Early patterning of the prospective midbrain–hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos

Mechanisms of Development 109 (2001) 225–239 www.elsevier.com/locate/modo

Early patterning of the prospective midbrain–hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos Jun Shinga a,b, Mari Itoh a,b, Koichiro Shiokawa a, Sumiko Taira a, Masanori Taira a,b,* a

b

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan Received 14 November 2000; received in revised form 9 July 2001; accepted 16 August 2001

Abstract The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain–hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 was detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm that is demarcated by the expression of the gene Xbra. Expression of XHR1 was detected much earlier than that of other known MHB genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra altered the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhanced En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, we propose that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Xenopus; Neuroectoderm; Central nervous system; Patterning; Midbrain–hindbrain boundary; Basic helix–loop–helix ; Hairy; Enhancer of split; HES; Xenopus HES-related 1

1. Introduction In vertebrates, the brain region arises from the anterior portion of the neural plate, and is induced from the dorsal ectoderm by the underlying dorsal endomesoderm, a region named the Spemann organizer in the case of amphibians (Spemann and Mangold, 1924). Recent molecular studies have revealed various regulatory factors responsible for neural induction, neural and neuronal differentiation, and patterning along the anteroposterior (AP) and dorsoventral (DV) axes (Doniach, 1995; Harland and Gerhart, 1997; Sasai, 1998; Chitnis, 1999; Gamse and Sive, 2000). In Xenopus, spatiotemporal expression analysis of these genes shows that neural patterning along the AP axis has already begun at the early gastrula stage (Gamse and Sive, 2000). The earliest genes known to be expressed for brain * Corresponding author. Tel.: 181-3-5841-4434; fax; 181-3-5841-4434. E-mail address: [email protected] (M. Taira).

patterning include Xotx2, which is expressed at early to mid gastrula stages and specifies the anterior part of the future brain. This region gives rise to the forebrain and midbrain at later stages (Blitz and Cho, 1995; Pannese et al., 1995). The region posterior to the Xotx2 domain appears to be specified by the posterior expression at the neurula stage of genes such as Xgbx-2 (von Bubnoff et al., 1996) as has been shown in the chick and mouse (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). Thus, the future brain region seems to be divided at least into the anterior and posterior portions at these early stages. However, it is not yet clear as to what extent the ANE is patterned at the early to late gastrula stages as well as at the neurula stage, nor is it clear how early one can trace in molecular terms the appearance of each of the brain regions that will form later. One of the reasons progress has been impeded in this research area is that there is an insufficient number of molecular marker genes identified

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0925-477 3(01)00528-7

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that define specific regions in the early neuroectoderm, where no morphologically defined compartments have yet formed at gastrula to neurula stages. To address this issue, we screened a cDNA library constructed with dissected anterior neuroectoderm (ANE) tissue from Xenopus late gastrulae on the basis of gene expression patterns and random sequencing, and identified a gene encoding a basic helix–loop–helix (bHLH) transcriptional repressor, named Xenopus Hairy and enhancer of split (HES)-related 1 (XHR1). XHR1 belongs to the prolinebHLH-WRPW gene family, and was found to be expressed specifically in the midbrain–hindbrain boundary (MHB) region at the tailbud stage. The MHB region is thought to function as an organizing center of neural patterning, since this region can induce the optic tectum in the midbrain and the cerebellum in the hindbrain (Wassef and Joyner, 1997). Thus, the formation of the MHB is likely to be a critical initial event in brain patterning. Several genes encoding transcription factors such as Pax-2/-5 and En-1/-2, and signaling molecules such as Wnt-1 and FGF8, have been shown to be specifically expressed in the MHB region, and are therefore implicated in the formation and maintenance of this region (McMahon and Bradley, 1990; Urbanek et al., 1994; Hanks et al., 1995; Lee et al., 1997; Lun and Brand, 1998). In this study, we analyzed the detailed spatiotemporal expression pattern of XHR1, and found that the presumptive MHB region is already demarcated by the expression of XHR1 at the early gastrula stage before the expression domain of Xotx2 comes into contact with that of Xgbx-2, contrary to general expectation from the studies of the chick and mouse embryos (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). We also performed functional analyses using mRNA injection, which suggest that XHR1 is involved in the early patterning of the presumptive MHB region at the early gastrula stage.

From the screening of a Xenopus gastrula cDNA library, we obtained three types of XHR1 clones of about 1.1 kb, each encoding a protein of 180 amino acids. We predicted the first ATG as the translation initiation codon based on its upstream sequence TTACCATG, which fits the Kozak’s rule (Kozak, 1987), though there is no in-frame stop codon in the 5 0 region upstream of this ATG. When these three clones were compared with the original clone initially isolated from the ANE cDNA library, they were found to have 100, 99.8, or 98.7% identity at the amino acid level, corresponding to 0, 1, or 5 amino acid substitutions, respectively. The three clones were named as XHR1-A, XHR1-A 0 , and XHR1-B, respectively (Fig. 2A). We assume that the difference of five amino acid residues between XHR1-A and XHR1-B reflects the pseudotetraploid nature of Xenopus laevis genome (Bisbee et al., 1977). The difference of one amino acid between XHR1-A and XHR1-A 0 may be due to polymorphism in the same allele. XHR1 has a well-conserved proline-bHLH domain (Fisher and Caudy, 1998), a loosely conserved ‘Orange’

2. Results 2.1. Isolation of a new member of the bHLH-WRPW family, XHR1 We constructed a cDNA library of Xenopus ANE, which was isolated from late gastrulae (stages 12–12.5) when the invaginated mesoderm was about to be segregated into prechordal and chordal areas (Fig. 1A; Nieuwkoop and Faber, 1967). After screening this library, we obtained several clones whose expression was restricted to the ANE at gastrula stages or to the anterior central nervous system (CNS) at tailbud stages. When cDNA inserts were isolated and sequenced, one of these genes, named XHR1, was found to encode a protein homologous to members of the HES family. By Northern hybridization analysis, XHR1 mRNA was detected as a single band of 1.2 kb in RNA from the ANE but not in samples from other regions of stages 12– 12.5 embryos (Fig. 1B).

Fig. 1. Northern hybridization analysis of XHR1. (A) Dissection of gastrulae (stages 12–12.5) performed for isolation of anterior neuroectodermal and prechordal plate regions. (B) Northern blot profiles. XHR1 was specifically expressed in the ANE. ANE, anterior neuroectodermal region; PCP, prechordal plate region including some adjacent endoderm; Carcass, the residual part of embryos after elimination of ANE and PCP; Whole, whole embryos (stages 12–12.5). The original XHR1 clone (0.7 kb) was used as probe. Ethidium bromide-stained 18S rRNA is a loading control.

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Fig. 2. Sequence analysis of XHR1. (A) Predicted amino acid sequences of XHR1-A and XHR1-B. bHLH domain, Orange domain, and WRPW sequence are shadowed. Dots in XHR1-B sequence indicate the same amino acids as in XHR1-A sequence. A proline residue at position 148 in XHR1-A (marked by an asterisk) is substituted to leucine in XHR1-A 0 . (B) Sequence alignment of XHR1 and other HES family members from Drosophila, zebrafish, Xenopus, and mouse. Amino acid numbers at the top refer to XHR1. Only bHLH domains, Orange domains, and WRPW motifs are shown. White letters in black, more than 85% identity in the column; shadowed letters, more than 50% similarity in the column; dashed lines, gaps for alignment; asterisk, conserved proline residues in the basic region. (C) Phylogram of bHLH domains of XHR1-A and other HES family members aligned in (B). (D) Structures of wild-type and mutant XHR1-A constructs for microinjection experiments. In XHR1-VP16, a region C-terminal to bHLH domain is substituted to VP16 transactivation domain (VP16 AD). In mb-XHR1, the basic region is point-mutated (depicted by an asterisk). In XHR1-DWRPWV, the C-terminal WRPWV sequence is deleted. The GenBank accession numbers for sequences used are: zebrafish HER-5 (X97627), Xenopus ESR-4 (AF137073), Xenopus ESR-5 (137072), mouse HES-1 (D16464), Xenopus hairy1 (U36194), Drosophila hairy (X16632), Drosophila deadpan (S48025), mouse HES-2 (AB009967), mouse HES-3 (D32200), mouse HES-5 (D32132), mouse Hey1/HRT1 (AF172286), Drosophila E(spl)m8 (X16553). The nucleotide sequences of XHR1-A, XHR1-A 0 , and XHR1-B have been deposited in DDBJ/GenBank/EMBL under accession numbers AB071432, AB071433, and AB071434, respectively.

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Fig. 3. Whole-mount in situ hybridization analysis of XHR1 expression during early development. (A) Dorsal view of a stage 10.5 embryo. The animal side is to the top. XHR1 expression is detected in a narrow area of the dorsal side. (B) Comparison of the expression domains of Sox-2 and XHR1. A stage 10.5 embryo was bisected sagittally into two hemispheres and was then hybridized with Sox-2 (left panel) or XHR1 (right panel). The animal side is to the top. The inset shows the higher magnification view of XHR1 expression in the sensorial layer. (C) Dorsal view of a stage 12 embryo. Anterior is to the top. The expression domain is sharpened and reduced in the midline, giving a V-shaped form. (D) Dorsal view of a stage 15 embryo. Anterior is to the top. XHR1 expression is confined to a limited area in the anterior neuroectoderm. (E) Dorsal view of a stage 17 embryo stained with XHR1 (brown) and En-2 (purple). Anterior is to the top. The expression of both XHR1 and En-2 overlaps. (F) Dorsoanterior view of a cleared stage 18 embryo. XHR1 is expressed in the prospective eye region (white arrowhead) and possible pineal gland region (white arrow) too. (G) Lateral view of a cleared stage 28 embryo. Anterior is to the left. XHR1 expression is restricted in the MHB region with slight expression in forebrain.

domain (Dawson et al., 1995), and a new variation of the WRPW motif (WRPWV), all of which are characteristic of the members of the HES family of bHLH transcriptional repressors (Fig. 2B,D). Based on the structural similarity to other HES family members, we assume that XHR1 is a transcriptional repressor. A phylogenetic analysis of bHLH domains showed that XHR1 belongs to a subfamily consisting of Xenopus ESR-4/-5, zebrafish Her-5, and mouse HES5, rather than to the other subfamily containing hairy/HES-1 (Fig. 2C). Among these, XHR1 is closest to zebrafish Her-5 (67.2% similarity and 62.1% identity in bHLH domains), which is expressed in the caudal region of the prospective midbrain during early embryogenesis (Muller et al., 1996),

and to Xenopus ESR-4 (67.2% similarity and 55.2% identity in bHLH domains), which is expressed in the forming somites (Jen et al., 1999). 2.2. Spatiotemporal expression pattern of XHR1 during development Whole-mount in situ hybridization was carried out to examine the expression pattern of XHR1 (Fig. 3). As shown in Fig. 3A,B, XHR1 expression was detected at the early gastrula stage (stage 10.5) in a very restricted region of the dorsal neuroectoderm, in comparison with the expression pattern of an early pan-neural marker Sox-2 (Fig. 3B,

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left panel). In the neuroectoderm, XHR1 was mainly detected in the inner layer (Fig. 3B, inset in the right panel). At late gastrula to neurula stages, the XHR1 expression domain became a band-shaped pattern, and the midline expression was reduced (Fig. 3C,D). Two-color in situ hybridization performed with XHR1 and En-2 probes revealed that the expression domains of these two genes overlap at the same level along the AP axis at the neurula stage (Fig. 3E), indicating that XHR1 is expressed in the prospective MHB region. At late neurula stages, XHR1 expression was also detected in the eye anlage and perhaps in the pineal gland anlage (Fig. 3F), but this expression was diminished at the tailbud stage (Fig. 3G). To determine whether the XHR1 expression domain corresponds to the morphologically defined MHB at late tailbud stages, embryos were hybridized with an XHR1 probe and the staining pattern compared to those of XFGF-8 and En-2 (Fig. 4). A narrow stripe of XHR1 expression was specifically detected in the anterior brain region at stage 38 (Fig. 4A), and horizontal sections showed that the

Fig. 4. Comparison of expression of XHR1 with that of XFGF-8 and En-2 at tailbud stages. (A–C) Lateral view of stages 36–37 embryos hybridized for XHR1, XFGF-8, and En-2. (A 0 –C 0 ) Horizontal sections of the embryos in (A–C). Anterior is to the left. Arrowheads, MHB; mb, midbrain; hb, hindbrain; ey, eye; ot, otic vesicle; st., developmental stage.

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expression domain was localized to a specific brain region recognized as an outward constriction of the inner wall of the neural tube (Fig. 4A 0 ). This region corresponded to the MHB as indicated by the expression domains of two known MHB marker genes, XFGF-8 and En-2 (Fig. 4B,B 0 ,C,C 0 ). Compared with the En-2 expression domain, which spans from the posterior midbrain to anterior hindbrain, the XHR1 expression was restricted to a narrower area at the MHB, similar to that of XFGF-8 (Fig. 4A 0 –C 0 ). 2.3. XHR1 demarcates the prospective MHB region at the early gastrula stage We analyzed in detail the spatiotemporal expression pattern of XHR1 during gastrula stages (stages 10.5–12). Since no appropriate MHB marker genes are available at this stage, at the early gastrula stage we analyzed the region of XHR1 expression in relation to the position of involuting mesoderm, and later it was analyzed in relation to the position of the presumptive notochord, the anterior tip of which roughly corresponds to the MHB. Thus, we compared the expression domain of XHR1 with that of Xbra at the stages from 10.5 to 12 (Fig. 5). At stages 10.5 and 11, Xbra was expressed in the mesoderm in a ring-shaped fashion around the blastopore, although the presumptive notochord was not yet formed on the dorsal side (Fig. 5A,B, right panel; Lerchner et al., 2000). At these stages, XHR1 was expressed in a portion of the dorsal neuroectoderm (Fig. 5A,B, left panel), in a region that was clearly more anterior than the region of Xbra expression. This pattern was confirmed by double hybridization with XHR1 and Xbra probes (Fig. 5A,B, middle panel). As gastrulation proceeded, the chordal area became apparent along the dorsal midline as visualized by Xbra expression, and extended anteriorly (Fig. 5C,D). Sagittal sections of double stained embryos (stages 11, 11.5, and 12) showed that the expression domain of XHR1 also moved anteriorly, with its center always corresponding to the anterior edge of Xbra expression (Fig. 5E–G). Furthermore, between the expression domains of XHR1 and Xbra lay the posterior neuroectodermal domain, probably the prospective hindbrain and spinal cord, expanding along the AP axis as gastrulation proceeded (Fig. 5E–G). These results demonstrate that the XHR1 expression domain encompasses the future MHB from the very beginning of its appearance. We assume that the onset of the regionalization of the prospective MHB takes place as early as stage 10.5, and this interpretation agrees with the reported fate map (Keller et al., 1992). It has been shown that the MHB is positioned in the expression border between Otx2 and Gbx2 along the AP axis in the chick and mouse (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). We therefore compared the expression domain of XHR1 with those of Xotx2 (Blitz and Cho, 1995; Pannese et al., 1995) and Xgbx-2 (von Bubnoff et al., 1996), the Xenopus orthologs of Otx2 and Gbx2, respectively, at the mid gastrula stages (Fig. 6). As

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Fig. 5. XHR1 expression in the prospective MHB region at gastrula stages. Expression of XHR1 was compared with that of Xbra during gastrula stages by whole-mount in situ hybridization and sections. (A–D) Gastrula embryos hybridized for XHR1 (left panel), for Xbra (right panel), and for both (middle panels). All embryos are dorsally viewed, and animal or anterior is to the top. (E–G) Sagittal sections of embryos hybridized for both XHR1 and Xbra. Only dorsal halves are shown, and animal is to the top. The boundary between the ectoderm and mesoderm is indicated by dotted lines, and was determined based on the differences in nuclear density of germ layers visualized by DAPI staining (lower panels). Arrowheads, delimitation of XHR1 expression; white arrowheads, delimitation of Xbra expression; st., developmental stage.

shown in Fig. 6A, hemisectioned embryos were hybridized for XHR1 (left panel) and Xotx2 (right panel). Fig. 6A 0 shows that Xotx2 was expressed mainly in the Spemann organizer region in the mesoderm layer, and also in the overlying ectoderm (right panel). This ectodermal Xotx2 expression appeared to partially overlap with the anterior part of XHR1 expression, however, the major XHR1 expression domain apparently corresponds to an Xotx2negative region (Fig. 6A 0 ), suggesting that Xotx2 is not involved in XHR1 expression. In addition, Xgbx-2 expression was first detected in the dorsolateral ectoderm with a wide gap at the midline at mid gastrula stages (Fig. 6B,C, right panels), also suggesting that the induction of XHR1 expression is independent of Xgbx-2 expression. Double in situ hybridization for XHR1 and Xgbx-2 confirmed that the expression of these two genes did not overlap with each other at stage 11 (Fig. 6B, middle panel) and that, at stage 11.5, both expression domains partially overlapped but were still distinct from each other (Fig. 6C, middle panel). Thus, XHR1 begins to be expressed before the formation of the expression border between Xotx2 and Xgbx-2, suggesting that the presumptive MHB region is initially demarcated independent of Xotx2 and Xgbx-2 expression, contrary to what has generally been expected. To examine the mechanism governing early patterning of the presumptive MHB region, we focused on the expression domain of Xbra in relation to that of XHR1, since there are no known genes which are specifically expressed in posterior neuroectoderm and mesoderm at the gastrula stage besides Xbra. To modulate Xbra expression, we used a dominant-negative FGF receptor, XFD, and eFGF, since Xbra expression is regulated by a positive feedback loop through eFGF and FGF receptor signaling (Isaacs et al., 1994). To reduce Xbra expression, mRNA for XFD was injected with nb-gal mRNA as a lineage tracer into a single dorsal blastomere at the four- to eight-cell stages. As shown in Fig. 6E, in response to XFD injection, the XHR1 expression domain was shifted posteriorly to the blastopore, where Xbra expression was dramatically reduced (12/12). It is noted that XFD expressing embryos show gastrulation arrest when compared to control embryos at the equivalent stage (Fig. 6D). Ectopic expression of eFGF by a DNA expression construct led to gastrulation arrest, similar to XFD, but induced Xbra expression in the ectoderm. This ectopic Xbra expression altered the position of the XHR1 expression domain, keeping a certain distance between their expression domains (13/14) (Fig. 6F, left panel; indicated by a thick arrow). Interestingly, when Xbra was ectopically expressed in the middle of the dorsal ectoderm, XHR1 expression was separated into right and left domains, and a gap was present between the Xbra and XHR1 expression domains (Fig. 6F, right panel). These data suggest the possibility that signal(s), perhaps posteriorizing factor(s) such as FGFs, Wnts, and retinoic acid, which are emitted from the Xbra-expressing posterior dorsal mesoderm, regulate XHR1 expression.

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2.4. XHR1 is required for MHB marker gene expression

Fig. 6. (A–C) Comparison of expression of XHR1 with that of Xotx2 and Xgbx-2 at gastrula stages. (A) A stage 11 embryo was bisected sagittally into two hemispheres and was then hybridized for XHR1 (left panel) or Xotx2 (right panel). Animal is to the top. (A 0 ) Higher magnification of (A). The expression domain of XHR1 does not correspond to that of Xotx2 in the ectoderm. (B, C) Embryos at stages 11 or 11.5 hybridized for XHR1 (left panel), for Xgbx-2 (right panel), and for both (middle panel). All embryos are dorsally viewed, and animal is to the top. (D–F) Possible regulation of XHR1 expression by the Xbra-expressing region. Embryos were injected with mRNA or DNA as indicated together with nb-gal mRNA (30 pg/ embryo) for a lineage tracer. Double in situ hybridization with XHR1 and Xbra probes was performed after nb-gal staining. (D) Injection of globin mRNA as negative control. (E) Injection of XFD mRNA (60 pg/embryo). Xbra expression was reduced in a nb-gal-stained region as indicated by an arrowhead. (F) Injection of CSKA-eFGF (10 pg/embryo). In situ staining in nb-gal-positive region means Xbra expression as indicated by asterisks. Two typical phenotypes are shown. Dotted lines, the boundary between ectoderm and mesoderm that was determined based on the differences in nuclear density of germ layers visualized by DAPI staining (data not shown; see Fig. 5E–G); st., developmental stage; thick arrows, shifted XHR1 expression; thin arrows, XHR1 expression; white asterisks, Xbra expression.

To examine the role of XHR1 in the early patterning of the presumptive MHB region, we constructed three types of dominant-negative mutants, mb-XHR1, XHR1-DWRPWV, and XHR1-VP16 (Fig. 2D), and tested the effect of their overexpression on MHB marker gene expression. Since the Hairy and HES-1 proteins reportedly bind to the N box (CANNAG) as a homodimer (Sasai et al., 1992; Ohsako et al., 1994; Bae et al., 2000), we expected that mb-XHR1, in which the DNA-binding basic region was point-mutated, dimerizes with the endogenous XHR1, but does not bind to DNA (see Strom et al., 1997). XHR1-DWRPWV, in which the C-terminal WRPWV was deleted, was expected to bind to DNA as a homodimer, but not to repress its target genes because it lacks the WRPW motif needed to bind to the corepressor Groucho (Paroush et al., 1994; Fisher et al., 1996; Grbavec and Stifani, 1996). XHR1-VP16, in which the Orange and WRPW domains were replaced with the VP16 activation domain, could activate target genes instead of repressing them, thereby perhaps exerting stronger antagonizing effects than the derepressors, mbXHR1 and XHR1-DWRPWV. We injected mRNAs for the dominant-negative constructs and nb-gal as a lineage tracer in the left-side dorsal blastomere near the midline at the four- to 16-cell stages, at a point which should give rise to the future MHB region. This injection point was determined empirically to be approximately 45–608 from the animal pole. We first collected embryos which had nb-gal positive cells in the ANE, then examined the embryos by whole-mount in situ hybridization for XPax2 and En-2 expression as MHB markers (Hemmati-Brivanlou et al., 1991; Heller and Brandli, 1997). Injection of globin mRNA as negative control did not alter the expression patterns of XPax-2 and En-2 (Fig. 7A–C). In embryos injected with XHR1-VP16 RNA, however, the expression of both XPax-2 and En-2 was dramatically reduced, and this effect was not seen in the uninjected half of the embryo (Fig. 7D–F). Tables 1 and 2 summarize the cumulative data obtained from these injection experiments. It can be seen that XHR1-VP16-injected embryos showed reduced expression of XPax-2 and En-2 with high frequency (100–71% for XPax-2; 57–75% for En-2). When embryos were injected with mb-XHR1 or XHR1-DWRPWV, reduction of XPax-2 and En-2 expression was also observed, but this occurred only transiently at stages 13–15, and the reduction became less pronounced at stage 16 (Fig. 7G–L). In Table 1, it is indicated that the reduced expression of XPax-2 by mbXHR1 was observed in 84% of injected embryos at stage 13/14, but only 20% at stage 16. Similarly, reduced expression of XPax-2 by XHR1-DWRPWV occurred in 29% of embryos at stage 13/14, but no embryos showed such a reduction at stage 16. The recovery of expression at later stages was also observed for En-2 expression in embryos injected with mb-XHR1 and with XHR1-DWRPWV (Table 2). Thus, XHR1-VP16 appeared to have stronger inhibitory effects on marker gene expression than the other two, as expected.

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Fig. 7. Dominant-negative XHR1 constructs downregulate MHB markers, XPax-2 and En-2. Injected embryos were stained with nb-gal (red) and then hybridized for XPax-2 (purple) or En-2 (purple). Typical embryos with severe defect are shown with the incidence (see Tables 1 and 2 for details). (A–C) globin. (D–F) XHR1-VP16. (G–I) mb-XHR1. (J–L) XHR1DWRPWV. (M–P) Wild-type XHR1. Cleared embryos are shown in lower panels of (M–O), and in (P). Injected RNAs are indicated on the left side of panels. Probe RNAs and developmental stages (st.) are indicated on the top of panels. (Q) Horizontal section of a stage 35 embryo injected with globin RNA and hybridized for En-2. (R) Horizontal section of a stage 31 embryo injected with XHR1-VP16 RNA and hybridized for En-2. Arrows, expression beneath the outgrowth of epidermis (see text); asterisks, reduced expression; arrowheads, MHB; mb, midbrain; hb, hindbrain; ey, eye; ot, otic vesicle.

These three types of XHR1 mutants caused essentially the same phenotype, a reduction of XPax-2 and En-2 expression, suggesting that the mutants function as dominant-negative forms of XHR1 and that disruption of the endogenous XHR1 function causes inhibition of MHB marker gene expression. Since the MHB can be recognized morphologically at tailbud stages as shown in Fig. 4, we examined the effects of XHR1-VP16 on the formation of the MHB. As shown in Fig. 7Q,R, the globin-injected control embryo exhibited normal En-2 expression in the morphologically defined MHB region, whereas XHR1-VP16 diminished En-2 expression and disrupted the morphological feature of the MHB on the gal-stained left side. These results suggest that XHR1 is involved in the expression of MHB genes as well as the formation of the MHB. We next examined the effects of the overexpression of wild-type XHR1 on MHB marker expression. Unexpectedly, some embryos injected with wild-type XHR1 RNA showed reduced expression of XPax-2 (Fig. 7M, 10/15; Table 1, 67%) and En-2 (Table 2, 43%) at stages 13–15. In addition, the injected embryos showed epidermal outgrowth in the nbgal-stained region at the later stage and, beneath the thickened epidermis, most embryos showed normal expression of XPax-2 (Fig. 7N, 98/121; Table 1, 81%) and En-2 (Fig. 7O, 97/137; Table 2, 71%). Nevertheless, in some of the wildtype XHR1-overexpressing embryos in which the nb-gal positive region was close to the MHB, we did observe an expansion of the En-2 expressing region (Table 2; see also Table 3). We therefore tested whether lower doses of wildtype XHR1 RNA might lead to expansion of En-2 expression. As shown in Fig. 7P, injection of XHR1 RNA at 10 pg/ embryo resulted in the expansion of En-2 expression (6/61). This result was reproducible (see Tables 2 and 3), although the incidence was not high. Therefore, we assume that wildtype XHR1 has the activity that is opposite to the dominantnegative constructs. This gain-of-function data also supports and strengthens the conclusion that XHR1 is involved in MHB marker gene expression. In order to further examine why, under some conditions, wild-type XHR1 reduces the expression of MHB genes rather than increasing their expression, we examined the expression of a pan-neural marker nrp-1 (Richter et al., 1990). We found that expression of nrp-1 was inhibited in the nb-gal positive region of wild-type XHR1-injected embryos (25/40) (Fig. 8A,B), whereas XHR1-VP16 did not exert such effect (87/88) (Fig. 8C,D). This data suggests that the reduction of the MHB markers by injection of wildtype XHR1 RNA, particularly at earlier stages (Tables 1 and 2), is due to inhibition of neuralization of dorsal ectoderm, although the reason for this is still not clear (see Section 3). 2.5. Reduction of MHB marker expression by XHR1-VP16 is rescued by coinjection with wild-type XHR1 Rescue experiments were carried out to examine whether the reduced expression of MHB markers by the

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Table 1 Effects of overexpression of XHR1 and its mutated constructs on XPax-2 expression a RNA injected (pg/embryo)

Stage

Number of embryos with XPax-2 expression (%) n

Not changed

Reduced

Expanded

Exp. no.

b-globin 100 100

13,14 16,18

22 75

22 (100) 75 (100)

0 (0) 0 (0)

0 (0) 0 (0)

3 1–3

XHR1 100 100,40

13,14 16,18

15 121

5 (33) 98 (81)

10 (67) 23 (19)

0 (0) 0 (0)

3 1–3

XHR1-VP16 100 100

13,14 16,18

15 112

0 (0) 33 (29)

15 (100) 79 (71)

0 (0) 0 (0)

3 1–3

mb-XHR1 100 100,40

13,14 16

31 44

5 (16) 35 (80)

26 (84) 9 (20)

0 (0) 0 (0)

3 2,3

XHR1-DWRPWV 100 100,40

13,14 16

28 48

19 (68) 48 (100)

8 (29) 0 (0)

1 (3) 0 (0)

3 2,3

a Phenotypes were scored with embryos showing nb-gal-positive cells adjacent to or over the marker gene expression domains at the stages indicated. Reduced, less than about 50% reduction compared to the level on the right side; expanded, more than about two-fold expansion compared to the level on the right side; n, total number of scored embryos; Exp. no., experiment number.

dominant-negative XHR1 mutants was a specific phenotype resulting from the loss of endogenous XHR1 function. For this purpose, we chose XHR1-VP16 for its strong and long-lasting effects on XPax-2 and En-2 expression. As shown in Table 3, the reduction of En-2 expression by XHR1-VP16 was partially suppressed by coinjection with wild-type XHR1 from 66 to 29 or 22%, dose-dependently. In turn, the reduction of En-2 expres-

sion by wild-type XHR1 was also suppressed by XHR1VP16 from 40 to 22%. Thus, the effects of XHR1-VP16 and wild-type XHR1 on En-2 expression were canceled out by their coexpression, rather than producing an additive enhancement of inhibition. This suggests that inhibition of MHB marker expression by XHR1-VP16 is brought about by the specific inhibition of the endogenous XHR1 function.

Table 2 Effects of overexpression of XHR1 and its mutated constructs on En-2 expression a RNA injected (pg/embryo)

Stage

Number of embryos with En-2 expression (%) n

Not changed

Reduced

Expanded

Exp. no.

b-globin 100,40 100,50,40

14,15 16,18,24

47 150

44 (94) 149 (99)

3 (6) 1 (1)

0 (0) 0 (0)

4,6 1–3,5,6

XHR1 40,20 100,50,40

14,15 16,18,24

21 137

12 (57) 97 (71)

9 (43) 39 (28)

0 (0) 1 (1)

4 1,2,5,6

XHR1-VP16 100,40 100,50,40

14,15 16,18,24

30 182

13 (43) 46 (25)

17 (57) 136 (75)

0 (0) 0 (0)

4,6 1–3,5,6

mb-XHR1 100 100,40

14,15 16

25 49

0 (0) 23 (47)

25 (100) 26 (53)

0 (0) 0 (0)

4,6 5,6

XHR1-DWRPWV 100 100,40

14,15 16

15 44

2 (13) 42 (95)

13 (87) 2 (5)

0 (0) 0 (0)

4,6 5,6

a

Experimental procedure and phenotype scoring were the same as in Table 1. n, total number of scored embryos; Exp. no., experiment number.

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Table 3 Effects of coinjection with XHR1 and XHR1-VP16 RNAs on En-2 expression a RNA injected

b-globin alone XHR1-VP16 alone 1 XHR1 1 XHR1 XHR1 alone

pg/embryo

40 20 20 40 40

Number of embryos with En-2 expression (%) n

Not changed

Reduced

Expanded

54 53 62 64 40

54 (100) 18 (34) 44 (71) 49 (77) 22 (55)

0 (0) 35 (66) 18 (29) 14 (22) 16 (40)

0 (0) 0 (0) 0 (0) 1 (1) 2 (5)

a

Effects of coinjection with XHR1 and XHR1-VP16 RNAs on En-2 expression. Experimental procedures were the same as in Table 1. RNAs were injected into one animal dorsal blastomere of the eight-cell stage embryos. Embryos were scored at stage 16. The data represent the sum of two independent experiments. n, total number of scored embryos.

3. Discussion We have isolated and characterized the Xenopus prolinebHLH-WRPW transcriptional repressor XHR1, which is expressed specifically in the MHB region and transiently in the eye anlage. Several MHB-specific genes have been isolated and characterized in Xenopus, as well as in other vertebrates, and include XPax-2, XPax-5 (Heller and Brandli, 1999), and En-2 (Hemmati-Brivanlou et al., 1991) for transcription factors, XFGF-8 (Christen and Slack, 1997) and Xwnt-1 (Wolda et al., 1993) for secreted signaling factors, and xGCNF for nuclear orphan receptors (Song et al., 1999). All of these genes are first expressed in the prospective MHB region at late gastrula to neurula stages in Xenopus, suggesting that these genes act in concert to

establish the MHB. In contrast, the expression of XHR1 starts much earlier than these genes, and is apparent in the dorsal neuroectoderm at the early gastrula stage (stage 10.5). According to a fate map of the Xenopus early gastrula, the prospective MHB is located next to a narrow and long area consisting of the future hindbrain and spinal cord region that in turn exists adjacent to the involuting dorsal marginal zone (Keller et al., 1992). Therefore, there is a narrow gap between the prospective MHB region and the involuting mesoderm. Using double staining with Xbra, which demarcates the involuting mesoderm and later chordal mesoderm, we verified that the early expression domain of XHR1 is in good agreement with the prospective MHB region. Thus, we propose that the onset of regionalization of the MHB takes place as early as at stage 10.5.

Fig. 8. nrp-1 expression in XHR1 or XHR1-VP16 RNA-injected embryos. Injected embryos at the tailbud stage (stages 24/25) were stained with nb-gal (red) and then hybridized for nrp-1 (purple). RNA, 100 pg, for wild-type XHR1 (A, B) or 50 pg of RNA for XHR1-VP16 (C, D) were injected. (B, D) Sections of stained embryos. Right panel, staining with DAPI. Sections are at the levels indicated by solid bars in (A, C). Solid bar (B, D), midline; broken line, the boundary between neural and mesodermal tissues; bracket, an area lacking nrp-1 expression in the neural tissue; nt, notochord; ey, eye; cg, cement gland.

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In mice and chick, the MHB has recently been shown to be positioned along the AP axis at the posterior limit of Otx2 expression, which is regulated by posterior Gbx2 expression (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). We compared the expression of XHR1 with that of Xotx2 and Xgbx-2 at gastrula stages to test whether the expression domain of XHR1 straddles or is adjacent to the expression boundary of Xotx2 and Xgbx-2. In Xenopus, the expression of Xgbx-2 has been reported to be first detected at the mid gastrula stage in the dorsolateral region of ectoderm with a wide gap at the dorsal midline (von Bubnoff et al., 1996). We showed that the early expression of Xgbx-2 did not overlap with that of XHR1, indicating that Xgbx-2 is not involved in the onset of XHR1 expression. Later in development, however, the anterior edge of Xgbx-2 expression corresponded to the MHB (von Bubnoff et al., 1996) and overlapped with XHR1 expression (data not shown). These results suggest that the prospective MHB region is ‘prepatterned’ as the XHR1-expressing domain, and this occurs before the expression boundary between Xotx2 and Xgbx-2 is formed on the dorsal side. It has been suggested that the patterning of the neuroectoderm along the AP axis by posterior transformation parallels neural induction by the organizer at gastrula stages (see reviews Doniach, 1995; Gamse and Sive, 2000). We have shown previously that Xbra can transform neural tissue from an anterior to a posterior character (Taira et al., 1997). In addition, several lines of evidence have shown that Wnt, FGF, and retinoic acid signaling, which emanate from the trunk organizer, are involved in the posteriorization of neural tissue (Moon et al., 1997; Taira et al., 1997; Gamse and Sive, 2000). The posteriorizing activity of Xbra may be mediated by eFGF and Xwnt-11, which act downstream of Xbra in the chordal region (Isaacs et al., 1994; Tada and Smith, 2000). Thus, the idea that initial neural patterning is governed by the posteriorizing effect of the trunk organizer is also supported by our data that the expression domain of XHR1 in the prospective MHB region at gastrula stages is likely to be positioned in relation to that of Xbra (Fig. 6D–F). It will be therefore interesting to examine further how the upregulation of the XHR1 gene is restricted to the prospective MHB region at the early gastrula stage, since this may be an initial step in AP patterning during neural development. Based on the structural similarities between XHR1 and the proline-bHLH-WRPW proteins, it is reasonable to expect that XHR1 functions as a transcriptional repressor. Several types of dominant-negative mutants have been successfully used for bHLH-WRPW factors: fusion constructs with a transcriptional activation domain (Jimenez et al., 1996; Jen et al., 1999), non-DNA binding forms (Deblandre et al., 1999; Jen et al., 1999; Strom et al., 1997; Takke et al., 1999; Koyano-Nakagawa et al., 2000), and WRPW-deleted forms (Giebel and Campos-Ortega, 1997; Takke et al., 1999; Koyano-Nakagawa et al., 2000). We tested these three types of dominant-negative XHR1, XHR1-VP16, mb-XHR1, and XHR1-DWRPWV and

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obtained consistent results in that all of these inhibit the expression of the MHB markers, XPax-2 and En-2 (Fig. 5; Tables 1, 2). While XHR1-DWRPWV has the weakest effect among the three, apparent inhibition of the endogenous XHR1 by this construct suggests the involvement of the corepressor Groucho for XHR1 function. In Xenopus, several genes encoding Groucho-related proteins have been identified (Choudhury et al., 1997; Roose et al., 1998), and among these XGrg-4 has been shown to interact with XTcf-3 and to act as a transcriptional corepressor (Roose et al., 1998). It is likely that XHR1 interacts with XGrg-4 or some other Groucho-related corepressor in Xenopus. In addition, we have shown that the effects of XHR1VP16 can be reversed by coexpression of wild-type XHR1 (Table 3), further supporting the specificity of this dominant-negative construct. Inhibition of MHB marker gene expression by the dominant-negative forms of XHR suggests the possibility that wild-type XHR1 represses transcription of a target gene whose product represses transcription of XPax-2 and En-2. Conversely, overexpression of wild-type XHR1 led to expansion of the MHB marker En-2 (Table 2). However, the incidence of this expansion is low, and ectopic expression of En-2 was not observed in a region distant from the prospective MHB. We interpret these data as indicating that XHR1 activity alone is not enough to initiate the expression of the MHB marker genes. In this context, expansion of En2 expression may be explained by a situation in which some transcriptional activators for XPax-2 and En-2 genes are present only in a narrow region surrounding the prospective MHB. It was reported that known MHB marker genes are expressed after the MHB is positioned by Otx2 and Gbx2 (Broccoli et al., 1999; Millet et al., 1999; Katahira et al., 2000). In addition, there is a relatively large time lag between the onset of XHR1 expression (stage 10.5) and that of XPax-2 and En-2 expression (stage 12 and later). Thus, XHR1 may regulate several steps including the boundary formation of Xotx-2 and Xgbx-2 before leading to induction of XPax-2 and En-2. Regarding the outgrowth of epidermis and the inhibition of neural tissue formation by XHR1 overexpression, one possible explanation is that the overexpressed wild-type XHR1 forms heterodimers with other bHLH proteins which are involved in neural tissue formation. This possibility also provides an alternative explanation for our results: the dominant-negative forms of XHR1 could also form heterodimers artificially, this would give rise to unusual neural tissue for some reason, and, as a result, reduce MHB marker gene expression. At present, we cannot exclude this possibility, because we do not know if the yet unidentified XHR1 dimerization partners and targets are present in the early embryo when the exogenous XHR1 constructs were overexpressed. Nevertheless, we interpret the reduction in MHB marker gene expression resulting from injection of the dominant-negative constructs, and rescued by the wild-type construct, as showing the specifi-

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city of XHR1 function in the prospective MHB region. We are planning to circumvent these obstacles in the near future using, for example, targeted disruption of endogenous XHR1 RNA by double-stranded XHR1 RNA or stage-specific expression of XHR1 by glucocorticoid receptor-fused constructs. Studies in chick have shown that transplantation of Fgf8soaked beads in the prospective diencephalon results in the transformation of diencephalon to the optic tectum (Crossley et al., 1996; Funahashi et al., 1999), and that misexpression of Pax-2, Pax-5, or En also induces ectopic optic tectum in the prospective diencephalon (Araki and Nakamura, 1999; Funahashi et al., 1999; Okafuji et al., 1999). Targeted gene disruption studies in mice have demonstrated that En2, Pax2, Pax5, and Fgf8 are required for formation of tectum and cerebellum (Joyner et al., 1991; Urbanek et al., 1997; Meyers et al., 1998). Thus, those MHB genes have been shown to play important roles in the organizing center of neural patterning. In Xenopus, however, it was reported that misexpression of Fgf8 or implantation of Fgf8-soaked beads induces some MHB genes but does not induce ectopic mesencephalic structures, whereas implantation of MHB grafts does (Riou et al., 1998). Thus, FGF8 seems to be insufficient for the organizing activity of the MHB in Xenopus. In the case of xGCNF, overexpression of the wild-type gene does not cause enhanced or ectopic expression of En-2, although the dominant-negative construct xGCNF-EnR inhibits En-2 expression in the MHB region (Song et al., 1999). Similarly, overexpression of Ol-eng2 at early stages in medaka has been shown to activate MHB genes only in the diencephalon but not in other regions (Ristoratore et al., 1999). These data, combined with our data on XHR1 (see Fig. 7P), suggests that a whole set of MHB genes does not seem to be upregulated easily in a given region by overexpression of any single MHB gene alone. We, therefore, assume that the integration of several signaling cascades is required for the initiation and establishment of MHB formation. To date, the bHLH transcriptional repressor genes that are expressed in the MHB region during development include mouse HES-3b and zebrafish her-5, while HES-3b is also expressed in the rhombomeres 2, 4, 6, and 7 (Muller et al., 1996; Lobe, 1997; Hirata et al., 2000). her-5, which is closest to XHR1, is reportedly expressed early in the midbrain anlage and later in the MHB region (Muller et al., 1996). However, the precise location of the early expression domain of her-5, as well as its function in MHB formation, have not been determined. In zebrafish, one of the mutations affecting the formation of the MHB is no isthmus (noi), which has been mapped to the pax2.1 locus. In noi mutants, engrailed expression is reduced, but the onset of her-5 expression is not affected (Lun and Brand, 1998). These data suggest that, in vertebrates, there is a common requirement for a bHLH-WRPW transcriptional repressor upstream of Pax-2 and En-2 in the formation of the MHB. The genetic cascade involving bHLH-WRPW repressors

as downstream components of Delta-Notch signaling is well conserved in invertebrates and vertebrates (Kageyama and Nakanishi, 1997). In Xenopus early embryogenesis, XNotch-1 is expressed maternally and later ubiquitously at gastrula stages (Coffman et al., 1993), whereas X-Delta-1 expression is first detected at late gastrula (stage 12) (Chitnis et al., 1995). Thus, XHR1 does not seem to be a downstream target of Delta-Notch signaling, and rather appears to function in early regional patterning of the neuroectoderm, much like the prepattern gene hairy in Drosophila (Fisher and Caudy, 1998). 4. Experimental procedures 4.1. Embryo manipulation Wild-type and albino Xenopus embryos were obtained by artificial fertilization. Embryos were dejellied, reared in 0.1 £ Steinberg’s solution with 50 mg/ml gentamicin (Peng, 1991), and staged according to Nieuwkoop and Faber (1967). 4.2. Construction of a Xenopus ANE cDNA library and library screen To obtain anterior halves of Xenopus neuroectoderm tissue, anterodorsal tissue was dissected from stages 12– 12.5 embryos and separated into the ectoderm region and the underlying prechordal and endodermal region by digestion with 1–2 mg/ml collagenase type I (Sigma) at room temperature for 10 min (Fig. 1A). Total RNA was extracted from about 600 pieces of ANE by guanidine-isothiocyanate method (Chomczynski and Sacchi, 1987). An ANE cDNA library was constructed using total RNA, Uni-ZAP XR vector, and a cDNA synthesis kit (Stratagene). To eliminate house-keeping genes and genes with broad and/or strong expression at later stages, the library was negatively screened with cDNA probes from total RNA extracted from the trunk region of tailbud stage embryos (stages 26–28). Further screening of the negative clones was performed on the basis of either cDNA sequence information or expression pattern by whole-mount in situ hybridization. For both types of screening, the negative clones were amplified by polymerase chain reaction (PCR) using the SK (5 0 -TCTAGAACTAGTGGATC-3 0 ) and T7-25 primers (5 0 CCGGATCCTAATACGACTCACTATA-3 0 ), and PCR products obtained were purified using MultiScreen 96Well Filtration Plates (Millipore). Briefly, PCR products were mixed with saturated KI solution, centrifuged in MultiScreen FB plates, washed with 70% ethanol/30% 0.1 M NaCl, and eluted with TE buffer. Eluates were filtrated with MultiScreen HV plates pre-loaded with Sephadex G50 Superfine (Pharmacia). Purified PCR products were sequenced using ABI PRISM 310 Genetic Analyzer (Perkin Elmer) or used as templates for digoxigenin-labeled antisense RNA probes for whole-mount in situ hybridization

J. Shinga et al. / Mechanisms of Development 109 (2001) 225–239

(Section 4.4). Longer cDNA clones were obtained by screening 6 £ 10 5 pfu of a cDNA library from Xenopus gastrulae (stages 10.5 and 11.5 mixed) with the PCR products as probes. Probes were labeled with [a- 32P]dCTP using Prime-It II Random Primer Labeling Kit (Stratagene), hybridized at 658C (Church and Gilbert, 1984) and washed at 658C with 0.1 £ SSPE/0.1% sodium dodecyl sulfate (SDS) (Sambrook et al., 1989). The cDNA clones obtained were sequenced on both strands with Thermo Sequenase Cycle Sequencing Kit (Amersham) and LONG READIR 4200 (LI-COR). Sequence alignment and generation of phylogram of XHR1 and other HES family members were performed using the ‘Pileup’, ‘Distances’, and ‘Growtree’ programs of the Wisconsin Package Version 10.0 (Genetics Computer Group, Madison, WI, USA). 4.3. Northern blot analysis Total RNA (4 mg) was electrophoresed on a 1% agarose/ formaldehyde gel and blotted onto a nylon membrane (Nytran, Schleicher and Schuell). cDNA probes were prepared and labeled with [a- 32P]dCTP as described above. Blots were hybridized at 658C, washed with 2 £ SSPE/0.1% SDS, and analyzed by BAS-2500 (Fuji Film). 4.4. Whole-mount in situ hybridization Whole-mount in situ hybridization was performed according to Harland (1991), manually or with an automated system (Automated ISH System AIH-101, Aloka). For expression pattern screening, RNA probes were synthesized using DIG RNA Labeling Mix (Boehringer Mannheim), T7 RNA polymerase (Boehringer Mannheim), and purified PCR products as templates containing T7 promoter sequence, followed by purification with MultiScreen HV plates pre-loaded with Sephadex G-50 Superfine. Digoxigenin- or fluoresceinlabeled antisense RNA probes were transcribed from Sox-2 (Mizuseki et al., 1998), En-2 (Hemmati-Brivanlou et al., 1991), XFGF-8 (Endo et al., 2000), Xbra (Cunliffe and Smith, 1992), Xotx2 (Pannese et al., 1995), XPax-2 (Heller and Brandli, 1997), and nrp-1 (Richter et al., 1990) plasmid templates. Digoxigenin-labeled antisense RNA probe for Xgbx-2 (nucleotide numbers 319-1341; accession number L47990) was transcribed from PCR products amplified with primers, 5 0 ATGAGTGCAGCCTTTCAG-3 0 and 5 0 ccggatcctaatacgactcactatagggTCAAGGTCTTGCTTGCTC-3 0 (lower case indicates an extra-sequence for T7 promotor), and a Xenopus stage 17–18 cDNA library. BM Purple or INT/BCIP (Boehringer Mannheim) was used for chromogenic reactions. After staining, embryos were fixed with Bouin’s fixative. When necessary, embryos were bleached and cleared with benzyl benzoate/benzyl alcohol. Some stained embryos were sectioned at 10 or 15 mm and stained with DAPI.

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4.5. Plasmid constructs and mRNA/DNA microinjection The coding region of XHR1-A (see Fig. 2A) was PCRamplified and inserted into BamHI/XbaI sites of pCS2 1 vector (Rupp et al., 1994), generating a wild-type XHR1 construct (pCS2 1 XHR1). To obtain a dominant-negative construct (mb-XHR1), three amino acid residues (Glu-22, Lys-23, Arg-26) in the basic region were mutated to alanine using an in vitro site-directed mutagenesis system (GeneEditor, Promega). The mutated primer used to generate mbXHR1 was 5 0 -AGA AAG CTA TTA AAG CCG TTG GTG GCG GCA AGG CGG GCG GAG AGG ATA AAT AAC AGC CTG GAG-3 0 (changing from EKRRRE to AARRAE). Also a deletion construct (XHR1-DWRPWV) was obtained by eliminating the C-terminal WRPWV sequence by sitedirected mutagenesis. The primer used here was 5 0 -TTT CAC TTA CCA AAG CTT CAA GAC CTG ATC TAG AAC TAT AGT GAG TCG TAT TAC-3 0 . XHR1-VP16 was constructed by ligating two cDNA fragments encoding the N-terminal region of XHR1 (amino acids 1–75) and the VP16 transactivation domain (amino acids 413–490; accession number X03141) with EcoRI sites. Both fragments were PCR-amplified with primers containing EcoRI sites so that XHR1-VP16 had two extra codons, AAT and TCT, translated into Asn–Ser between the two fragments. Synthetic mRNAs for wild-type or mutant forms of XHR1 as well as a dominant-negative FGF receptor, XFD, were generated using MEGAscript kit (Ambion) and a CAP analog 7mG(5 0 )ppp(5 0 )G (New England Biolabs) as described (Taira et al., 1994), except that RNA was dissolved in water. mRNA for the nuclear-localized form of b-galactosidase (nb-gal), which was transcribed from linearized pCS2 1 nbgal, was coinjected as lineage tracer, and nb-gal was visualized using Red-Gal (Research Organics). XHR1 mutant constructs used for injection experiments are shown in Fig. 2D. All constructs were verified by nucleotide sequencing. A DNA expression construct of eFGF (CSKA-eFGF) was also injected with nb-gal mRNA.

Acknowledgements We thank the following generous contributors for reagents: Bruce Blumberg (a Xenopus gastrula cDNA library), David Turner (pCS2 1 and pCS2 1 nbgal), Yoshiki Sasai (Sox-2), Richard Harland (En-2), Hiroyukii Ide (XFGF-8), Jim Smith (Xbra), Maria Pannese and Edoardo Boncinelli (Xotx2), Enrique Amaya (XFD), Harry Isaacs (CSKA-eFGF), Andre´ Bra¨ ndli (XPax-2), and Peter Good (nrp-1). We are grateful to Ryoichiro Kageyama for discussions, Shin-ya Ohmori for cDNA sequencing, Hirofumi Ono for his initial contribution to this study, and Marcia O’Connell for critical reading of the manuscript. This work was supported in part by a Grant in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, by Toray Science

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Foundation, Japan and by a grant from Japan Society for the Promotion of Science Fellowship (J.S.).

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