Mechanisms of dorsal-ventral patterning in noggin

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We have investigated mechanisms of dorsal-ventral pat- terning of neural tissue, using Xenopus ectoderm neural- ized by noggin protein. This tissue appears to ...
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Development 124, 2477-2488 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV3669

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

SUMMARY We have investigated mechanisms of dorsal-ventral patterning of neural tissue, using Xenopus ectoderm neuralized by noggin protein. This tissue appears to be patterned dorsoventrally; 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-treated explants (Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M. (1995) Development 121, 1927-1936). Here we show further evidence that this pattern represents a partial dorsal-ventral organization. Additionally, we test two mechanisms that could account for this pattern: a dosedependent response to a gradient of noggin protein within the explant, and regulative cell-cell interactions. We show that noggin exhibits concentration-dependent effects,

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

INTRODUCTION

with noggin protein, the tissue is directly converted into anterior neural tissue (Lamb et al., 1993). Since these explants contain little epidermis and no mesoderm, and thereby lack these sources of patterning signals, one would expect the explants to be unpatterned dorsoventrally. Surprisingly, we observed gene expression patterns that suggested dorsalventral organization; cpl-1, a gene expressed in the dorsal brain, and etr-1, a neural marker largely excluded from the dorsal brain, are expressed in separate regions in noggintreated explants (Fig. 1A,B; Knecht et al., 1995). Here, using additional markers, we provide further evidence that this expression pattern represents partial dorsal-ventral patterning. We proposed three mechanisms that could be responsible for this pattern. First, explants might exploit a cryptic prepattern in blastula ectoderm. There is a dorsal bias in neural competence (Sharpe et al., 1987; Otte et al., 1991; London et al., 1988), which may be established by signaling from dorsal mesoderm (Savage and Phillips, 1989; Otte and Moon, 1992). However, using ventralized embryos which lack dorsal mesoderm (reviewed by Gerhart et al., 1989), we found that dorsal-ventral patterning of explants does not require prepatterning by dorsal mesoderm (Knecht et al., 1995). Secondly, patterning of explants may result from unequal exposure to noggin. Cells in different locations within the explant are likely to have differential access to noggin protein. Animal caps contain several cell layers, and the outer epidermal layer is thought to be impermeable to growth factors (Cooke et al., 1987); thus noggin can only diffuse into the tissue through the inner surface. Also, noggin binds tightly to

Over the last decade, significant advances have been made in identifying tissue interactions and molecules that pattern the neural tube dorsoventrally. Ventrally, the neural tube is patterned by signals from the notochord and floorplate (von Straaten et al., 1988; Smith and Schoenwolf, 1989; Yamada et al., 1991), with sonic hedgehog being a strong candidate for the 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 an interaction between neural tissue and prospective epidermis (Moury and Jacobson, 1989; Dickinson et al., 1995; Selleck and Bronner-Fraser, 1995). Liem et al. (1995) suggested a mechanism for this induction: they found that Bone Morphogenetic Proteins (BMPs) 4 and 7 are expressed in the epidermis and are capable of dorsalizing neural tissue. How BMPs mediate this patterning is unknown. However, a BMP homolog in Drosophila, decapentaplegic (dpp), has been shown to act as a morphogen, inducing different fates at different concentrations (Ferguson and Anderson, 1992, Nellen et al., 1996). Thus vertebrate BMPs may also act as morphogens, with highest concentrations inducing epidermis (Wilson and Hemmati-Brivanlou, 1995), slightly lower concentrations inducing dorsal neural fates, and still lower concentrations producing more ventral neural fates. A convenient way to study neural patterning is to use neural tissue induced by noggin. When ectodermal explants, or ‘animal caps,’ are dissected from Xenopus blastulae and treated

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

2478 A. K. Knecht and R. M. Harland heparin (L. Zimmerman and R. M. H., unpublished data), so it may not diffuse well through some extracellular matrices. Unequal exposure to noggin in explants could generate dorsal/ventral pattern in two ways. First, if noggin does not penetrate the explant completely, some cells might remain epidermal, while others would be induced to form neural tissue. The resulting interaction between neural tissue and epidermis would dorsalize neural tissue at the boundary, as is thought to occur normally at the boundary of the neural plate. Secondly, noggin may play a more direct role in dorsal-ventral patterning: noggin may act as a morphogen, inducing different fates at different locations in the explant, depending upon the local concentration of noggin protein. Furthermore, noggin’s morphogenetic effects could be explained through its inhibition of BMP-4, which is normally present in animal caps (Dale et al., 1992), and which may also act as a morphogen, as described above. Zimmerman et al. (1996) have shown biochemically that noggin binds to BMP-4 and prevents it from binding to its receptor; they therefore suggest that noggin acts by antagonizing BMP signaling. Since BMP-4 can dorsalize neural tissue, one would predict that high doses of noggin would block the formation of dorsal neural fates. Lastly, neural tissue may organize itself, by the classical process of regulation. Although poorly understood at a molecular level, regulative cell-cell interactions are a well known property of neural fields (Jacobson and Sater, 1988). In noggin-induced neural tissue, such interactions may involve the downregulation of dorsal potential except in one region of each explant, in a mechanism similar to lateral inhibition (reviewed by Muskavitch, 1994). Here we show that cells respond to noggin in a concentration-dependent manner, but regulation also occurs, involving a dramatic change in cell motility. Therefore we suggest that both mechanisms participate in the dorsal-ventral organization of noggin-treated explants, and are likely to be important in vivo. However, an additional process, changing competence, determines exactly how cells respond to these two mechanisms. The response of Xenopus ectoderm to different signals changes continuously and autonomously, with neural competence declining at the mid-gastrula stage (Kintner and Dodd, 1991; Servetnick and Grainger, 1991). Interestingly, we have found that neural competence increases dramatically in dissociated cells between stages 8 and 10. Furthermore, during that same period, neuralized explants lose their ability to regulate pattern. These experiments strongly suggest an important role for changing competence, as well as concentration-dependent responses to noggin and BMP, and regulative cell-cell interactions, in dorsal-ventral patterning of neural tissue. MATERIALS AND METHODS Preparation of embryos and noggin-treated explants Pigmented or albino Xenopus eggs were obtained and fertilized in vitro, as described previously (Condie and Harland, 1987). Embryos were staged according to the normal table of Nieuwkoop and Faber (1967). Animal cap ectoderm was dissected at stages 8, 9 and 10, as described by Lamb et al. (1993). Explants were dissected in 75% NAM solution (Slack and Forman, 1980), then transferred into very low calcium and magnesium Ringer’s solution (vLCMR; 65.5 mM NaCl, 0.925 mM KCl, 0.185 mM CaCl2, 0.095 mM MgCl2, 1.2 mM NaHCO3, 2.5 mM Hepes, pH 7.2, and 50 mg/ml gentamycin; Lamb et al., 1993) to prevent

explants from healing closed prior to treatment. Explants were treated in low calcium and magnesium Ringer’s (LCMR; Hemmati-Brivanlou et al., 1990) + 0.5% protease-free BSA (Sigma #A-3294), with or without 1 µg/ml purified human noggin (a gift from Regeneron Pharmaceuticals). After 30 minutes of treatment, explants were washed extensively and cultured in 75% NAM until stage 22-23. Cell dissociation and reaggregation Cells were dissociated following the methods of Green and Smith (1990), with the following variations. Throughout the procedure, we used petri dishes and pipettes coated with 50 mg/ml poly HEMA (Polysciences, Inc.) dissolved in 95% ethanol. After all explants were dissected as described above, vLCMR was replaced with calcium- and magnesium-free medium (CMFM; Sargent et al., 1986). Cells were dispersed 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 reaggregated cell mass was subsequently split into ten smaller reaggregates. For noggin and BMP experiments, cells were dissociated and treated with varying concentrations of purified noggin protein, as above. After 15 minutes of treatment, cells were washed extensively with CMFM, and half of the cells in each treatment were transferred into CMFM + 0.5% BSA containing 1 ng/ml purified human BMP-2 (a gift from Genetics Institute). After 15 minutes, all samples were washed extensively in 75% NAM and allowed to reaggregate. Continuously dissociated cells were washed with CMFM after noggin 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 in CMFM. Just prior to harvesting, dissociated cells were pelleted by centrifugation for 1 minute at 110 g. Five-explant recombinants Explants were dissected as described above. To hold explants open until recombination, explants were treated in vLCMR + 0.5% BSA, with or without 1 µg/ml noggin protein. Recombinants were made by placing five explants together in a small well in an agarose-coated Petri dish. For aging experiments, explants were cultured in vLCMR until sibling embryos reached the appropriate stage. β-galactosidase lineage tracing followed the method of Smith and Harland (1991). One-cell stage embryos were microinjected in the animal pole with 0.5 ng of synthetic mRNA encoding nuclearlocalized β-galactosidase. Animal caps were dissected from injected or uninjected embryos, treated as described above, and combined, one injected cap with four uninjected caps. β-galactosidase activity was visualized by magenta-gal (Biosynth AG) staining. Fixation and whole-mount in situ hybridization All samples were fixed for 1 hour and then processed according to the method of Harland (1991), using modifications and the double in situ hybridization 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 purple AP-substrate first, then alkaline phosphatase substrate kit II (Vector Laboratories). Samples were photographed in methanol (Fig. 2) or cleared in BBBA (2:1 benzyl benzoate/benzyl alcohol) (Figs 7-10). Reverse transcription-PCR analysis RNA was harvested as described (Condie and Harland, 1987). RTPCR was carried out following the protocol of Wilson and Melton (1994). Primer sets were described for NCAM by Hemmati-Brivanlou and Melton (1994); for cardiac actin, EF-1α, and epidermal keratin by Wilson and Melton (1994); and for XAG-1 by Blitz and Cho (1995). We designed primers for cpl-1: upstream, GTCTTAG-

Neural D/V patterning by noggin 2479 GCAAGTGGTAC (211-218); downstream, ATCATCAGCGAGTCCTTG (601-618). (Numbers correspond to sequence in the GenBank database, accession number X84414.)

RESULTS

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

Partial dorsal-ventral pattern in noggin-treated explants We wondered whether the gene expression patterns observed in noggin-treated explants indicate dorsal-ventral patterning. Previously we showed that the neural markers cpl-1 and etr-1 are 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 normally expressed exclusively in the dorsal brain, while etr-1 is expressed broadly throughout the nervous system, but excluded from regions of the dorsal brain (Fig. 1A; Knecht et al., 1995). Therefore we suggested that the separate regions of expression in noggin-treated explants might represent dorsal and ventral domains. Here, we explore this interpretation using additional dorsal or ventral forebrain markers. To see whether expression of cpl-1, a lipocalin, indeed indicates dorsal fates, we analyzed explants for expression of XBF-1, a transcription factor expressed in the dorsal forebrain (Fig. 1C; Papalopulu and Kintner, 1996). XBF-1 shows the same expression pattern as cpl-1 in noggin-treated explants: both genes are expressed separately from etr-1, and both frequently coincide with pits or protrusions (Fig. 1D). Thus these structures express two very different types of genes which both mark the dorsal forebrain. We also analyzed explants for the presence of ventral forebrain using the gene XeNK-2, which is expressed strictly in the ventral neural tube (Fig. 1E; Saha et al., 1993). Unlike the more general marker etr-1, XeNK-2 is not expressed in noggin-treated explants (Fig. 1F). Therefore we conclude that noggin-treated explants contain a partial dorsal-ventral pattern: the most ventral fates, marked by XeNK-2, are absent; however, dorsal forebrain is Fig. 1. Whole-mount in situ hybridizations showing expression of dorsal and ventral forebrain localized to one region, separate from markers in stage 23 embryos and noggin-treated explants. (A) Triple staining of cpl-1 (blue), the remaining tissue which is presumetr-1 (purple) and XAG-1 (a light blue ring marking the cement gland), in the head. Expression ably intermediate between dorsal and of cpl-1 in the dorsal brain overlaps some etr-1 expression, though etr-1 is mainly expressed ventral. Overlapping expression of noggin and cpl-1 in the anterior neural folds We subsequently investigated the mechanisms responsible for restricting the dorsal cpl-1-expressing cells to one

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ventrally in the brain. (B) Double staining of cpl-1 (blue) and etr-1 (purple) in separate territories in noggin-treated explants. cpl-1 is expressed in several protrusions (arrowheads). (C) Double staining of XBF-1 (blue) and etr-1 (brown) in the head. XBF-1 expression is restricted to the dorsal forebrain. (D) Double staining of XBF-1, in blue, and etr-1, in purple, in noggin-treated explants. The pattern of expression of XBF-1 is identical to that of cpl-1, in B. (E) Side view of expression of XeNK-2 in the ventral neural tube. (F) XeNK-2 is not expressed in noggin-treated explants. Scale bars in A, B, and C represent 0.5 mm. D-F are the same magnification as B.

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Fig. 2. Dorsoanterior views of the overlapping expression of noggin and cpl-1 in stage 13 embryos. All embryos were processed together for double in situ hybridization; however, the brown-staining substrate was omitted from the embryos in A, and the blue substrate was omitted from the embryos in B. (A) noggin expression (blue), at the anterior edge of the neural plate, and in the prechordal plate and notochord. (B) cpl-1 expression (brown), also in the anterior neural folds. (C) An embryo double stained for noggin and cpl-1, showing overlapping expression in the anterior neural folds. (The two stains together appear bluish-brown; compare with the blue nogginexpressing notochord). (D) Higher magnification view of C. Scale bars in A and D represent 0.5 mm. B and C are the same magnification as A.

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cpl-1 is induced by noggin in a dose-dependent manner Noggin’s patterning of explants may be mediated by unequal exposure to noggin protein within the intact tissue, with different effective concentrations of noggin inducing different fates. We tested dose-dependent effects of noggin, using a dissociated cell assay (Green and Smith, 1990) to ensure equal noggin exposure. Cells were treated with different concentrations 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 induced by noggin in a dose-dependent manner, with weakly neuralizing doses of noggin inducing cpl-1 most strongly. Neuralization was assessed by analyzing expression of the general neural marker NCAM (Kintner and Melton, 1987). Dissociated cells express some NCAM at 1 ng/ml noggin; at 10 ng/ml noggin NCAM levels peak, and the epidermal marker epidermal keratin (Jonas et al., 1989) is completely turned off, as cells are converted from prospective epidermis into neural tissue (Fig. 3). cpl-1 is most strongly induced at this epidermal/neural threshold (1-10 ng/ml noggin); its expression declines with higher noggin concentrations (Fig. 3; see also Fig. 7). Since cells treated with high noggin doses are still neural, as shown by NCAM expression, declining expression of the dorsal marker cpl-1 suggests that cells are being converted into a more

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

ventral type of neural tissue. Noggin also has a dose-dependent effect on the induction of cement gland (Fig. 3), an ectodermal structure marked by XAG-1 (Sive et al., 1989). XAG-1 is induced by dissociation alone, but is strongly upregulated by the lowest neuralizing dose of noggin, 1 ng/ml. At higher noggin concentrations, XAG-1 is turned off, because cells are completely neuralized. Thus noggin exhibits concentrationdependent effects on induction of both dorsal forebrain and cement gland. Neural competence and cpl-1 induction vary with stage In dissociated cells, the concentration of noggin that induces cpl-1 most strongly varies considerably, depending upon when animal caps were dissected. This variation is due to a dramatic increase in neural competence between stages 8 (early blastula) and 10 (early gastrula). Cells from explants dissected and dissociated at stage 8 are induced to express NCAM by only a high dose of noggin (1 µg/ml; Fig. 4). However, stage 9 cells express NCAM weakly when treated with only 1 ng/ml noggin, and stage 10 cells are neuralized by dissociation alone (Fig. 4). This last result is consistent with previous findings that dissociation can 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 (see control lane, Fig. 3). Dissociation does seem to sensitize cells to noggin, since dissociated cells are neuralized by much lower doses of noggin (1 ng/ml noggin; Fig. 3) than is required for intact explants (1 µg/ml; Lamb et al., 1993). Since cpl-1 is induced at the epidermal/neural threshold, its induction also changes over time. At stage 8, cpl-1 is induced weakly by 1 µg/ml noggin (Fig. 4). At stage 9 it is induced strongly by 1 ng/ml noggin and repressed by 1 µg/ml noggin,

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cpl-1 XAG-1 Ep. Keratin EF-1α muscle actin Fig. 4. cpl-1 induction shifts with increasing neural competence. RTPCR analysis of noggin dose-response, using explants dissected and dissociated at stage 8, stage 9, or stage 10. For each stage, cells were untreated (‘control’), treated with 1 ng/ml noggin protein, or 1 µg/ml noggin (N, noggin protein). All samples were cultured until stage 2223. Controls and primers are the same as described for Fig. 3.

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

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 neuralization, this shift in dose-response correlates with a shift in the epidermal/neural threshold. Without BMP-2, the epidermal markers (XAG-1 and epidermal keratin) are turned off, and NCAM is turned on, at 10 ng/ml noggin. However, in the presence of BMP-2, 100 ng/ml noggin is required to suppress epidermal markers and induce NCAM. Thus BMP-2 is able to antagonize all the effects of noggin in this assay, in support of the idea that noggin acts by inhibition of BMP signaling.

cpl-1 is upregulated in continuously dissociated cells Our results suggest that noggin, via its inhibition of BMPs, can have a direct effect on patterning. However, the results could also be explained by the model of neural patterning via interactions between neuralized and non-neuralized cells. If dissociated cells are heterogeneous in their neural competence, a mixture of neural and epidermal cells could result at a threshold concentration of noggin. Subsequent reaggregation would allow epidermal cells to contact and dorsalize neural cells, thereby inducing a high level of cpl-1. We tested the requirement for cell-cell contact by comparing reaggregated cells with cells that were continuously dissociated. Long term dissociation exacerbates the weak neuralizing effect of dissociation, such that untreated cells express NCAM weakly and express reduced levels of epidermal markers epidermal keratin and XAG-1(Fig. 6). cpl-1 is not significantly induced in any of the reaggregated cells, presumably because the critical dose of noggin was not tested. (Since cpl-1 induction varies considerably with stage of dissection (Fig. 4), it was not possible to predict exactly which doses of noggin would induce cpl-1.) Surprisingly, cpl-1 is greatly upregulated in all continuously dissociated treatments. These results show that cell-cell contact is not required for induction of cpl-1,

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Addition of BMP antagonizes the effects of noggin Zimmerman et al. (1996) showed that noggin can block BMP4 signaling. If noggin mediates dorsal-ventral patterning in our assays by inhibiting the dorsalizing effects of BMPs, then application of BMP protein should antagonize the effects of noggin, causing a shift in ng/ml noggin embryo the dose-response profile toward higher control + 1 ng/ml BMP2 ng/ml noggin doses of noggin. To test this idea within a reasonable range of noggin concentrations, we used cells dissociated at late stage 9/early stage 10, which possess maximal neural NCAM competence (see Fig. 4). We also used a very low dose of purified BMP-2 (1 ng/ml), since cpl-1 higher BMP doses have been shown to completely block neuralization (Wilson and Hemmati-Brivanlou, 1995). (Although XAG-1 BMP-2 is not expressed during gastrulation and early neurulation (Clement et al., 1995), Ep. Keratin it is used in these studies to substitute for BMP-4, a more likely candidate for the neural dorsalizer, since BMP-2 and -4 have EF1α identical biological activities in early muscle actin Xenopus development (Clement et al., 1995; Hemmati-Brivanlou and Thomsen, 1995).) Fig. 5. Noggin dose-response profiles are shifted in the presence of BMP-2. RT-PCR In the presence of added BMP-2, higher analysis of cells dissociated at late stage 9/early stage 10 and treated with a range of doses of noggin are required to elicit the concentrations of noggin protein, as indicated. Cells were then divided into two pools; same effects observed with noggin alone. In one pool was untreated, and the other was additionally treated with 1 ng/ml purified the absence of BMP-2, cpl-1 is induced BMP-2. All samples were harvested at stage 22. Controls and primers are the same as described for Fig. 3. maximally by dissociation alone, 1 ng/ml

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NCAM cpl-1 XAG-1 Ep. Keratin EF-1α muscle actin Fig. 6. Continously dissociated cells express high levels of cpl-1. RTPCR analysis of noggin dose-response in dissociated cells, treated as in Fig. 4 (N, noggin protein). After treatment, one-third of the cells were reaggregated (‘reagg.’), and the rest were kept dissociated (‘dissoc.’). All samples were cultured until stage 20. Controls and primers are the same as described for Fig. 3.

thereby discounting the neural/non-neural interaction hypothesis. Moreover, the upregulation of cpl-1 in the absence of cellcell contact suggests that communication between cells normally acts to repress cpl-1 expression, perhaps by a process similar to lateral inhibition. We have further investigated the ability of cells to self-organize in the following experiments.

trusions (Fig. 7C,D), reminiscent of the cpl-1-expressing protrusions in intact explants. These protrusions are rarely seen with higher noggin concentrations (Fig. 7E-H); instead, these aggregates exhibit large pits (arrowhead, Fig. 7F), producing a cup-like shape (arrowhead, Fig. 7H). These morphological changes and the partial localization of cpl-1 both indicate that these cells are capable of self-organization. We also tested the effects of changing competence on cpl1 induction and localization (Fig. 8). The dissection of explants at progressively later stages has a dramatic effect on overall levels of cpl-1, which is induced by lower doses of noggin as neural competence increases (as shown in Fig. 4). However, reaggregates that express cpl-1 strongly are generally equally capable of localizing cpl-1 expression, regardless of stage (Fig. 8E,G,H). cpl-1 expression in stage 8 cells is diffuse (Fig. 8C), but that may be due to the weakness of the signal. Protrusions were not observed in this experiment, but since only two concentrations of noggin were tested, we may have missed the critical dose for protrusion formation. 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 and self-organize. Regulation occurs in five-explant recombinants, but varies with stage To further explore this process of regulation, we devised an assay in which five animal caps were treated with noggin and then allowed to heal together into one recombinant (Fig. 9A). If regulation occurs, recombinants should express cpl-1 in one patch. However, if each explant retains its own patterning information, five cpl-1 patches should result.

Regulation of cpl-1 expression in reaggregated cells We hypothesized that the localized expression of cpl-1 in intact explants occurs because of unequal exposure to noggin, with cpl-1 being expressed by only the cells seeing a parA B C D ticular dose of noggin. If this mechanism is solely responsible for cpl-1 localization, then one would predict that dissociated cells, with equal exposure to noggin, should express cpl-1 uniformly. We tested this prediction by performing in situ hybridizations for cpl-1 on dissociated cell reaggregates. The patterns of cpl-1 expression demonstrate the ability of these cells to E F G H self-organize. Overall expression levels verify our RT-PCR results (Fig. 3 for example), showing declining cpl-1 expression at higher noggin doses (Fig. 7). The weak expression of cpl-1 at high noggin doses (100 ng/ml and 1 µg/ml; Fig. 7G,H) is observed as diffuse staining throughout the explants. However, the strong expression at low doses (1 and 10 ng/ml; Fig. 7C-F) is Fig. 7. Partially localized cpl-1 expression in stage 22 cell reaggregates. Dissociated cells were extremely non-uniform, with little untreated (A), or treated with the following noggin protein concentrations: 0.1 ng/ml (B), 1 staining in some regions within the ng/ml (C, shown at higher magnification in D), 10 ng/ml (E, shown at higher magnification in aggregates, and concentrated staining in F), 100 ng/ml (G), or 1 µg/ml (H). Darkly stained protrusions are observed at 1 ng/ml noggin other regions. Strikingly, at 1 ng/ml (C,D). Pits are observed at higher noggin concentrations (E-H, see arrowheads in F and H). noggin these concentrations of Scale bars in A and D represent 0.5 mm. F is the same magnification as D; all others are the expression are often associated with pro- same magnification as A.

Neural D/V patterning by noggin 2483 We found that regulation occurs but is very stage-dependent. Explants dissected at stage 8 regulate, expressing cpl-1 in one large patch which is almost always associated with a pit (arrowhead, Fig. 9Ba). However, early stage 9 explants occasionally show additional patches of cpl-1 (Fig. 9Bb), and late stage 9 explants exhibit such extensive cpl-1 staining that discrete patches cannot be identified (Fig. 9Bc). Therefore, over time, explants lose their ability to regulate the cpl-1 pattern. One explanation for this observation is that later explants, while still in the embryo at stage 9, receive additional patterning information which prevents subsequent regulation. In this case, the stage of dissection is critical. Alternatively, the stage at which explants are combined may be important, since it establishes when cell-cell signaling can begin. Lastly, the stage of noggin treatment may be crucial, as we have established that cells exhibit changing competence to respond to noggin during this period. To test whether time of dissection is critical, we dissected explants at stage 8, early stage 9, and late stage 9, but aged earlier explants in culture to late stage 9 before treatment and combination. This aging causes early explants to lose their ability to regulate, such that all recombinants express multiple patches of cpl-1 (Fig. 9Bd,e), as do late stage 9 explants that were not aged (Fig. 9Bc,f). This experiment shows that time of dissection is unimportant, and embryonic signaling is not required for loss of ability to regulate, since early explants can lose this ability in vitro. Secondly, to distinguish between time of treatment and time of combination, we dissected explants at the same three stages and control treated with noggin immediately after dissection. After A treatment, however, earlier explants were aged until late stage 9, when all recombist. 8 nants were made. This treatment rescues the ability of stage 8 explants to regulate cpl-1 expression to one patch (Fig. 9Bg). Thus D timing of noggin treatment is critical in determining the properties of the resulting st. 9 tissue, with only early treated tissue being able to regulate. Early, regulating recombinants exhibit greater cell movement We wished to investigate the mechanism by which this pattern regulation occurs. One possibility is that dorsal localization occurs via localized specification of dorsal fates within the neural field. A second possibility is that dorsal cell fates

G

are specified at multiple locations, but dorsal cells later come together by cell movement. To test the latter possibility, we used a lineage tracing technique diagrammed in Fig. 10A. Animal caps were dissected, at stage 8 or late stage 9, from embryos injected with RNA encoding β-galactosidase (β-gal), and from uninjected embryos. All explants were immediately treated with noggin; then one β-gal-injected explant was combined with four uninjected explants. After culturing, the position of cells from the injected explant was determined by β-gal staining with magenta-gal. Additionally, by performing in situ hybridizations for cpl-1 (stained blue), we could relate the location of β-gal-stained cells to the location of the dorsal patch. Noggin-treated stage 8 recombinants, which regulate to express one patch of cpl-1, show remarkably extensive cell movement (Fig. 10B,C). Furthermore, this movement only occurs in recombinants undergoing regulation, since we observed no evidence of cell movement in non-regulating late stage 9 recombinants (Fig. 10E), or untreated stage 8 recombinants (Fig. 10D). The movement observed in regulating recombinants appears to include expansion of the entire population of labeled cells, often into large arcs (Fig. 10C), and not just migration of a dorsally specified subpopulation. Thus it seems unlikely that cell movement can solely account for cpl1 localization, if dorsally specified cells are initially widely dispersed. Nonetheless, these explants are clearly undergoing considerable reorganization, and the strong correlation with regulation suggests that cell movement may contribute to the localization of dorsal fates.

1 ng/ml noggin 1 µg/ml noggin B C

E

F

H

I

st. 10

Fig. 8. Partially localized cpl-1 expression in reaggregated cells, dissociated at stage 8 (A-C), stage 9 (D-F), or stage 10 (G-I). Cells were untreated (A,D,G), treated with 1 ng/ml noggin protein (B,E,H), or 1 µg/ml noggin (C,F,I), then cultured until stage 20. Neuralized reaggregates often appear cup-shaped (arrowhead in H). Scale bar in A represents 0.5 mm.

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

which ultimately determines how cells respond to patterning signals.

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

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

A = regulation in situ for cpl-1

=

+ noggin

B

stage 8

early stage 9

late stage 9

treated and combined immediately Fig. 9. Regulation in five-explant recombinants, cultured until stage 22-23. (A) Recombinants were made by allowing five noggin-treated explants to heal together. If regulation occurs, one large patch of cpl-1 expression should result; otherwise, each explant should express cpl-1 independently, such that five separate patches result. (B) cpl-1 expression in five-explant recombinants, made from explants dissected at stage 8 (a,d,g), early stage 9 (b,e,h), or late stage 9 (c,f,i). (a-c) Explants were dissected at the stages indicated, then treated and combined immediately. Stage 8 recombinants regulate to form one patch of cpl-1 expression (a), which often contains a large pit (arrowhead in a); later recombinants (b,c) exhibit multiple, fused patches of cpl-1. (d-f) Explants were dissected at the stages indicated, but aged until late stage 9 before treatment and combination. All recombinants exhibit multiple cpl-1 patches (d-f). (g-i) Explants were treated immediately after their dissection, but all were aged to late stage 9 before combination. Stage 8 recombinants regulate to form one cpl-1 patch (g), as in a. Scale bar in a represents 0.5 mm.

no regulation

a

b

c

d

e

f

g

h

i

treated and combined at late st. 9

treated immediately, combined at late st. 9

Neural D/V patterning by noggin 2485 (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 represents telencephalon outgrowth. While dorsal fates are restricted to one patch in noggintreated explants, the remaining tissue is not ventral, since it does not express XeNK-2. Ericson et al. (1995) have shown that a homolog of this gene, Nkx-2.1, can be induced in chick forebrain treated with sonic hedgehog. It is therefore likely that exposure to sonic hedgehog is required for the proper formation of ventral fates. Since noggin-treated explants lack sonic hedgehog, the non-dorsal tissue probably adopts an intermediate neural tube fate. Concentration-dependent effects of noggin in dorsal-ventral patterning We have considered several mechanisms that might generate dorsal-ventral pattern in explants. First, we proposed that cells within explants may have unequal exposure to noggin, which could then generate pattern either directly or indirectly. In a direct model, a noggin gradient would be established in the explant, with different concentrations of noggin inducing different dorsal-ventral fates. The critical requirement for this

A

model is that noggin must act in a concentration-dependent manner. Alternatively, noggin may pattern indirectly by incompletely neuralizing the explant; the subsequent interaction between neural and non-neural cells could cause localized dorsalization (Moury and Jacobson, 1989; Dickinson et al., 1995; and Selleck and Bronner-Fraser, 1995). An important aspect of this indirect model is that cell-cell contact is required for dorsal induction. Using dissociated cells, we have found that noggin does elicit concentration-dependent effects. Low doses of noggin induce cpl-1, but higher doses repress it, presumably because cells are adopting more ventral neural fates (Fig. 3). Similarly, the cement gland marker XAG-1 is strongly induced by very low doses of noggin, but turned off by higher doses, as cells are converted into neural tissue. Furthermore, these inductions do not require contact between neural and non-neural cells, since continuously dissociated cells are still capable of expressing cpl-1 and XAG-1(Fig. 6). These concentration-dependent effects support the idea that a noggin gradient could generate dorsal-ventral patterning in intact noggin-treated explants. How does noggin mediate this patterning? Previous results suggested that noggin acts by antagonizing signaling by BMP4 (Zimmerman et al., 1996). Our results confirm one prediction of this model: since BMP-4 is known to dorsalize neural

lacZ RNA

no cell = movement Fig. 10. Cell movement in stage 20 five-explant recombinants. (A) Explants were dissected from embryos injected at the one cell stage with β-gal RNA. Recombinants were made by combining one labeled explant with four unlabeled explants. (B) Stage 8 noggin-treated recombinants, stained for β-gal activity, in magenta, and cpl-1 expression, in blue. All recombinants form one patch of cpl-1, and all patches contain some β-gal-labeled cells. Several explants show extreme elongation of the labeled patches. (C) Higher magnification view of two recombinants from B, showing labeled cells extending into the cpl-1 pit. (D) In untreated stage 8 recombinants, labeled cells remain in round clumps. (E) Late stage 9 noggin-treated recombinants also show little dispersion of labeled cells, and cpl-1 expression is not regulated to one patch. Scale bars all represent 0.5 mm.

cell = movement

B

C

D

E

2486 A. K. Knecht and R. M. Harland tissue (Liem et al., 1995), high doses of noggin should block the most dorsal fates; likewise, we have found that high doses of noggin repress cpl-1 (Fig. 3). Furthermore, we have verified that BMP-2 can antagonize noggin in our assays, since higher doses of noggin are required to induce cpl-1 (and neural tissue in general) in the presence of BMP-2 protein (Fig. 5). These results, combined with previous biochemical data showing a direct interaction between noggin and BMP-4 (Zimmerman et al., 1996), make a strong case for the model of noggin action via BMP-4 inhibition. The observed concentration-dependent effects of noggin then lead to the important conclusion that BMP-4 may also act morphogenetically. According to this model, high concentrations of BMP-4 induce epidermis, as shown by Wilson and Hemmati-Brivanlou (1995). Slightly lower concentrations (or high concentrations combined with low concentrations of a BMP antagonist like noggin) induce cement gland (see XAG1 induction in Fig. 3 for example). Still lower concentrations of BMP-4, or higher concentrations of an antagonist, induce dorsal neural tissue (see cpl-1 induction in Fig. 3, e.g.). Finally, the lowest BMP-4 concentrations, or highest concentrations of an antagonist, give rise to more ventral types of neural tissue. Although we cannot say when these patterning events are occurring in vivo, one possible period is the late gastrula/early neurula stages. During these stages BMP-4 is expressed in the non-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 a gradient may exist across this boundary. This gradient may be maintained or modified by the weak expression of noggin at the anterior edge of the neural plate. Noggin’s dorsal expression seems at first to be contradictory with its activity as a neural ventralizer at high doses (though this activity is consistent with its expression in the notochord). However, our results show that a low dose of noggin actually induces cpl-1, presumably by reducing activity of the morphogen BMP-4 to the appropriate level for neural dorsalization. The juxtaposition of BMP expression to noggin at the neural plate boundary may help provide a steep gradient of BMP activity and a resulting sharp focus of tissue fates. The role of regulation in dorsal-ventral patterning We found that cpl-1 is strongly upregulated in continuously dissociated cells, indicating that all cells have adopted a dorsal fate (Fig. 6). This result is particularly surprising since one might predict that prolonged dissociation would more effectively remove BMPs from the surface of cells, causing them to be ventralized relative to cells which are only briefly dissociated. The extent of this effect would depend upon several parameters, including the substantial affinity of BMPs for the cell surface (Koenig et al., 1994). However, our observation of the opposite result, that continuously dissociated cells are strongly dorsalized relative to reaggregated cells, indicates that reaggregation itself causes downregulation of cpl-1. We suggest that this repression of dorsal fates is due to cell-cell communication, which may act to restrict dorsal fates to one small region by a regulative process analogous to lateral inhibition. Furthermore, although noggin gradients probably contribute to patterning of intact noggin-treated explants, unequal exposure is not strictly required for localization of dorsal fates.

In reaggregates of dissociated cells, with equal exposure to noggin, cpl-1 is highly expressed in some regions and only weakly expressed in others (Figs 7 and 8). Also, these reaggregates form pits and protrusions, structures which must require the coordinated movements of many cells. We therefore conclude that cells possess a regulative capacity allowing them to organize themselves dorsoventrally. Not only can this regulation occur in the absence of additional patterning information, as in dissociated cells (Figs 7 and 8), but it can also override previous patterning information, as in five-explant recombinants (Figs 9 and 10). In vivo, this mechanism may play an important role in refining and maintaining the dorsalventral axis established by growth factors. Although regulation is a widespread phenomenon in development, its molecular mechanisms are largely unknown. We suggested two ways that dorsal fates could be localized: by localized specification of dorsal fates, and by movement of dorsally specified cells. Interestingly, we did observe considerable cell movement in five-explant recombinants that regulate, and no cell movement in non-regulating recombinants (Fig. 10). This observation represents a novel discovery about the behavior of neuralized cells, and the strong correlation with regulation suggests that cell movement may play an important role in regulation, perhaps also mediating the cell reorganization that gives rise to pits and protrustions. Such movements are likely to refine the organization of the neural tube in vivo. However, cell movements alone cannot explain the partial localization of cpl-1 in reaggregates of dissociated cells (Figs 7 and 8), since in the absence of a noggin gradient, all cells should express equal levels of cpl-1. Regulative cell-cell interactions probably mediate the repression of cpl-1 in some regions of the reaggregates, as is also suggested by the repression of cpl-1 in reaggregated versus continuously dissociated cells (Fig. 6). Thus cell-cell signaling contributes to dorsal specification, with cell movement refining this process by allowing dorsally specified cells in a general area to converge. The central role of competence The ectoderm loses its response to neural inducers during late gastrulation (Kintner and Dodd, 1991; Servetnick and Grainger, 1991). Here we have shown that the competence of cells to be neuralized increases dramatically at the onset of gastrulation (Figs 4 and 8). Thus the effect of different doses of noggin can be mimicked by application of the same doses at different times. In addition, we have found that early application of noggin to animal caps increases the ability of explants to regulate localized expression of cpl-1(Fig. 9). Early application of noggin also increases the motility of cells, contributing to refinement of localized cpl-1 expression (Fig. 10). Therefore the age of the responding cells has effects on cell fate, secondary cell-cell signaling and cell movement. Although we do not understand the basis for the changing response of cells to noggin over time, these findings highlight the important contribution of changes in competence to cell fate. Morphogens induce different cell fates at different concentrations; however, a substance may mimic a morphogen by acting at the cell surface at different times. Consequently, future models of dorsal-ventral patterning of neural tissue must take into account not just signals like BMP-4 and noggin, and regulative cell-cell interactions, but also the timing with which these signals act.

Neural D/V patterning by noggin 2487 The authors thank Nancy Papalopulu for providing XBF-1 prior to publication; Regeneron Pharmaceuticals for the gift of purified noggin protein; Genetics Institute for the gift of purified BMP-2 protein; and David Hsu for critical reading of the manuscript. This work was supported by NIH grants to R. M. H. and a Howard Hughes Medical Institute predoctoral fellowship to A. K. K.

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