Xenopus brain factor-2 controls mesoderm ... - Semantic Scholar

Mechanisms of Development 80 (1999) 15–27

Xenopus brain factor-2 controls mesoderm, forebrain and neural crest development Jose´ Luis Go´mez-Skarmeta a, Elisa de la Calle-Mustienes a, Juan Modolell b, Roberto Mayor a ,* b

a Laboratorio de Biologıá del Desarrollo, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile Centro de Biologıá Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı´ficas and Universidad Autońoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 9 September 1998; revised version received 29 September 1998; accepted 30 September 1998

Abstract The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurula forebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.  1999 Elsevier Science Ireland Ltd. All rights reserved Keywords: Xbf2; Mesoderm; Neural induction; Neural crest; Xenopus

1. Introduction Families of transcription factors present in restricted territories within embryos generate positional identity and control morphogenesis of different tissues. The ‘winged helix’ or ‘forkhead’ family of transcription factors comprise a large and growing group whose members are defined by a common 110 amino acid DNA binding domain (reviewed in Kaufmann and Kno¨chel, 1996). The determination of the crystal structure of the DNA-binding domain of one of its members (HNK-3g) has revealed that this region is a variant of the helix-turn-helix motif (Clark et al., 1993). A 20 amino acid region N-terminal to helix 3 has been shown to be important for binding site specificity (Overdier et al., 1994).

* Corresponding author. Tel.: +34-562-678-7271; fax: +34-562-2712983.

0925-4773/99/$ - see front matter PII S0925-4773 (98 )0 0190-7

Since its original discovery as a region of homology between the rat hepatocyte nuclear factor 3 (HNF-3; Lai et al., 1991) and the Drosophila forkhead gene (Weigel and Ja¨ckle, 1990), the forkhead domain has been found in more than 60 proteins encoded by genes from species ranging from yeast to human (reviewed in Kaufmann and Kno¨chel, 1996). Analysis of expression patterns as well as the phenotypes observed under gain and loss of function conditions have implicated these genes in pattern formation during embryogenesis. Several members of the winged helix gene family are expressed in the central nervous system in different organisms (Ha¨cker et al., 1992; Tao and Lai, 1992; Clevidence et al., 1993; Kaestner et al., 1993; Hatini et al., 1994; Murphy et al., 1994; Pierrou et al., 1994; Lef et al., 1996; Yuasa et al., 1996). The expression of brain factor 1 (bf1) and brain factor 2 (bf2), two members of this family, occupy adjacent domains within the developing mammalian and avian retina and forebrain (Tao and Lai, 1992; Hatini et al., 1994). Loss of function experiments of

 1999 Elsevier Science Ireland Ltd. All rights reserved

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J.L. Go´mez-Skarmeta / Mechanisms of Development 80 (1999) 15–27

bf1 and 2 indicate that they participate in forebrain and kidney development, respectively (Xuan et al., 1995; Hatini et al., 1996). A search for forkhead related genes in Xenopus revealed the existence of a multigene family, all of whose members share the 110 amino acid conserved domain albeit at varying degrees of similarity. They are called Xenopus forkhead domain related genes or XFD (Kno¨chel et al., 1992). Each XFD has a specific spatial pattern of expression and these patterns partially overlap with one another (Dirksen and Jamrich, 1992; Kno¨chel et al., 1992; Ruiz i Altaba and Jessell, 1992). These and other data obtained with several organisms suggest that members of this family participate in complex networks required for pattern formation and tissue differentiation during early embryogenesis. The forkhead region of XFD-6 and -9 share a high identity with that of mouse and chick Brain Factor 2 (BF2). However, outside of this domain they diverge from each other and from BF2 proteins, which suggests that they belong to a BF2 subfamily of forkhead, but are not true homologues of BF2. In addition, a partial cDNA fragment encoding a Xenopus homologue of bf1 has also been identified. This gene is expressed in the prospective telencephalon (Papalopulu and Kintner, 1996). However, no functional analyses have been carried out. In this paper we report the cloning of the Xenopus homologue of the chicken and mouse brain factor 2. We call it Xenopus brain factor 2 (Xbf2). Xbf2 is expressed during early embryonic development in mesoderm, anterior neural plate and neural crest cells. Overexpression experiments in embryos and in isolated tissues allowed us to discern possible functions of Xbf2. Dorsolateral mesoderm expression appears important for patterning of the mesoderm, as it represses ventral mesoderm genes and promotes the expression of dorsolateral genes such as XmyoD. Anterior neural plate expression of Xbf2, which represses Xbf1, could be necessary for patterning of the forebrain. Overexpression and grafting experiments suggest that Xbf2 represses the migration of neural crest cells. All of these functions may be mediated by an interference with BMP-4 signaling.

2. Results 2.1. Molecular cloning of Xbf2 In a low stringency screen of a Xenopus gastrula (stages 10.5–11.5) cDNA library we identified a 1.8 kb cDNA clone which corresponded to a gene with strong expression in the region of the neural folds of stage 17 embryos (see below). This cDNA encoded a protein that was a new member of the winged helix or forkhead family of transcription factors. Moreover, the primary structure of this protein outside of the winged helix domain indicated that, most likely, it was the Xenopus homologue of BF2 from

mouse and chicken (Hatini et al., 1994; Yuasa et al., 1996). Comparison between MBF2, CBF2 and XBF2 showed 97% identity and 99% similarity within the forkhead domain and 50% identity and 70% similarity outside this domain (Fig. 1). 2.2. Xbf2 gene is expressed in mesoderm, anterior neural plate and prospective neural crest cells The earliest detectable expression of Xbf2, as detected by whole-mount in situ hybridization to developing embryos, occurs at gastrula stage 10 (Niewkoop and Faber, 1967) in a dorso-lateral gradient at the marginal zone (Fig. 2A). This expression continues in the prospective uninvoluted mesoderm (Fig. 2B), but it becomes undetectable during gastrulation once the mesoderm has involuted. No expression is detected in the ventral marginal zone. At neurula stage 12.5, expression occurs in the anterior neural plate and is localized close to the dorsal midline, which corresponds to the prospective diencephalon and eye field (Fig. 2C, arrow). At this stage faint expression is also observed in the anterior neural folds (Fig. 2C, arrowhead). As neurulation proceeds, Xbf2 expression becomes stronger in the anterior neural plate and in the anterior neural folds (Fig. 2D,E). At stage 17, the subdivision, within the cephalic neural crests, of the prospective mandibular, hyoid and branchial arches can be distinguished (Fig. 2E, arrowhead). At a more advanced stage, the expression of Xbf2 becomes detectable in the optic vesicle (Fig. 2F, arrow), while the gene is switched off in the prospective neural crest at the time these cells begin migration (Fig. 2F). At this stage, expression also takes place in the paraxial mesoderm (Fig. 2F, arrowhead). 2.3. Ectopic expression of Xbf2 induces a partial secondary axis and enlarges the neural plate To analyze the function of Xbf2 in Xenopus development, we injected 1- or 2-cell stage embryos with different amounts of Xbf2 mRNA. Injection of 4 ng of mRNA in 1cell stage embryos induced formation of a partial secondary axis and a significant reduction of the head (Fig. 3A). When the same amount of mRNA was injected in one blastomere of a 2-cell stage embryo, a partial secondary axis was the most frequent outcome. Injection of 0.25 ng of mRNA in one cell of a 2-cell stage embryo produced a small expansion of the neural plate in approximately half of the embryos (not shown) and, with a low frequency (5%), a secondary axis. A reduction of head size was also observed in most embryos when this amount of mRNA was injected at the 1cell stage. The secondary axis produced under this condition expressed weak but detectable levels of neural crest markers such as Xtwist (Hopwood et al., 1989b) and Xslug (Mayor et al., 1995) (Fig. 3B,C, arrowheads). The expanded neural plate was recognized by the expression of the neural plate marker Xsox2 (Mizuseki et al., 1998) (Fig. 3D, arrowhead). High amounts of injected mRNA (4 ng) lead to an expanded


ventral ectopic expression of Xsox2, which was apparently associated with the duplicated axis present in most of these embryos (Fig. 3E, arrow). The ectopic axis lacked the anterior-most tissues, such as eyes or cement gland (not shown).

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In addition to neural markers, Xbf2 overexpression also affected the paraxial mesodermal marker XMyoD (Hopwood et al., 1989a), as its domain of expression was expanded (Fig. 3F, arrow).

Fig. 1. Comparison of the sequences of mouse, chicken and Xenopus BF2 proteins (accession numbers Q61345, U47276, AJO11652). The forkhead domain is under and overlined. Note the high similarity between all BF2 proteins outside the forkhead domain.

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2.4. Expression of Xbf2 in the ventral marginal zone confers on it neural inducer activity The ectopic expression of neural plate markers and the induction of a partial secondary axis in Xbf2 injected embryos might be due to a dorsalization of the ventral meso-

derm, which could endow it with neural inducer activity, and/or to a direct neuralization of the ectoderm. To investigate the first possibility, we analyzed whether the expression of Xbf2 in ventral mesoderm was sufficient to allow this tissue to induce neural plate in naive ectoderm in vitro. Animal caps and dorsal and ventral marginal zones were

Fig. 2. Expression pattern of Xbf2 in Xenopus embryos at different developmental stages. (A–E) Dorsal views, anterior is up; (F) lateral view, anterior is up. (A) At stage 10 Xbf2 is initially detected in the dorso-lateral prospective mesoderm. (B) Stage 11 embryo showing Xbf2 expression in the uninvoluted dorsolateral prospective mesoderm. (C) Stage 12.5; at this stage expression appears in the anterior neural plate (arrow) and in the anterior neural folds (arrowhead). (D,E) At stages 14 and 17, respectively, Xbf2 expression becomes stronger in the anterior neural plate (arrows) and in the anterior neural folds (arrowheads). At stage 17, Xbf2 is clearly expressed in the segmented premigratory cephalic neural crest (arrowhead). (F) Stage 20 embryo showing Xbf2 expression in the eye (arrow) and in the paraxial mesoderm (arrowhead). Note that at this stage the expression in the migratory neural crest has disappeared.


dissected at stage 10, were conjugated in different combinations and were cultured until the equivalent of stage 17, when expression of the neural plate marker Xsox2 was analyzed. Animal caps cultured alone (Fig. 4A) or conjugated with ventral marginal zone (Fig. 4C) never expressed

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Xsox2; but animal caps conjugated with ventral marginal zone taken from embryos injected with 4 ng of Xbf2 mRNA, did express Xsox2. (Fig. 4D). This inducing activity was similar, although not as strong, as the expression induced by dorsal marginal zone (Fig. 4B). As expected,

Fig. 3. Effect of Xbf2 overexpression on embryonic development. Embryos were injected at the 1-cell stage (A) or 2-cell stage (B–F) with different concentrations of Xbf2 mRNA and 0.3 ng of lacZ mRNA as a lineage marker (blue dots). Embryos were cultured until stage 16 or 25, when the expression of different genes was analyzed by in situ hybridization. Views are from the dorsal side, anterior is up. (A) Reduced head and absence of anterior structures, such as eyes or cement gland (arrow) and partial secondary axis (arrowhead) in an embryo injected with 4 ng of Xbf2 mRNA (95%, n = 22). (B–D) Low level of Xtwist (B) or Xslug (C) expression in the secondary axis of an embryo injected with 0.25 ng of Xbf2 mRNA. This duplicate axis is clearly detected by Xsox2 (D) expression (arrowheads point to the double axis that were found in about 5% of the injected embryos, n = 32, 43 and 54, respectively). (E) Ectopic expression of Xsox2 close to the ventral region of an embryo injected with 4 ng of Xbf2 mRNA (arrow; 93%, n = 54). (F) Embryos injected with 4 ng of Xbf2 mRNA also showed expansion of the XMyoD domain in the injected side (arrow; 75%, n = 40).

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ventral marginal zones taken from injected embryos and cultured alone did not express Xsox2 (Fig. 4D, inset). These results indicate that expression of Xbf2 in the prospective ventral mesoderm changes its inducing capacity, possibly by transforming it to a more dorsal mesoderm. As shown below, Xbf2 can also directly neuralize the ectoderm. 2.5. Xbf2 inhibits ventral mesodermal genes We examined whether the expression of several mesodermal genes was affected by the injection of 4 ng of Xbf2 mRNA in the equatorial region of one blastomere of the 2-cell stage embryo (arrowheads in Fig. 5 point to the injected sites in all embryos). The expression of the dorsal mesodermal genes gsc (Cho et al., 1991) (Fig. 5A) and Xlim 1 (Taira et al., 1992) (Fig. 5B) was not affected. However,

XMyoD expression, which is normally transcribed at a more lateral position, was strengthened in the injected side (Fig. 5C) and clearly expanded at later stages (Fig. 3F). Ventral and lateral injections of Xbf2 mRNA did not affect the expression of Xvent2, a slightly more ventral mesodermal gene (Onichtchouk et al., 1996) (Fig. 5D). However, dorsal injections weakly inhibited Xvent2 expression but only at its more dorsal domain (not shown). In contrast, the more ventral genes Xwnt8 (Christian et al., 1991; Smith and Harland, 1991) and Xvent1 (Gawantka et al., 1995) were strongly inhibited in the injected side (Fig. 5E,F, respectively). Since Xvent1 and Xvent2 are downstream genes of BMP-4 (Gawantka et al., 1995; Onichtchouk, et al., 1996), we analyzed the expression of BMP-4 in the Xbf2 injected embryos. Xbf2 overexpression apparently reduced the expression of BMP-4 (Fig. 5G) although not as strongly as that of other ventral genes. Similarly, Xmsx1, another BMP4 downstream gene (Maeda et al., 1997; Suzuki et al., 1997), was also inhibited at the injected site when Xbf2 was overexpressed (Fig. 5 H). Taken together, these results suggest that Xbf2 participates in the specification of the lateral prospective mesoderm (XMyoD domain), and that its overexpression leads to a lateralization of the ventral mesoderm. 2.6. Direct neural induction by Xbf2

Fig. 4. Xbf2 affects the neural inducer capacity of the mesoderm. Different tissues were dissected from control or Xbf2 injected stage 10 embryos, conjugated and cultured until the equivalent of stage 17, when Xsox2 expression was analyzed. (A) Control animal caps showed no Xsox2 expression (n = 15). (B) All the conjugates of animal cap and dorsal marginal zone strongly expressed Xsox2 (n = 14). (C) Animal caps conjugated with ventral marginal zone did not express Xsox2 (n = 17). (D) Most of the conjugates of animal cap and ventral marginal zone taken from embryos injected with 4 ng of Xbf2 mRNA expressed Xsox2 (70%, n = 13), although at lower levels than conjugates of animal cap and dorsal marginal zone. In contrast, the ventral marginal zone alone taken from injected embryos did not express Xsox2 (inset; n = 10).

We tested whether Xbf2 could neuralize ectodermal cells directly, that is, in the absence of inducing signals emanating from the mesoderm. Embryos were injected at the 1-cell stage with either 0.25 ng or 4 ng of Xbf2 mRNA. Animal caps were dissected at stage 9 and cultured until the equivalent of stage 17, when Xsox2 expression was analyzed. Uninjected animal caps (Fig. 6A, inset) or caps injected with 0.25 ng of Xbf2 mRNA did not express Xsox2, while caps from embryos injected with 4 ng of Xbf2 mRNA strongly expressed this neural plate marker (Fig. 6A). These caps did not express the mesodermal marker XmyoD (not shown). Since neural induction apparently requires the suppression of BMP-4 signals in the ectoderm (reviewed in Hemmati-Brivanlou and Melton, 1997), we examined whether expression of Xmsx1 was affected in embryos injected with 4 ng of Xbf2 in one cell of the 2cell stage. At stage 14, its expression was suppressed in the posterior ventral region, where it should be expressed very strongly (not shown), and at the neurula stage, when this gene is mainly expressed in the neural fold area, its expression was also strongly reduced (Fig. 6B, arrowhead). The inhibition of Xmsx1 expression suggests that XBF2 inhibits BMP signaling. As a consequence, ectodermal cells are neuralized. 2.7. Patterning the forebrain by mutual inhibition of Xbf1 and Xbf2 In mouse and chicken, bf1 and bf2 genes are expressed in adjacent domains within the developing retina and forebrain


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Fig. 5. Effect of Xbf2 mRNA on mesodermal development. Embryos were injected in one cell at the 2-cell stage in the equatorial region with 4 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA and cultured until stage 10.5–11.5, when the expression of different mesodermal markers were analyzed. Arrowheads point to the injected sites. Neither gsc (A) nor Xlim1 (B) expression were affected by Xfb2 overexpression (100%, n = 18 and 13, respectively). (C) XMyoD expression was strengthened in the lateral mesoderm in the injected side (60%, n = 34). (D) Ventral or lateral Xbf2 injections did not affect Xvent2 expression (94%, n = 48). In contrast, overexpression of Xbf2 strongly inhibited the expression ofXwnt8 (E; 95%, n = 45) and Xvent1 (F; 92% n = 23) and weakly that of BMP-4 (G; 80% n = 15) and Xmsx1 (H; 58%, n = 12) at the injection site.

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(Tao and Lai, 1992; Hatini et al., 1994). We find that in the Xenopus forebrain region these genes are also expressed in adjacent domains, that of Xbf1 being more anterior than that of Xbf2 (Fig. 7A). This complementary pattern of expression suggests a mutually inhibitory regulation between these two genes. To test this, we injected one blastomere of 2-cell stage embryos with low amounts of Xbf2 mRNA (0.25 ng, to avoid secondary axis induction) and we examined the expression of Xbf1 (Papalopulu and Kintner, 1996). The injected side showed a strong reduction or elimination of Xbf1 expression (Fig. 7C, arrowhead). Similarly, injection of 0.5 ng of Xbf1 mRNA inhibited Xbf2 expression (Fig. 7B, arrowhead). This mutual inhibition could help patterning the forebrain. We also analyzed whether other forebrain-expressed genes were affected by Xbf2 overexpression. Xcpl1, a gene expressed at the anterior-most region of the neural fold (anterior to Xbf1, Good et al., 1990), was also inhibited (Fig. 7D, arrowhead). However, Xotx2, a gene expressed in a broad anterior area that includes the regions of expression of Xbf1, Xbf2 and Xcpl1, was not affected (not shown). The inhibition of Xbf1 and Xcpl1 suggests that the overexpression of Xbf2 could transform the telencephalic territory into a diencephalic region, while it maintains an anterior character, as determined by Xotx2 expression. 2.8. Xbf2 inhibits neural crest migration Xbf2 is expressed in the premigratory neural crest cells (Fig. 2C–E) and its expression is completely extinguished once these cells start to migrate. Thus, we examined whether Xbf2 extinction was required for migration of neural crest cells by analyzing the effect of Xbf2 overexpression on this process. Embryos injected in one blastomere at the 2-cell stage with 0.25 ng of Xbf2 mRNA were analyzed for the neural crest marker Xtwist once the neural crest cells began migrating (stage 25). At the uninjected side, expression of Xtwist and migration of the crest cells were normal (Fig. 8A, arrowhead). However, at the injected side, levels of Xtwist expression were normal but migration did not occur (Fig. 8B, arrow). To rule out the possibility that the overexpression of Xbf2 was affecting the migratory path of the crest cells, stage 10 ectoderm injected with lacZ mRNA, as a lineage marker, with or without Xbf2 mRNA was grafted into the prospective neural crest region of normal stage 11 embryos. Crest cells from the grafts migrated normally when they contained only the lineage marker (Fig. 8C, arrowhead), but did not do so when they contained in addition Xbf2 mRNA (Fig. 8D, arrow). Moreover, Xsox2 expression was not detected in these grafts (Fig. 8D, inset) or in grafts cultured in isolation (not shown), indicating that the graft expressing Xbf2 was not transformed into neural plate. These results thus suggest that extinction of Xbf2 expression is a requisite for the migration of the neural crest cells.

3. Discussion 3.1. Expression of Xbf2 Avian and mouse bf1 and bf2 genes play important roles in patterning the forebrain, retina and kidney (Xuan et al., 1995; Hatini et al., 1996). In the forebrain, they probably specify the positional identity of the telencephalic neuroepithelium and determine its proliferative potential during brain development (Hatini et al., 1994; Xuan et al., 1995). We now find that the expression of the Xenopus homologue Xbf2 in the anterior neural plate is compatible with this function. However, Xbf2 is also expressed in two other regions, which suggests additional functions. The expression in premigratory neural crest cells is similar to the expression of other Xenopus forkhead genes (XFD-3, -6, 9, -10; Lef et al., 1996), while the early expression in prospective mesoderm is a novel one for a brain factor gene. Our data suggest that XBF2 acts as a transcriptional repressor. Indeed other forkhead proteins, such as Qin, Genesis, CWH-1, CWH-3 and CWH-3, behave as transcriptional repressors (Li et al., 1995; Sutton et al., 1996; Freyaldenhoven et al., 1997). 3.2. Xbf2 participates in mesodermal development The dorso-ventral patterning of the mesoderm is a complex process in which dorsal signals (e.g. noggin, chordin, Frzb-1) interact directly with ventral signals (BMP-4, Wnt8) (Holley et al., 1996; Piccolo et al., 1996; Zimmerman et al., 1996; Leyns et al., 1997; Wang et al., 1997; reviewed in Hemmati-Brivanlou and Melton, 1997 and Moon et al., 1997). As a result, dorso-ventral gradients of BMP-4 and Xwnt8 are established. These locally activate transcription factors which specify distinct mesodermal territories. Some of these factors are encoded by the Xmsx1, Xvent1 and Xvent2 genes and have ventralizing activities (Gawantka et al., 1995; Onichtchouk et al., 1996; Dosch et al., 1997; Maeda et al., 1997; Suzuki et al., 1997; Onichtchouk et al., 1998). It has been proposed that the combination of Xvent1 and Xvent2 functions specifies different mesodermal cells (Onichtchouk et al., 1998). Thus, in ventral mesoderm both genes are expressed and this suppresses notochord and muscle differentiation. In dorsolateral mesoderm only Xvent2 is expressed and this inhibits notochord but allows muscle differentiation. The overexpression of Xbf2 inhibits strongly Xvent1 but very weakly Xvent2, and this only at its dorsal-most domain of expression. As a consequence, the territory that expresses Xvent2 alone is expanded and this should be compatible with muscle differentiation. Indeed, the domain of expression of XMyoD is expanded in the Xbf2 injected embryos. In contrast, Xbf2 overexpression does not induce ectopic expression of dorsal genes such as gsc and Xlim1. A possible interpretation of these results is that Xbf2, which is expressed in the dorsal and dorso-lateral meso-


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Fig. 6. Direct neuralization of the ectoderm by Xbf2 expression. Animal caps were dissected from stage 9 embryos and cultured until the equivalent of stage 17, when Xsox2 expression was analyzed. (A) Animal caps taken from embryos injected with 4 ng of Xbf2 mRNA at the 1 cell stage. Xsox2 is expressed in these caps (58%, n = 12). Note also that these caps are not elongated as could be expected if dorsal mesoderm was induced. Inset: control animal caps showing no Xsox2 expression (100%, n = 13). (B) Embryos were injected in one blastomere at the 2-cell stage with 4 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA. Xmsx1 expression was analyzed at stage 16 embryos. Notice the absence of Xmsx1 expression at the injected side (arrowhead; 83%, n = 18). Fig. 7. Xbf2 participates in patterning of the forebrain. All the embryos are shown from anterior side. The weaker expression of Xbf2 in neural crest cells is not visible in A and B due to low staining development. (A) Double in situ hybridization of an stage 17 embryo. Blue (arrow) corresponds to Xbf2 expression and purple (arrowhead) denotes Xbf1 expression. (B) Xbf2 expression in an stage 17 embryo injected in one blastomere of a 2-cell stage embryo with 0.5 ng of Xbf1 mRNA and 0.3 ng of lacZ mRNA. Notice the complete inhibition of Xbf2 expression at the injected side (58%, n = 12). (C) Embryo injected in one blastomere of a 2-cell stage embryo with 0.25 ng of Xbf2 mRNA and 0.3 ng lacZ mRNA and cultured until stage 17. Xbf1 expression is suppressed in the injected side (72%, n = 22). (D) Similar embryo as in C, but expression of Xcpl1 was analyzed. Notice the inhibition of Xcpl1 in the injected side (53%, n = 15).

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Fig. 8. Overexpression of Xbf2 blocks neural crest migration. (A,B) One blastomere of a 2-cell stage embryo was injected with 0.25 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA, was cultured until stage 25 and the expression of Xtwist was analyzed. (A) Right side view of the embryo shown in (B). (A) View of uninjected right side. Notice the normal expression of Xtwist and the migration of the neural crest cells (arrowhead). (B) View of injected left side. Notice that the cells still express Xtwist but they do not migrate normally, most of them remaining in the dorsal side of the embryo (arrow; 58%, n = 17). (C,D) Grafts of ectodermal tissue, taken from stage 10 embryos previously injected with 0.3 ng of lacZ mRNA without (C) or with (D) 0.25 ng of Xbf2 mRNA, were placed in the prospective neural crest region of a stage 11 embryo. The embryos were cultured until stage 28 and b-galactosidase was analyzed. (C) Control embryo that received a graft containing lacZ mRNA. Note in C the normal migration of the grafted cells (arrowhead; 87%, n = 15) and in D the permanence of the labeled cells in the original site of the graft (arrow; 87%, n = 15). Inset: a similar, but more lateral (to better distinguish b-galactosidase staining from Xsox2 expression) graft, does not show Xsox2 expression (100%, n = 12).

derm, specifies the dorso-lateral mesoderm but is incapable of inducing a more dorsal fate by itself. The dorsalizing activity of Xbf2 could then be due to an interference with BMP-4 and/or Xwnt8. The observation that XBF2 inhibits the expression of BMP-4 and of its downstream genes and induces neuralization in caps suggests that XBF2 antagonizes BMP-4 signaling. The inhibition of Xwnt8 caused by Xbf2 overexpression could then be due to this interference with BMP-4 signaling, since a dominant negative BMP-4 receptor suppresses Xwnt8 expression (Schmidt et al., 1995; Hoppler and Moon, 1998). In contrast to our observed inhibition of Xwnt8 together with an expansion of the XMyoD domain, it has been shown that a dominant negative form of Xwnt8 inhibits XMyoD (Hoppler et al., 1996). This apparent discrepancy may be explained if this dominant negative has a broader than

expected range of activity and also antagonizes other Xwnt molecules (Hoppler et al., 1996) that may participate in XMyoD activation. In addition, XBF2 may activate XMyoD directly or via another gene required for its transcription. 3.3. Xbf2 and anterior neural plate development In chicken and mouse, bf1 is expressed in the telencephalon and in the rostral half of the retina, adjacent to the domains of bf2 expression within the diencephalon and posterior retina (Tao and Lai, 1992; Yuasa et al., 1996). Similarly, Xbf1 and Xbf2 have adjacent domains of expression within the forebrain. Our results suggest that, within the forebrain, each Xbf gene is required to specify the territory where it is expressed, as an expansion of the domain of one


gene leads to the reduction of the domain of the other, probably by mutual repression. In addition, Xotx2 expression is not affected in Xbf2 injected embryos. This suggests that the anterior neural plate could be transformed into a more posterior territory, although still within the Xotx2 expression domain.

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mesoderm, anterior neural plate and neural crest development.

4. Materials and methods 4.1. Molecular cloning of the Xbf2 gene

3.4. Xbf2 interferes with neural crest cell migration Xbf2 is expressed in the neural crest region up to the time crest cells begin to migrate. Our results, obtained in overexpression and grafting experiments, suggest that Xbf2 can repress neural crest migration. As XBF2 exhibits a repressor activity in other tissues, it may also inhibit genes which are necessary for neural crest cell migration. This could explain the switching off of Xbf2 before crest cells start to migrate. This migration requires an epithelial to mesenchymal transition of the cells, and although this process has been extensively studied and many molecules have been proposed to be involved in it, not much is known about the signal that triggers such change (see review in Mayor et al., 1998). Thus, it is possible that XBF2 represses a gene which is required for the initiation of the epithelial to mesenchymal transition that precedes neural crest migration. 3.5. Xbf2 and BMP signaling Ectopic neural development upon Xbf2 overexpression can be due to the simultaneous dorso-lateralization of the ventral mesoderm and the neuralization of ectoderm. Both effects can be explained by XBF2 interference with BMP-4 signaling. Note that the combined action of organizing molecules (noggin, chordin and follistatin), which interfere with BMP-4 by direct binding, and XBF2, which represses transcription of BMP-4, may help establish the dorso-ventral gradient of BMP-4 activity necessary for proper development of the mesoderm. The complementary domains of expression of Xbf1 and Xbf2 may also be related to BMP-4 signaling. In fact, it has been found that the cement gland, an anterior marker, is induced at slightly higher levels of BMP activity than those required for neural plate induction (Wilson et al., 1997). Thus, different levels of BMP-4 signaling may establish the adjacent territories of Xbf1 and Xbf2 expression. Subsequently, their mutual repression and the interference of XBF2 with the BMP-4 pathway may help refine their domains of expression. Finally, it is of interest that different BMPs are expressed during neural crest specification, migration and development (Liem et al., 1995; Schmidt et al., 1995). It is possible that XBF2 represses some BMPs and/or their downstream factors and that this repression is required for proper development and migration of the neural crest cells. Thus, it is possible that XBF2 blocks BMP-4 signaling in all tissues where it is expressed and this interference is necessary for

Approximately 106 phages from a Xenopus gastrula (stages 10.5–11.5) UniZap-XR cDNA library (Cho et al., 1991) were screened at low stringency with a Drosophila abrupt probe, in hybridization buffer (40% formamide, 4× SSC, 5× Denhartd’s, 0.1% SDS, 20 mM phosphate buffer (pH 6.8), 0.1 mg/ml salmon sperm DNA and 0.1% sodium pyrophosphate) at 42°C. Filters were washed with 2× SSC, 0.1% SDS at 42°C. Seven positives were obtained, were purified and the corresponding plasmids were excised. Unexpectedly, one of the cDNAs encoded the Xenopus homologue of the mouse and chicken brain factor 2 genes. 4.2. DNA sequencing Sequencing was performed in an automatic DNA sequencer using ABI chemistry and T3, T7 and custom synthesized oligonucleotides (ISOGEN Bioscience BV, Maarsesen, The Netherlands) as primers. Consensus sequences were assembled and analyzed with the University of Wisconsin GCG software packages (Devereux et al., 1984). 4.3. Whole-mount in situ hybridization and X-Gal staining Antisense RNA probes were prepared from Xbf-2 cDNA and Xslug (Mayor et al., 1995), gsc (Cho et al., 1991), Xlim1 (Taira et al., 1992), Xsox2 (kindly provided for Dr. R.M. Grainger), Otx-2 (Blitz and Cho, 1995; Pannese et al., 1995), Xtwist (Hopwood et al., 1989b), XMyoD (Hopwood et al., 1989a), Xvent1 (Gawantka et al., 1995), Xvent2 (Onichtchouk et al., 1996), Xcpl1 (Good et al., 1990), Xmsx1 (Maeda et al., 1997; Suzuki et al., 1997), Xwnt8 (Christian et al., 1991; Smith and Harland, 1991) and BMP-4 (Schmidt et al., 1995) cDNAs using digoxigenin or fluoresceine as labels. Specimens were prepared, hybridized and stained by the method of Harland (1991) with modifications (Mancilla and Mayor, 1996). Double in situ hybridizations were made as described in Go´mez-Skarmeta et al. (1998). 4.4. In vitro RNA synthesis and plasmid constructs All the vectors were linearized and transcribed as described by Harland and Weintraub (1985) with GTP cap analog (New England Biolabs). SP6, T3 or T7 RNA polymerases were used. After DNase treatment, RNA was extracted with phenol-chloroform and precipitated with ethanol. mRNA to be injected was resuspended in water.

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4.5. Embryos, explants, conjugates and microinjections Xenopus embryos were obtained as described previously (Mayor et al., 1993) and staged according to Niewkoop and Faber (1967). Animal caps, marginal zone, conjugates and grafts were performed as previously described (Mancilla and Mayor, 1996; Mayor et al., 1997). Synthetic mRNA was injected at the 1- or 2-cell stage embryos in 8–12 nl volume as described (Mayor et al., 1993).

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