sonic hedgehog. It is therefore likely thatexposure to sonic hedgehog is required for the properformation of ventral fates. Since noggin-treated explants lacksonic hedgehog, the non-dorsal tissue probably adopts an inter-mediate neural tube fate.

Concentration-dependent effects of noggin indorsal-ventral patterningWe have considered several mechanisms that might generatedorsal-ventral pattern in explants. First, we proposed that cellswithin explants may have unequal exposure to noggin, whichcould then generate pattern either directly or indirectly. In adirect model, a noggin gradient would be established in theexplant, with different concentrations of noggin inducingdifferent dorsal-ventral fates. The critical requirement for this

A

B

D

lacZRNA

Fig. 10. Cell movement in stage20 five-explant recombinants.(A) Explants were dissectedfrom embryos injected at theone cell stage with β-gal RNA.Recombinants were made bycombining one labeled explantwith four unlabeled explants.(B) Stage 8 noggin-treatedrecombinants, stained for β-galactivity, in magenta, and cpl-1expression, in blue. Allrecombinants form one patch ofcpl-1, and all patches containsome β-gal-labeled cells.Several explants show extremeelongation of the labeledpatches. (C) Highermagnification view of tworecombinants from B, showinglabeled cells extending into thecpl-1 pit. (D) In untreated stage8 recombinants, labeled cellsremain in round clumps. (E)Late stage 9 noggin-treatedrecombinants also show littledispersion of labeled cells, andcpl-1 expression is notregulated to one patch. Scalebars all represent 0.5 mm.

model is that noggin must act in a concentration-dependentmanner. Alternatively, noggin may pattern indirectly by incom-pletely neuralizing the explant; the subsequent interactionbetween neural and non-neural cells could cause localized dor-salization (Moury and Jacobson, 1989; Dickinson et al., 1995;and Selleck and Bronner-Fraser, 1995). An important aspect ofthis indirect model is that cell-cell contact is required for dorsalinduction.

Using dissociated cells, we have found that noggin doeselicit concentration-dependent effects. Low doses of noggininduce cpl-1, but higher doses repress it, presumably becausecells are adopting more ventral neural fates (Fig. 3). Similarly,the cement gland marker XAG-1 is strongly induced by verylow doses of noggin, but turned off by higher doses, as cellsare converted into neural tissue. Furthermore, these inductionsdo not require contact between neural and non-neural cells,since continuously dissociated cells are still capable of express-ing cpl-1 and XAG-1(Fig. 6). These concentration-dependenteffects support the idea that a noggin gradient could generatedorsal-ventral patterning in intact noggin-treated explants.

How does noggin mediate this patterning? Previous resultssuggested that noggin acts by antagonizing signaling by BMP-4 (Zimmerman et al., 1996). Our results confirm one predic-tion of this model: since BMP-4 is known to dorsalize neural

C

E

no cellmovement

cell movement

=

=

2486 A. K. Knecht and R. M. Harland

tissue (Liem et al., 1995), high doses of noggin should blockthe most dorsal fates; likewise, we have found that high dosesof noggin repress cpl-1 (Fig. 3). Furthermore, we have verifiedthat BMP-2 can antagonize noggin in our assays, since higherdoses of noggin are required to induce cpl-1 (and neural tissuein general) in the presence of BMP-2 protein (Fig. 5). Theseresults, combined with previous biochemical data showing adirect interaction between noggin and BMP-4 (Zimmerman etal., 1996), make a strong case for the model of noggin actionvia BMP-4 inhibition.

The observed concentration-dependent effects of nogginthen lead to the important conclusion that BMP-4 may also actmorphogenetically. According to this model, high concentra-tions of BMP-4 induce epidermis, as shown by Wilson andHemmati-Brivanlou (1995). Slightly lower concentrations (orhigh concentrations combined with low concentrations of aBMP antagonist like noggin) induce cement gland (see XAG-1 induction in Fig. 3 for example). Still lower concentrationsof BMP-4, or higher concentrations of an antagonist, inducedorsal neural tissue (see cpl-1 induction in Fig. 3, e.g.). Finally,the lowest BMP-4 concentrations, or highest concentrations ofan antagonist, give rise to more ventral types of neural tissue.

Although we cannot say when these patterning events areoccurring in vivo, one possible period is the late gastrula/earlyneurula stages. During these stages BMP-4 is expressed in thenon-neural ectoderm and excluded from the neural plate(Fainsod et al., 1994; Hemmati-Brivanlou and Thomsen, 1995;Schmidt et al., 1995), so it is not difficult to imagine that agradient may exist across this boundary. This gradient may bemaintained or modified by the weak expression of noggin atthe anterior edge of the neural plate. Noggin’s dorsalexpression seems at first to be contradictory with its activity asa neural ventralizer at high doses (though this activity is con-sistent with its expression in the notochord). However, ourresults show that a low dose of noggin actually induces cpl-1,presumably by reducing activity of the morphogen BMP-4 tothe appropriate level for neural dorsalization. The juxtapositionof BMP expression to noggin at the neural plate boundary mayhelp provide a steep gradient of BMP activity and a resultingsharp focus of tissue fates.

The role of regulation in dorsal-ventral patterningWe found that cpl-1 is strongly upregulated in continuouslydissociated cells, indicating that all cells have adopted a dorsalfate (Fig. 6). This result is particularly surprising since onemight predict that prolonged dissociation would more effec-tively remove BMPs from the surface of cells, causing them tobe ventralized relative to cells which are only briefly dissoci-ated. The extent of this effect would depend upon severalparameters, including the substantial affinity of BMPs for thecell surface (Koenig et al., 1994). However, our observation ofthe opposite result, that continuously dissociated cells arestrongly dorsalized relative to reaggregated cells, indicates thatreaggregation itself causes downregulation of cpl-1. Wesuggest that this repression of dorsal fates is due to cell-cellcommunication, which may act to restrict dorsal fates to onesmall region by a regulative process analogous to lateral inhi-bition.

Furthermore, although noggin gradients probably contributeto patterning of intact noggin-treated explants, unequalexposure is not strictly required for localization of dorsal fates.

In reaggregates of dissociated cells, with equal exposure tonoggin, cpl-1 is highly expressed in some regions and onlyweakly expressed in others (Figs 7 and 8). Also, these reag-gregates form pits and protrusions, structures which mustrequire the coordinated movements of many cells. We thereforeconclude that cells possess a regulative capacity allowing themto organize themselves dorsoventrally. Not only can this regu-lation occur in the absence of additional patterning informa-tion, as in dissociated cells (Figs 7 and 8), but it can alsooverride previous patterning information, as in five-explantrecombinants (Figs 9 and 10). In vivo, this mechanism mayplay an important role in refining and maintaining the dorsal-ventral axis established by growth factors.

Although regulation is a widespread phenomenon in devel-opment, its molecular mechanisms are largely unknown. Wesuggested two ways that dorsal fates could be localized: bylocalized specification of dorsal fates, and by movement ofdorsally specified cells. Interestingly, we did observe consid-erable cell movement in five-explant recombinants thatregulate, and no cell movement in non-regulating recombinants(Fig. 10). This observation represents a novel discovery aboutthe behavior of neuralized cells, and the strong correlation withregulation suggests that cell movement may play an importantrole in regulation, perhaps also mediating the cell reorganiza-tion that gives rise to pits and protrustions. Such movementsare likely to refine the organization of the neural tube in vivo.However, cell movements alone cannot explain the partiallocalization of cpl-1 in reaggregates of dissociated cells (Figs7 and 8), since in the absence of a noggin gradient, all cellsshould express equal levels of cpl-1. Regulative cell-cell inter-actions probably mediate the repression of cpl-1 in someregions of the reaggregates, as is also suggested by the repres-sion of cpl-1 in reaggregated versus continuously dissociatedcells (Fig. 6). Thus cell-cell signaling contributes to dorsalspecification, with cell movement refining this process byallowing dorsally specified cells in a general area to converge.

The central role of competenceThe ectoderm loses its response to neural inducers during lategastrulation (Kintner and Dodd, 1991; Servetnick andGrainger, 1991). Here we have shown that the competence ofcells to be neuralized increases dramatically at the onset of gas-trulation (Figs 4 and 8). Thus the effect of different doses ofnoggin can be mimicked by application of the same doses atdifferent times. In addition, we have found that early applica-tion of noggin to animal caps increases the ability of explantsto regulate localized expression of cpl-1(Fig. 9). Early appli-cation of noggin also increases the motility of cells, contribut-ing to refinement of localized cpl-1 expression (Fig. 10).Therefore the age of the responding cells has effects on cellfate, secondary cell-cell signaling and cell movement.

Although we do not understand the basis for the changingresponse of cells to noggin over time, these findings highlightthe important contribution of changes in competence to cellfate. Morphogens induce different cell fates at different con-centrations; however, a substance may mimic a morphogen byacting at the cell surface at different times. Consequently,future models of dorsal-ventral patterning of neural tissue musttake into account not just signals like BMP-4 and noggin, andregulative cell-cell interactions, but also the timing with whichthese signals act.

2487Neural D/V patterning by noggin

The authors thank Nancy Papalopulu for providing XBF-1 prior topublication; Regeneron Pharmaceuticals for the gift of purified nogginprotein; Genetics Institute for the gift of purified BMP-2 protein; andDavid Hsu for critical reading of the manuscript. This work wassupported by NIH grants to R. M. H. and a Howard Hughes MedicalInstitute predoctoral fellowship to A. K. K.

REFERENCES

Blitz, I. L. and Cho, K. W. Y. (1995). Anterior neurectoderm is progressivelyinduced during gastrulation: the role of the Xenopus homeobox geneorthodenticle. Development 121, 993-1004.

Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal,H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in micelacking Sonic hedgehog gene function. Nature 383, 407-413.

Clement, J. H., Fettes, P., Knöchel, S., Lef, J. and Knöchel, W. (1995). Bonemorphogenetic protein 2 in the early development of Xenopus laevis. Mech.Dev. 52, 357-370.

Condie, B. G. and Harland, R. M. (1987). Posterior expression of a homeoboxgene in early Xenopus embryos. Development 101, 93-105.

Cooke, J., Smith, J. C., Smith, E. J. and Yaqoob, M. (1987). The organizationof mesodermal pattern in Xenopus laevis: experiments using a Xenopusmesoderm-inducing factor. Development 101, 893-908.

Dale, L., Howes, G., Price, B. M. J. and Smith, J. C. (1992). Bonemorphogenetic protein 4: a ventralizing factor in early Xenopusdevelopment. Development 115, 573-585.

Dickinson, M., Selleck, M., McMahon, A. and Bronner-Fraser, M. (1995).Dorsalization of the neural tube by the non-neural ectoderm. Development121, 2099-2106.

Eagleson, G. W. and Harris, W. A. (1990). Mapping of the presumptive brainregions in the neural plate of Xenopus laevis. J. Neurobiol. 21, 427-440.

Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon,J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family ofputative signaling molecules, is implicated in the regulation of CNS polarity.Cell 75, 1417-1430.

Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T.(1995). Sonic hedgehog induces the differention of ventral forebrainneurons: a common signal for ventral patterning with the neural tube. Cell 81,747-756.

Fainsod, A., Steinbeisser, H. and De Robertis, E. M. (1994). On the functionof BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J.13, 5015-5025.

Ferguson, E. L. and Anderson, K. V. (1992). decapentaplegic acts as amorphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell71, 451-461.

Gerhart, J. C., Danilchik, M., Doniach, T., Roberts, S., Rowning, B. andStewart, R. (1989). Cortical rotation of the Xenopus egg: consequences forthe anteroposterior pattern of embryonic dorsal development. Development107 Supplement, 37-51.

Godsave, S. F. and Slack, J. M. W. (1989). Clonal analysis of mesoderminduction in Xenopus laevis. Dev. Biol. 134, 486-490.

Green, J. B. A. and Smith, J. C. (1990). Graded changes in dose of a Xenopusactivin A homologue elicit stepwise transitions in embryonic cell fate. Nature347, 391-394.

Grunz, H. and Tacke, L. (1989). Neural differentiation of Xenopus laevisectoderm takes place after disaggregation and delayed reaggregation withoutinducer. Cell Diff. Dev. 28, 211-217.

Gurdon, J. B., Fairman, S., Mohun, T. J. and Brennan, S. (1985). Activationof muscle-specific actin genes in Xenopus development by an inducerbetween animal and vegetal cells of a blastula. Cell 41, 913-922.

Harland, R. M. (1991). In situ hybridization: an improved whole mountmethod for Xenopus embryos. In Methods in Cell Biology Vol. 36 (ed. B. K.Kay and H. B. Peng), pp. 685-695. Academic Press, San Diego.

Hemmati-Brivanlou, A., Stewart, R. M. and Harland, R. M. (1990).Region-specific neural induction of an engrailed protein by anteriornotochord in Xenopus. Science 250, 800-802.

Hemmati-Brivanlou, A. and Melton, D. A. (1994). Inhibition of activinreceptor signalling promotes neuralization in Xenopus. Cell 77, 273-281.

Hemmati-Brivanlou, A. and Thomsen, G. H. (1995). Ventral mesodermalpatterning in Xenopus embryos: expression patterns and activities of BMP-2and BMP-4. Dev. Genet. 17, 78-89.

Jacobson, A. G. and Sater, A. K. (1988). Features of embryonic induction.Development 104, 341-359.

Jonas, E. A., Snape, A. M. and Sargent, T. D. (1989). Transcriptionalregulation of a Xenopus embryonic epidermal keratin gene. Development106, 399-405.

Kintner, C. R. and Melton, D. A. (1987). Expression of Xenopus N-CAMRNA in ectoderm is an early response to neural induction. Development 99,311-325.

Kintner, C. R. and Dodd, J. (1991). Hensen’s node induces neural tissue inXenopus ectoderm. Implications for the action of the organizer in neuralinduction. Development 113, 1495-1505.

Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M. (1995). Dorsal-ventral patterning and differentiation of noggin-induced neural tissue in theabsence of mesoderm. Development 121, 1927-1936.

Koenig, B. B., Cook, J. S., Wolsing, D. H., Ting, J., Tiesman, J. P., Correa, P.E., Olson, C. A., Pecquet, A. L., Ventura, F., Grant, R. A., Chen, G.-X.,Wrana, J. L., Massague, J. and Rosenbaum, J. S. (1994). Characterizationand cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells. Mol.Cell. Biol. 14, 5961-5974.

Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionallyconserved homolog of the Drosophila segment polarity gene hh is expressedin tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.

Krieg, P. A., Varnum, S. M., Wormington, W. M. and Melton, D. A. (1989).The mRNA encoding elongation factor 1-alpha (EF-1alpha) is a majortranscript at the midblastula transition in Xenopus. Dev. Biol.133, 93-100.

Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. N.,Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993). Neuralinduction by the secreted polypeptide noggin. Science 262, 713-718.

Liem, K. F. J., Tremml, G., Roelink, H. and Jessell, T. M. (1995). Dorsaldifferentiation of neural plate cells induced by BMP-mediated signals fromepidermal ectoderm. Cell 82, 969-979.

London, C., Akers, R. and Phillips, C. (1988). Expression of Epi 1, anepidermis-specific marker in Xenopus laevis embryos, is specified prior togastrulation. Dev. Biol. 129, 380-389.

Moury, J. D. and Jacobson, A. G. (1989). Neural fold formation at newlycreated boundaries between neural plate and epidermis in the axolotl. Dev.Biol.133, 44-57.

Muskavitch, M. A. (1994). Delta-notch signaling and Drosophila cell fatechoice. Dev. Biol.166, 415-430.

Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-rangeaction of a DPP morphogen gradient. Cell 85, 357-368.

Nieuwkoop, P. D. and Faber, J. (1967). Normal table of Xenopus laevis(Daudin). North-Holland Publishing Company, Amsterdam.

Otte, A. P., Kraner, I. M. and Durston, A. J. (1991). Protein kinase C andregulation of the local competence of Xenopus ectoderm. Science 251, 570-573.

Otte, A. P. and Moon, R. T. (1992). Ectopic induction of dorsal mesoderm byoverexpression of Xwnt-8 elevates the neural competence of Xenopusectoderm. Dev. Biol. 152, 184-187.

Papalopulu, N. and Kintner, C. (1996). A posteriorising factor, retinoic acid,reveals that anteroposterior patterning controls the timing of neuronaldifferentiation in Xenopus neuroectoderm. Development 122, 3409-3418.

Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P.A. and Jessell, T. M. (1995). Floor plate and motor neuron induction bydifferent concentrations of the amino-terminal cleavage product of sonichedgehog autoproteolysis. Cell 81, 445-455.

Saha, M. S., Michel, R. B., Gulding, K. M. and Grainger, R. M. (1993). AXenopus homeobox gene defines dorsal-ventral domains in the developingbrain. Development 118, 193-202.

Sargent, T. D., Jamrich, M. and Dawid, I. B. (1986). Cell interactions and thecontrol of gene activity during early development in Xenopus laevis. Dev.Biol. 114, 238-246.

Savage, R. and Phillips, C. R. (1989). Signals from the dorsal blastopore lipregion during gastrulation bias the ectoderm toward a nonepidermal pathwayof differentiation in Xenopus laevis. Dev. Biol. 133, 157-168.

Schmidt, J. E., Suzuki, A., Ueno, N. and Kimelman, D. (1995). LocalizedBMP-4 mediates dorsal/ventral patterning in the early Xenopus embryo. Dev.Biol. 169, 37-50.

Selleck, M. A. J. and Bronner-Fraser, M. (1995). Origins of the avian neuralcrest: the role of neural plate/epidermal interactions. Development 121, 525-538.

Servetnick, M. and Grainger, R. M. (1991). Changes in neural and lenscompetence in Xenopus ectoderm: evidence for an autonomousdevelopmental timer. Development 112, 177-188.

2488 A. K. Knecht and R. M. Harland

Sharpe, C. R., Fritz, A., De Robertis, E. M. and Gurdon, J. B. (1987). Ahomeobox-containing marker of posterior neural differentiation shows theimportance of predetermination in neural induction. Cell 50, 749-758.

Sive, H. L., Hattori, K. and Weintraub, H. (1989). Progressive determinationduring formation of the anteroposterior axis in Xenopus laevis. Cell 58, 171-180.

Slack, J. M. W. and Forman, D. (1980). An interaction between dorsal andventral regions of the marginal zone in early amphibian embryos. J. Embryol.Exp. Morph. 56, 283-299.

Smith, J. and Schoenwolf, G. C. (1989). Notochordal induction of cellwedging in the chick neural plate and its role in neural tube formation. J. Exp.Zool. 25, 49-62.

Smith, W. C. and Harland, R. M. (1991). Injected Xwnt-8 RNA acts early inXenopus embryos to promote formation of a vegetal dorsalizing center. Cell67, 753-765.

Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a newdorsalizing factor localized to the Spemann organizer in Xenopus embryos.Cell 70, 829-840.

von Straaten, H. W. M., Hekking, E. J. L. M., Wiertz-Hoessels, F. T. andDrukker, J. (1988). Effects of the notochord on the differentiation of a floorplate area in the neural tube of the chick embryo. Anat. Embryol. 177, 317-324.

Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis andinhibition of neural fate by Bmp-4. Nature 376, 331-333.

Wilson, P. A. and Melton, D. A. (1994). Mesodermal patterning by an inducergradient depends on secondary cell-cell communication. Current Biol. 4,676-686.

Yamada, T., Placzek, M., Tanaka, H., Dodd, J. and Jessell, T. M. (1991).Control of cell pattern in the developing nervous system: polarizing activityof the floor plate and notochord. Cell 64, 635-647.

Zimmerman, L. B., DeJesús-Escobar, J. M. and Harland, R. M. (1996). TheSpemann organizer signal noggin binds and inactivates Bone MorphogeneticProtein 4. Cell 86, 599-606.

(Accepted 7 April 1997)