Induction of neural crest in Xenopus by AP2␣

Ting Luo*, Young-Hoon Lee†, Jean-Pierre Saint-Jeannet†, and Thomas D. Sargent*‡

*Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and †Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104

Communicated by Igor B. Dawid, National Institutes of Health, Bethesda, MD, November 27, 2002 (received for review September 23, 2002) We report experiments with Xenopus laevis, using both intact recently been described (29). Homologs of AP2␣ have been embryos and ectodermal explants, showing that the transcription reported to be expressed in the early stages of NC development factor AP2␣ is positively regulated by bone morphogenetic of mouse, fish, and frog, as well as in other tissues including (BMP) and Wnt signaling, and that this activation is an essential epidermis (26, 30–33). In this article we have adopted the name step in the induction of neural crest (NC). Ectopic expression of AP2␣ to denote the single AP2 isoform that has been described AP2␣ is sufficient to activate high-level expression of NC-specific to date in Xenopus, which is most closely homologous to mam- such as Slug and Sox9, which can occur as isolated domains malian AP2␣ and thus probably the homolog of this (34). within the neural plate as well as by expansion of endogenous NC In the mouse, gene targeting of AP2␣ resulted in a perinatal territories. AP2␣ also has the property of inducing NC in isolated lethal phenotype characterized by absence or severe reduction of ectoderm in which Wnt signaling is provided but BMP signaling is cranial bones of NC origin, delayed neural fold elevation, minimized by overexpression of chordin. Like other NC regulatory extensive failure of neural tube closure, failure of ventral body factors, activation of AP2␣ requires some attenuation of endoge- wall closure, lateral displacement of facial primordia, as well as nous BMP signaling; however, this process occurs at a lower limb and other defects (35, 36). These results implicated AP2␣ threshold for AP2␣. Furthermore, AP2␣ expression domains are in NC development, at least in the rostral compartment. How- larger than for other NC factors. Loss-of-function experiments with ever, some NC-derived cranial structures persisted, including the antisense AP2␣ morpholino oligonucleotides result in severe re- hyoid bone, Meckel’s cartilage, and mandibular tissue. In addi- duction in the NC territory. These results support a central role for tion, the NC marker genes Twist and Pax3 continued to be AP2␣ in NC induction. We propose a model in which AP2␣ expres- expressed in homozygous knockout mice (35), making it difficult sion, along with inactivation of NC inhibitory factors such as Dlx3, to assign a specific role for AP2␣. establish a feedback loop comprising AP2␣, Sox9, and Slug, leading To further elucidate this potential role for AP2␣, we have to and maintaining NC specification. turned to Xenopus laevis embryos, which are ideally suited for gene regulatory and cell signaling studies at early gastrula and bone morphogenetic protein ͉ Wnt ͉ Sox9 ͉ Slug ͉ morpholino antisense neurula stages. We present the results of both gain-of-function oligonucleotides and loss-of-function experiments in X. laevis, supporting an essential role for AP2␣ gene function at the earliest stage of NC ␣ eural crest (NC) cells are a distinguishing feature of verte- development. Furthermore, key differences between AP2 and Nbrate embryos. They arise from the boundary between the other NC regulators reveal interesting features of the response dorsal͞neural plate and ventral͞epidermal territories as the to BMP and Wnt signaling in this process. result of inductive interactions, both between these tissues and Materials and Methods with other embryonic cell types (1, 2). Evidence obtained Embryo Manipulation. Embryos were obtained from adult X. laevis primarily from Xenopus strongly suggests that two independent by hormone-induced egg laying and artificial fertilization with signals are necessary for NC induction: a bone morphogenetic standard methods and staged according to Nieuwkoop and protein (BMP) signal, which must be partially modulated (3–5), Faber (37). For ectodermal explants, full-length capped tran- and a separate input that can be either a canonical Wnt signal scripts encoding AP2␣ (31), chordin (Chd) (38), or Xwnt-3a (39) (6–9), retinoic acid (10), or fibroblast growth factor signal (11). were injected into two sites in the animal hemisphere of the During neurulation, NC cells undergo a classical epithelial- one-cell embryos. After injection, the embryos were cultured in mesenchymal transition and commence an elaborate migration 4% Ficoll͞1ϫ modified Ringer’s solution (MR) for 90 min and throughout the body, following prescribed routes, and subse- then transferred to 0.3ϫ MR. Ectodermal explants were re- quently differentiate into a host of terminal cell types including moved at stages 7 and 8 and cultured until stages 14 and 15 then craniofacial bones and cartilage, elements of the peripheral processed for Northern blot analysis. For whole-embryo exper- nervous system, and melanocytes (12, 13). iments, embryos were injected into one cell at the two-cell stage The molecular basis for NC specification is still incompletely or into a single animal hemisphere cell at the 8- to 16-cell stage understood. A diverse assortment of regulatory factors has been with either plasmid DNA (to avoid axis duplication) encoding implicated in the early steps in this process. Among these are zinc Xwnt-1 or Xwnt-3a, or RNA encoding GSK␤,AP2␣ or mor- finger factors Snail (14), Slug (15, 16), Zic3 and Zic5 (17, 18), the pholino oligonucleotides (MOs) (GeneTools, Philomath, OR), helix–loop–helix factor Twist (19), the winged helix factor mixed with ␤-galactosidase RNA as a lineage tracer. The AP2␣ FoxD3 (20, 21), the paired class homeodomain Pax3 (22), the MO sequence was 5Ј-GGC GAT CCT GCC ATT CCC CCA high mobility group factor Sox9 (23), and the translational TTT T-3Ј (translational start codon underlined), and the con- initiation factor eIF4AII (24). To varying degrees these factors trol, which was mismatched at four positions (lowercase), was are either capable of inducing NC marker gene expression when 5Ј-GGC cAT CgT GCC ATT CCC gCA TAT T-3Ј. Embryos expressed at ectopic locations or are required for NC as shown were fixed for in situ hybridization at mid neural plate stage in by gene targeting or other loss-of-function studies. The transcription factor AP2 comprises a family with four members in the , with functional and develop- Abbreviations: NC, neural crest; BMP, bone morphogenetic protein; MO, morpholino mental expression data available on the AP2␣ (25, 26), AP2␤ oligonucleotide; Chd, chordin. (27), and AP2␥ (28) genes. The fourth member, AP2␦, has only ‡To whom correspondence should be addressed. E-mail: [email protected].

532–537 ͉ PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237226100 Downloaded by guest on September 29, 2021 Fig. 1. Comparison of neurula stage expression of AP2␣ and other NC marker genes. (A) Whole-mount in situ hybridization with probes for AP2␣, Slug, and Sox9 are shown in dorsal and lateral views. AP2␣ transcripts appear to extend more laterally than for the other two genes. This is shown more clearly in cross section in B, where the bracket indicates the Slug domain and the arrows point to the more extensive lateral AP2␣. Note that under the conditions used here ␣ the epidermal expression of AP2 , which is considerably lower than for NC, is Fig. 2. NC expression of AP2␣ depends on Wnt signaling. Whole-mount in ϫ ϫ not apparent. The notochord is indicated by n. (Magnifications: A, 7; B, 42.) situ hybridizations with NC markers Sox9, Slug, and AP2␣ to embryos injected into one cell at the two-cell stage with 100 pg of plasmid DNA encoding Xenopus Xwnt-1 (Left) and Xwnt-3A (Center), or 1 ng RNA encoding GSK3␤ 4% paraformaldehyde and stained for ␤-galactosidase activity by (Right), along with a ␤-galactosidase lineage tracer (red staining). In all cases using 5-bromo-4-chloro-3-indolyl-␤-D-galactoside (GIBCO͞ the injected side is oriented to the right (red staining). Both Wnt treatments BRL) or 6-chloro-3-indolyl-␤-D-galactoside (Research Organ- resulted in lateral and posterior expansion of NC territory, including expres- ics). Coupled in vitro transcription͞translation reactions with sion of AP2␣ as well as the other NC markers. The reciprocal treatment with ␤ AP2␣ plasmid DNA were carried out according to the manu- GSK3 , which interferes with downstream Wnt signaling (50), had the oppo- site effect: AP2␣, Sox9 and Slug all were repressed. (Magnification: ϫ15.) facturer’s instructions (Promega).

Northern Blot Hybridization. RNAs were isolated and analyzed by Several lines of evidence suggest that induction of NC in using denaturing methylmercury hydroxide RNA gels as de- ␣ vertebrate development depends on multiple signals, including scribed (40). Probes for expression of AP2 (31), Slug (16), Sox9 BMPs and Wnt-class factors (5–9, 46–48). To test whether the (23), Xtwi (19), (41), Otx2 (42), Dlx3 (43), and Dlx5 (44) NC-specific expression of AP2␣ conformed to this pattern with were labeled with 32P-dCTP by primer extension (Amersham BIOLOGY respect to Wnt signaling, embryos were injected into one blas- Pharmacia). Staining with 18S ethidium bromide was used to DEVELOPMENTAL tomere at the two-cell stage with plasmid DNA encoding Xwnt-1 monitor equal loading of embryonic RNA samples. (49) or Xwnt-3A (39). DNA was used instead of RNA to circumvent axis duplication (7). As shown in Fig. 2, these In Situ Hybridization. Whole-mount in situ hybridization was carried out according to Harland (45), with some modifications treatments resulted in significant lateral expansion of the cranial (44). Antisense probes labeled with digoxigenin or fluorescein NC territory as indicated by hybridization with NC markers such as Sox9 (23) or Slug (16). Wnt signaling can be inhibited at the were synthesized by using a Roche Molecular Biochemicals RNA ␤ labeling kit with cDNA templates encoding AP2␣, Slug, Sox9, intracellular level by overexpression of GSK3 (50), which results in reduction or elimination of NC marker gene expression and Sox2. For histology, stained embryos were embedded into ␣ hydroxyethyl methacrylate, and 12-␮m sections were cut on a (Fig. 2). In both types of experiment, AP2 behaved in a manner rotary microtome and briefly counterstained with eosin. similar to that of Sox9 and Slug, indicating that like these other genes, AP2␣ expression in NC is under the positive control of Results Wnt signaling. Regulation of AP2␣ Expression in NC by Wnt Signaling. At open ␣ ␣ neural plate stages, Xenopus AP2␣ transcripts are highly abun- AP2 Is Required for NC Induction. Transgenic mice lacking AP2 dant in premigratory NC, particularly in the cranial region, but gene function have major craniofacial deficiencies, suggesting a ͞ are also evident in prospective trunk NC cells. Interestingly, the function for this factor in the formation and or subsequent rostral expression domain extends over a larger area than that of development of the cranial NC cells that give rise to facial bones other NC regulatory factors, i.e., Slug and Sox9 (Fig. 1A). This and cartilage (35, 36). To evaluate the importance of AP2␣ in the pattern is evident in cross sections comparing AP2␣ and Slug initial induction of NC, we used antisense MOs (51) to interfere expression (Fig. 1B). AP2␣ RNA is present at lower but signif- with AP2␣ translation in Xenopus embryos. As shown in Fig. 3A, icant levels in the epidermis, expression that also depends on the AP2␣ MO strongly repressed in vitro translation of synthetic BMP signaling (34). AP2␣ RNA has not been detected in any AP2␣ mRNA, whereas a control MO incorporating four mis- mesodermal or endodermal tissues through stage 20 (data not matches (of 25; see Materials and Methods) had much less effect. shown). When injected into one of the two blastomeres of a two-cell

Luo et al. PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 ͉ 533 Downloaded by guest on September 29, 2021 Fig. 3. Loss of function by MO antisense injections. (A) In vitro-coupled transcription͞translation reactions with plasmid encoding the AP2␣ ORF in the presence of either control (Co-mo) or perfect-match AP2␣ MO (AP2-mo; 50 ng; see Materials and Methods for sequences). (B) Neurula stage embryos injected into one cell at the two-cell stage with 30 ng of AP2␣ MO or the control MO along with ␤-galactosidase mRNA as a lineage tracer and probed for expression of NC markers Slug and Sox9 and the neural plate marker Sox2. The injected sides are on the right in these dorsal views (reddish ␤-galactosidase staining). The AP2␣ MO reduces NC and slightly expands neural plate laterally. (C) AP2␣-MO inhibits Slug expression in a dose-dependent manner (F). Values in parentheses indicate the number of embryos analyzed for each concentration of injected MO. Injection of 30 ng of a control MO had a minor effect on Slug expression (■). (D) Cross section of AP2␣ MO-injected embryo showing reduction in Slug. The injected side is to the right and the uninjected side is to the left (arrow). This also shows the reduced elevation of neural fold after AP2␣ MO injection. (Magnifications: B, ϫ7; D, ϫ42.)

embryo, the AP2␣ MO, but not the control MO, strongly induction of NC was not observed in the most posterior region inhibited expression of NC markers, such as Slug and Sox9 (Fig. of the neural plate. 3B). This effect may be accompanied by some lateral expansion of neural plate, as indicated by Sox2 expression (Fig. 3B Upper Differential Response of NC Gene Expression to a BMP Signaling Right), although this is difficult to evaluate because of morpho- Gradient. In Xenopus (5, 44, 53) and zebrafish (32), the degree of logical effects on neural fold elevation noted below. The inhib- BMP signal attenuation plays an important role in determining itory effect of the AP2␣ MO was dose dependent, as shown in the type of ectodermal tissue induced during gastrulation. To Fig. 3C, reaching nearly 100% at 40 ng injected. The control MO examine this in the context of NC induction, increasing doses of was not tested at all concentrations, but at 30 ng had little effect. RNA encoding Chd were injected, to simulate a gradient of BMP The reduction in NC gene expression was also accompanied by activity, along with a constant dose of Xwnt-3a RNA. Expression a suppression of neural fold elevation on the injected side (Fig. of AP2␣ and other regulatory factors was then monitored by 3D), an effect that has also been observed with loss-of-function Northern blot. The results, shown in Fig. 5A, indicate that AP2␣ experiments targeting Sox9 (23). We have obtained essentially differs from Sox9, Slug, and Xtwi with respect to the lower identical results by using overexpression of dominant negative threshold for activation; AP2␣ (which is expressed at low level in AP2␣ derivatives lacking an activation domain or fused to the uninjected ectoderm, presumably as part of the epidermal repressor domain of the Drosophila factor (data not program) is significantly up-regulated by the lowest (10 pg) dose shown). of Chd, whereas the other three genes were not similarly activated until 10 times more Chd RNA was injected. Expression Ectopic AP2␣ Transforms Neural Plate to NC. The preceding data of Dlx3, which we have reported to act as an antagonist of NC support the conclusion that AP2␣ is required early in NC induction (54) was reduced to an essentially constant baseline by development. To determine whether this factor is sufficient for the 10-pg Chd dose, whereas Dlx5 expression was reduced at the NC specification, we carried out ectopic expression experiments. lower Chd doses, then partially recovered at higher doses, similar RNA encoding AP2␣ was injected at the 8- to 16-cell stage into to what has been observed in the absence of Wnt overexpression blastomeres predicted from the Xenopus embryonic fate map to (44). Neural plate marker genes Sox2 and Otx2 were repressed include neural plate tissue among their progeny (52). The result in animal caps under all conditions. When BMP signaling was of this ectopic expression, shown in Fig. 4, was that strong inhibited to an even higher extent, by injection of more Chd activation of NC-specific gene expression (e.g., Slug and Sox9) RNA (Fig. 5B), the NC marker genes were down-regulated was elicited. Importantly, in many cases this conversion to NC compared with lower Chd doses, whereas the neural plate comprised isolated domains within the neural plate, which were marker Sox2 was activated. This finding supports the idea that physically separated from endogenous NC territories, as op- some level of BMP signaling is essential for NC induction, but posed to contiguous, lateral expansions of NC into the neural not, as revealed by the continued repression of the anterior plate and epidermis (which was also observed). Ectopic expres- neural marker Otx2, for posterior transformation of ectoderm by sion of AP2␣ was also found to repress neural plate gene activity, Wnt signaling. The effective elimination of BMP signaling in this as exemplified by Sox2 (Fig. 4 Bottom). In these experiments, experiment is reflected in the virtually complete repression of

534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237226100 Luo et al. Downloaded by guest on September 29, 2021 Fig. 5. Differential control of NC factors by BMP signal attenuation. Shown is Northern blot analysis of RNA isolated from animal caps derived from embryos injected at the one-cell stage with a mixture of RNAs encoding Chd, Xwnt-3A, and AP2␣. Uninjected (UI) embryo animal cap RNA and whole embryo (W) RNA are shown for comparison. Ethidium bromide staining of 18S ribosomal RNA is shown to confirm equal lane loading. All samples were cultured to stages 14 and 15. (A) Shallow gradient of Chd dosage ranging from 10 pg to 2.5 ng. AP2␣ up-regulation was elicited by as little as 10 pg of Chd RNA, whereas other NC markers (Sox9, Slug, and Xtwi) required more com- plete inhibition of BMP signaling (100–300 pg of Chd RNA). In all injected samples, the BMP-dependent gene Dlx3, and, to a lesser extent, Dlx5, was repressed, as were Sox2 and Otx2. (B) Steeper gradient of Chd Fig. 4. Ectopic expression of AP2␣ converts neural plate into NC. Whole- dosage resulting in complete inhibition of BMP signaling. The results are mount in situ hybridization of probes for Slug, Sox9 (NC), and Sox2 (neural similar to A, except the highest Chd dose (3 ng), which suppresses NC gene plate) to embryos injected at the 16-cell stage with 100 pg of RNA encoding expression, restores the general neural plate marker Sox2 expression but not AP2␣, along with ␤-galactosidase lineage tracer RNA (5-bromo-4-chloro-3- the anterior neural marker Otx2. The completeness of BMP signal inhibition indolyl-␤-D-galactoside, turquoise staining). Discrete domains of intense ec- can be judged by the reduction of Dlx5 transcripts to background levels. (C) topic expression of both NC marker genes was observed (arrows) within the Induction of NC gene expression in the absence of BMP signaling by AP2␣.A neural plate. Endogenous Slug and Sox9 expression is indicated by the arrow- very high Chd dose (4 ng) prevents BMP signaling, induces Sox2, and blocks NC heads. Concomitant repression of neural plate identity also occurred, as induction as in B, whereas coinjection of 100 pg of AP2␣ RNA results in indicated by the reduction in Sox2 expression (arrow). (Magnification: ϫ22.) reactivation of NC markers and suppression of Sox2. The anterior neural plate marker Otx2 continues to be repressed under these conditions, presumably because of Wnt signaling. Dlx5, which shows a lower sensitivity to BMP signal interference than Dlx3 (44, 54). The loss of NC under very high Chd͞very low BIOLOGY BMP signal conditions can be reversed by coinjection of AP2␣ mal expression of Sox9 and Slug initiates as part of the NC DEVELOPMENTAL mRNA, as shown in Fig. 5C. This restored expression of Sox9, pathway, and thus follows that of AP2␣. Another difference in Slug, and Xtwi (endogenous levels of AP2␣ were not measured expression is spatial, with a more lateral boundary for AP2␣, because of interference from injected RNA) and repressed Sox2, particularly in comparison to Slug (Fig. 1). This finding may be indicating that under these conditions AP2␣ was able to trans- at least due to differential regulation by a morphogenetic form neural plate tissue into NC, analogous to what was ob- gradient of BMP signaling. In the artificially generated gradient served in the intact embryo (Fig. 4). of BMP signaling in animal caps expressing Chd, AP2␣ was activated by Wnt signaling at higher BMP levels than Slug, Xtwi, Discussion or Sox9 (Fig. 5). AP2␣ was also able to induce Slug and Sox9 and The results presented here strongly support the conclusion that repress neural plate in the absence of BMP signaling (Figs. 4 and the transcription factor AP2␣ is essential for the specification of 5C). One interpretation of this finding is that AP2␣ expres- NC in Xenopus. Therefore the deficiencies in NC derivatives sion is the primary essential target of BMP signaling in NC found in AP2␣ knockout mice are most likely the result of an specification. initial failure to specify NC progenitors as opposed to subse- An attempt to systematically rationalize these observations is quent abnormalities in migration or differentiation. Comparing summarized in Fig. 6. This model proposes that naive ectoderm the properties and functions of AP2␣ to that of other factors requires BMP signaling to activate relatively low-level AP2␣ furthermore suggests that AP2␣ might occupy a distinct level in expression throughout the ectoderm at the beginning of gastru- the hierarchy of NC regulation. For instance, overexpression of lation, as part of the epidermal specification pathway (34) and ectopic AP2␣ regularly induces NC within the neural plate, that this AP2␣ expression is a prerequisite for subsequent NC whereas Slug (9) and to a lesser extent Sox9 (23) induce NC via induction. This requirement accounts for the observation that expansion of existing domains and do not induce NC within the AP2␣ can induce NC in the midline of the neural plate, but prospective CNS. During development, AP2␣ is initially acti- overexpression of Wnt factors or Sox9 and Slug typically does vated throughout the ectoderm at the beginning of gastrulation not, because the prerequisite AP2␣ levels are absent in this (34) and is subsequently up-regulated in NC, whereas ectoder- region (and bypassed by ectopic AP2␣ introduced into the neural

Luo et al. PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 ͉ 535 Downloaded by guest on September 29, 2021 BMP signal attenuation involves two steps: an initial BMP reduction that results in the silencing of the first of two NC inhibitory factors and, in conjunction with Wnt signaling, in up- regulation of AP2␣ from the basal, epidermal expression to that of NC. This step is followed by a second phase of BMP attenuation (see below). A candidate for the first inhibitor is the homeodomain gene Dlx3. As shown in Fig. 5, this factor is repressed to a baseline level by the same dose of Chd (10 pg), in conjunction with Xwnt-3a that triggers up-regulation of AP2␣. In addition, Dlx3 can antag- onize Wnt͞␤-catenin signaling in early development and prevent NC induction in vivo (54, 55). Transcripts of the Dlx3 gene are excluded from the NC, at least in cranial region, and the Dlx3 gene has an intermediate dependence on BMP signaling, becoming silenced under BMP attenuation conditions that do not inactivate AP2␣ expression (ref. 43; T.L. and T.D.S., unpublished data). After the increase and decrease, respectively, of AP2␣ and Dlx3, a secondary, more severe reduction in BMP signaling (also in conjunction with Wnt signaling) could then lead to activation of the remaining NC program. We have no candidate to mediate this secondary effect, but we predict the control will be negative in nature based on the paradigm of Dlx3 and suggest that another as yet unidentified NC antagonist exists, exhibiting less sensitivity to BMP attenuation compared with Dlx3. In this model, the critical function of Slug, which functions as a transcriptional repressor (56), is to stabilize the silencing of Dlx3 and the other NC inhibitors, which were extinguished as the result of BMP signal attenuation. This finding might account for the capacity of Slug to induce NC in ␣ Fig. 6. A model for NC induction. In the ectoderm, BMP signaling initiates explants when Wnt signaling is provided (9). Sox9, like AP2 ,isa and supports the expression of epidermal AP2␣, at a basal level, and NC transcriptional activator, so these factors could establish an oblig- inhibitors. Because BMP signaling is attenuated by antagonists (Chd), this atory feedback mechanism that is necessary for continued expres- sequentially results in down-regulation of two NC inhibitors, the first of which sion of all three regulatory genes. This process would then lead to may be the homeodomain gene Dlx3. With the first inhibitor removed, Wnt activation of additional, NC-specific target genes and specification ␣ signaling results in up-regulation of AP2 , and, after silencing of the second of this tissue. Identification of these downstream targets will be an inhibitor, induction of other factors such as Slug and Sox9. Slug reinforces the repression of the NC inhibitors, whereas Sox9 and AP2␣ positively regulate important goal of future research. each other and Slug. All three factors are required for full induction and͞or Finally, it is important to recognize that there are other maintenance of NC. See Discussion for details. regulatory factors that can induce or influence NC when exper- imentally expressed. For example, the RNA helicase eIF4AII has been reported to autonomously induce NC in ectodermal plate). For AP2␣ up-regulation and NC initiation to take place, explants (24). We would predict that such a function resides in addition to a Wnt signal, BMP signaling must be partially downstream from the Chd and Wnt signaling events, but could attenuated, symbolized by Chd in Fig. 6. In this model Wnt is either be upstream or downstream from the obligatory feed- meant as a shorthand for factors, including retinoids and fibro- back͞maintenance mechanism that we have portrayed as having blast growth factor signaling, in addition to the Wnt͞␤-catenin three components but almost certainly is more complicated, pathway, and Chd for the various BMP antagonists emanating including other factors that can induce NC in vivo. It should be from the organizer region. As shown in Fig. 5, injection of 10 pg possible to answer these kinds of questions with suitable coex- Chd RNA, along with 300 pg of Xwnt-3a RNA, results in pression experiments in Xenopus, which will continue to be a ␣ essentially complete up-regulation of AP2 . This treatment is productive approach to this complex and important problem. insufficient, however, to activate the other NC markers Sox9, Slug, or Twist, which require a 10-fold higher Chd RNA dose. In We thank Natasha Saint-Germain for technical help. J.-P.S.-J. is sup- view of the different thresholds for up-regulating AP2␣ com- ported by grants from the National Institutes of Health (DE014212) and pared with activation of other NC factors, we propose that this the March of Dimes.

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