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Developmental Biology 298 (2006) 71–86 www.elsevier.com/locate/ydbio

Neural induction in Xenopus requires inhibition of Wnt-β-catenin signaling ⁎ Elizabeth Heeg-Truesdell a, Carole LaBonne a,b,

a Department of Biochemistry, Molecular Biology and Cell Biology, , Evanston, Il 60208, USA b Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Il 60208, USA

Received for publication 27 February 2006; revised 5 June 2006; accepted 6 June 2006 Available online 14 June 2006

Abstract

Canonical Wnt signals have been implicated in multiple events during early embryogenesis, including primary axis formation, induction, and A–P patterning of the neural plate. The mechanisms by which Wnt signals can direct distinct fates in cell types that are closely linked both temporally and spatially remains poorly understood. However, recent work has suggested that the downstream transcriptional mediators of this pathway, Lef/Tcf family DNA binding proteins, may confer distinct outcomes on these signals in some cellular contexts. In this study, we first examined whether inhibitory mutants of XTcf3 and XLef1 might block distinct Wnt-dependent signaling events during the diversification of cell fates in the early embryonic ectoderm. We found that a Wnt-unresponsive mutant of XTcf3 potently blocks neural crest formation, whereas an analogous mutant of XLef1 does not, and that the difference in activity mapped to the C-terminus of the proteins. Significantly, the inhibitory XTcf3 mutant also blocked expression of markers of anterior-most cell types, including cement gland and sensory placodes, indicating that Wnt signals are required for rostral as well as caudal ectodermal fates. Unexpectedly, we also found that blocking canonical Wnt signals in the ectoderm, using the inhibitory XTcf3 mutant or by other means, dramatically expanded the size of the neural plate, as evidenced by the increased expression of early pan-neural markers such as Sox3 and Nrp1. Conversely, we find that upregulation of canonical Wnt signals interferes with the induction of the neural plate, and this activity can be separated experimentally from Wnt-mediated neural crest induction. Together these findings provide important and novel insights into the role of canonical Wnt signals during the patterning of vertebrate ectoderm and indicate that Wnt inhibition plays a central role in the process of neural induction. © 2006 Elsevier Inc. All rights reserved.

Keywords: Neural induction; Wnt; BMP; XTcf3; XLef1; Neural crest; Placode

Introduction koop, 1999). Later, the model of neural induction, referred to as the ‘default model’ proposed that BMP antagonists secreted During vertebrate embryogenesis, distinct regions of the from the organizer function as Nieuwkoop's “activator” signals ectoderm become specified to form epidermis, CNS progeni- and induce anterior neural fates, whereas the high level BMP tors, neural crest, and placodes. During this process information signaling characteristic of ectoderm further from the organizer with respect to position along the anterior–posterior (A–P) and instructs these cells to form epidermis (Hemmati-Brivanlou and dorso-ventral (D–V) axes is provided to each of these cell types Melton, 1997; Sasai and De Robertis, 1997; Wilson et al., (De Robertis and Kuroda, 2004; Gamse and Sive, 2000; Sasai 1997). While there is significant experimental evidence for the and De Robertis, 1997; Wilson and Maden, 2005). In a model default model in Xenopus, recent work in a number of model first proposed by Nieuwkoop (1952),an“activator” molecule organisms indicates that other signals in addition to BMP was thought to impart anterior neural character on cells that inhibition play important roles in neural induction (Stern, would otherwise form epidermis, while “transformer” signals 2005). subsequently generated more posterior neural fates (Nieuw- Based on the observation that they can confer more caudal identity to neural tissue induced by BMP antagonists such as ⁎ chordin or noggin, it has been suggested that canonical Wnt Corresponding author. Department of Biochemistry, Molecular Biology and “ ” Cell Biology, Northwestern University, Evanston, Il 60208, USA. signals may act as transforming signals during CNS pat- E-mail address: [email protected] (C. LaBonne). terning (Erter et al., 2001; Fekany-Lee et al., 2000; Kazanskaya

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.06.015 72 E. Heeg-Truesdell, C. LaBonne / 298 (2006) 71–86 et al., 2000; Kelly and Melton, 1995; Kiecker and Niehrs, 2001; In this study, we set out to examine whether XLef1 and Kudoh et al., 2002; McGrew et al., 1995). Wnt signaling is also XTcf3 play distinct roles during the patterning of the embryonic required for induction of the neural crest (Deardorff et al., 2001; ectoderm. We found that while a Wnt-unresponsive mutant of LaBonne and Bronner-Fraser, 1998; Saint-Jeannet et al., 1997; XTcf3 potently blocks neural crest formation, an analogous Wu et al., 2005), and cranial placodes (Bang et al., 1999; mutant of XLef1 did not; however, blocking XLef1 function did Honore et al., 2003; Saint-Germain et al., 2004; Stark et al., interfere with other effects of Wnt signaling. The constitutively 2000), two groups of cells that arise at the juncture of the neural repressing XTcf3 mutant also blocked formation of cement ectoderm and presumptive epidermis. Wnts are secreted gland and anterior placodes, a finding not predicted by the glycoproteins that bind to seven-pass transmembrane receptors, prevailing view of Wnts as posteriorizing factors. Importantly, termed Frizzleds, in cooperation with LDL family coreceptors we found that blocking canonical Wnt signals in the ectoderm, (He, 2003; Yanfeng et al., 2003). Canonical Wnt signaling using the inhibitory XTcf3 mutant or by other means, led to a centers on the stability of the transcriptional coactivator β- dramatic increase in the size of the neural plate, as evidenced by catenin (Pandur et al., 2002). In the absence of a Wnt signal, the the increased expression of early pan neural markers such as serine/threonine kinase GSK-3 phosphorylates β-catenin, Sox3 and Nrp1. By contrast, upregulation of canonical Wnt targeting it for ubiquitin-mediated proteosomal degradation. signals was found to inhibit neural plate formation, and this Receptor activation results in the inactivation of a degradation activity could be distinguished experimentally from Wnt- complex containing GSK-3, APC, Axin and other factors, mediated neural crest induction. Our findings demonstrate that resulting in an increase in β-catenin stability. Stabilized β- not only is activation of Wnt/β-catenin signaling required for catenin enters the nucleus and interacts with Lef/Tcf family neural crest induction, but also that inhibition of canonical Wnt DNA binding proteins to activate transcription of Wnt target signaling is an essential step in the process that leads to neural genes. In the absence of an interaction with β-catenin, Lef/Tcf plate formation. factors remain bound to the DNA and are thought to function as transcriptional repressors (Hurlstone and Clevers, 2002). The Materials and methods mechanisms whereby canonical Wnt signals can direct such diverse responses in cell types such as the neural crest, placodes, DNA constructs and neural plate, when their induction is so closely linked both temporally and spatially, remain poorly understood. However, The wild-type and mutant Lef and Tcf isoforms used in this study recent work has suggested that functional diversity amongst the (accession numbers AF287148 and X99308 respectively) were generated by low cycle number PCR using a high fidelity polymerase (TGO, Roche) and different Lef/Tcf family proteins may confer distinct outcomes verified by sequencing. ΔXLef1 (which consists of aa 110–373) and ΔXTcf3 on canonical Wnt signals in some cellular contexts. (which consists of aa 88–555) are based on previously described dominant Although Lef/Tcf factors are often depicted as equivalent negative mutants (Huber et al., 1996; Molenaar et al., 1996; Roel et al., 2002) in simplified models of canonical Wnt signaling, these and delete the β-catenin binding domain of the respective proteins. Both of proteins possess distinct structural attributes that are proving these constructs were cloned into a pCS2 expression vector (D. Turner) that incorporates six in frame N-terminal myc tags. β-CateninΔXLef1 and β- to be of functional significance. All Lef/Tcf factors have an cateninΔXTcf3 were constructed by inserting a portion of β-catenin (accession HMG-type DNA binding domain and an N-terminal β- number M77013; aa 737 to 868) in frame between the N-terminal myc tag and catenin binding motif (Brantjes et al., 2002), but outside the corresponding ΔXLef1 or ΔXTcf3 coding sequence (Hsu et al., 1998; Staal these regions, they differ significantly. For example, although et al., 1999). XLef1HMGEnR (aa 266 to 359) and XTcf3HMGEnR (aa 320– it is believed that all Lef/Tcf factors can bind Groucho/TLE 412) were created by placing the HMG domain into a pCS2 expression vector incorporating the engrailed repressor domain and an N-terminal myc tag (D. family corepressors (Brantjes et al., 2001; Cavallo et al., Turner). The β-catenin binding domain of XTcf3 (aa 2–87) and XLef1 (aa 2– 1998; Daniels and Weis, 2005; Levanon et al., 1998; Roose 97) was N-terminally myc tagged in pCS2 to create NTCF and NLEF et al., 1998), only certain Tcfs can recruit the unrelated respectively, and includes an NLS engineered in at the C-terminus corepressor, CtBP, through motifs present in their extended (PKKKRKV) (Hamilton et al., 2001; Molenaar et al., 1996). For ΔXLef1- Δ – C-terminus (Brannon et al., 1999). Such structural variations Tailswitch, the N-terminal portion of XLef1 (aa 110 344) was fused to the C-terminal portion of XTcf3 (aa 398–554). Δβ-Catenin was constructed by between Lef/Tcf factors are likely to underlie many of the removing the N-terminal region that confers instability (aa 1–167) (Yost et al., functional differences that have emerged from studies in 1996) and inserting the remaining reading frame into a pCS2 incorporating six Xenopus. For example, using constitutively repressing N-terminal myc tags. The dominant inhibitory FGFR1 (dnFGFR), constitu- mutants that are unable to bind β-catenin, Roel et al. tively active BMPR (caBMBR), and Neurogenin expression constructs have demonstrated that XTcf3 is required to mediate the Wnt- been previously described (Amaya et al., 1991; Candia et al., 1997; Ma et al., 1996). dependent dorsalization of the embryonic axis, whereas XLef1 is required for subsequent Wnt-mediated ventraliza- Embryo manipulations and morpholinos tion of the mesoderm (Roel et al., 2002). The presence or absence of “LVPQ” and “SXXSS” motifs in the region N- All results shown are representative of at least two independent terminal to the HMG domain was shown to underlie the experiments. Collection, injection, and in situ hybridization of Xenopus distinct activities displayed by these Lef/Tcf factors during embryos were as described (Bellmeyer et al., 2003; LaBonne and Bronner- Fraser, 1998). RNA for injection was produced in vitro from linearized plasmid mesodermal patterning (Liu et al., 2005), and these motifs templates using the Message Machine kit (Ambion). β-Catenin morpholino have been proposed to function in transcriptional repression (Heasman et al., 2000) was injected into one cell at the eight-cell stage. (Gradl et al., 2002). Embryos were staged according to Nieuwkoop and Faber (1967). For animal E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 73 cap assays, embryos were injected with the indicated RNA into the animal pole Results of both cells at the two-cell stage. In the case of the β-cateninΔXTcf3 experiment, embryos were injected with Chordin or Noggin at the two-cell stage and then with nβ-gal or nβ-gal+β-cateninΔXTcf3 into one cell at the Wnt unresponsive Lef/Tcf factors differ in their ability to inhibit four-cell stage. Ectodermal explants were removed from the embryo using hair neural crest formation knives at the blastula stage and cultured in 1xMMR until sibling embryos reached the stage indicated, at which time they were harvested and processed Given their distinct roles in the formation of mesodermal for in situ hybridization or RT-PCR. tissues (Liu et al., 2005; Roel et al., 2002), we wished to determine if functional differences between XLef1 and XTcf3 RT-PCR might contribute to the diversity of responses to canonical Wnt Total RNA was isolated from animal caps and whole embryos as previously signals observed during ectodermal patterning. To this end, we described (LaBonne and Bronner-Fraser, 1998). Reverse transcription was generated mutations in XLef1 and XTcf3 that delete the β- carried out on RNA from 10 animal cap equivalents using MMLV-RT (100U, catenin binding domain (ΔXLef1 and ΔXTcf3) and therefore USB) at 45°C for 1 h in a 20 μl reaction containing 1× First Strand Buffer render these proteins Wnt-unresponsive. Analogous mutations (USB), 2 mM DTT, 0.5 mM of each dNTP, 10 U RNAsin (Promega), and 0.5 have been used in numerous other studies to interfere with OD units oligo(dT) (Pharmacia). 2 μl of RT sample was utilized per PCR reaction. PCR reactions were carried out in a 25 μl volume in the presence of canonical Wnt signaling, as such mutations cause Lef/Tcf trace 32P[αdCTP] using an annealing temperature of 55°C and 22 cycles. Primer family proteins to function as constitutive repressors (Bang et pairs for PCR were as follows: L32: Up: 5′-AAAGCCACTTTTGCTGGCTA- al., 1999; Huber et al., 1996; Molenaar et al., 1996; Reya and 3′; Dn: 5′-CACCTTTCCCCAGATCACAC-3′; Xbra (LaBonne and Whitman, Clevers, 2005; Roel et al., 2002). mRNA encoding epitope- ′ ′ ′ 1994): Up: 5 -GGATCGTTATCACCTCTG-3 ; Dn: 5 -GTGTAGTCTGTAG- tagged ΔXLef1 or ΔXTcf3 was injected into one cell of two- CAGCA-3′; Sox3 (Penzel et al., 1997): Up: 5′-TGATGCAGGACCAG- TTGGGC-3′; Dn: 5′-TGAAGTGAAGGGTCGCTGGC-3′. cell stage Xenopus embryos, and the injected embryos were cultured to stages at which the effects on neural crest markers Western blots couldbeassayedusinginsitu hybridization. In these experiments, mRNA encoding β-galactosidase was coinjected Xenopus embryos, injected as described above, were lysed in 20 μl per as a lineage tracer and Western blots against the epitope tags embryo of 1× PBS 1% NP40. Lysates were cleared by centrifugation, resolved were used to ensure that the proteins were being expressed at by SDS Page, and blotted to nitrocellulose. Expressed proteins were detected equivalent levels in experiments in which their activities were with antibodies against the epitope tag (anti-myc 9E10, Santa Cruz Δ Biotechnology) or protein (anti-actin, Sigma) and HRP-conjugated secondary being compared. We found that expression of XTcf3 blocked antibodies (goat anti-mouse, Zymed), and were visualized by chemilumines- formation of neural crest precursor cells, as seen by the loss of cence (Pierce). Slug and Sox9 expression (Figs. 1Aii, iv). By contrast, ΔXLef1

Fig. 1. ΔXLef1 and ΔXTcf3 differ in their ability to influence neural crest induction. (A) Embryos injected with mRNA encoding β-gal and ΔXLef1 nMT (i, iii, v) or ΔXTcf3 nMT (ii, iv, vi) were examined by in situ hybridization for expression of Slug (i, ii); Sox9 (iii, iv); and Msx1 (v, vi), with “p.” denoting probe used. All embryos are positioned showing anterior/dorsal side, with their injected side to the right (arrow). ΔXTcf3 potently blocks expression of neural crest markers while ΔXLef1 does not. (B) Schematic representation of constructs utilized in these experiments compared to full-length coding regions. (C) Western blot demonstrating that ΔXLef1 and ΔXTcf3 were expressed at equivalent levels in these experiments; blot was stripped and re-probed for actin as a loading control. (D) Expression of either ΔXLef1 nMT or ΔXTcf3 nMT leads to an increase in Opl/Zic1 expression. Light blue staining is lineage tracer β-gal. 74 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 had either no effect or led to a modest increase in the expression of these markers (Figs. 1Ai, iii). Like these definitive neural crest markers, expression of the neural plate border marker Msx-1 has also been shown to be dependent on canonical Wnt signals (Bang et al., 1999; Monsoro-Burq et al., 2005). We therefore asked if ΔXLef1 and/or ΔXTcf3 could interfere with Msx-1 expression. As found for Slug and Sox9, expression of Msx-1 was blocked in embryos injected with ΔXTcf3 but not ΔXLef1 (Figs. 1Av, vi). Differences noted in the ability of these inhibitory mutants to prevent neural crest formation reflect true differences in the activity of these factors, as the proteins were expressed at similar levels as shown by Western blot (Fig. 1C). Importantly, the ΔXLef1 mutant is active in these assays; a significant lateral expansion of the expression domain of Opl/ Zic1 was noted in ΔXLef1 injected embryos, and here the effects were comparable to those mediated by ΔXTcf3 (Fig. 1D). Together these findings indicate that XTcf3 and XLef1 do not function equivalently during patterning of the embryonic ectoderm and suggest that the transcriptional response to neural crest inducing signals might differ depending upon which of these transcription factors is present and bound to DNA. While epitope tagged forms of ΔXTcf3 and ΔXLef1 were used in these experiments to allow precise control of expressed protein levels, untagged forms of these proteins have comparable effects (Supplemental Fig. 1).

ΔXTcf3-injected embryos have dramatically enlarged neural Δ plates Fig. 2. XTcf3 dramatically expands expression of neural plate markers. (A) Embryos injected with mRNA encoding β-gal and ΔXLef1 (i, iii) or ΔXTcf3 (ii, iv) were examined by in situ hybridization for expression of Sox3 (i, ii, iv) or The expanded expression of Opl/Zic1 in ΔXLef1 and Opl/Zic1 (iii). The lateral view of the embryo in ii is shown in iv. ΔXLef1 ΔXTcf3-injected embryos is noteworthy, as this factor is injected embryos show a modest increase in Sox3 and Opl/Zic1, whereas frequently described as a neural crest marker. However, Opl/ ΔXTcf3-injected embryos show dramatic increase in Sox3 expression. (B) Zic1 expression marks a region of the early ectoderm that DXTcf3 expands expression of neuronal progenitor markers Sox2 (i) and Nrp1 (ii). Red staining is from lineage tracer β-gal. Arrowheads indicate injected side. includes both neural crest, placodal, and dorsal CNS precursors, and in our hands, its expression is consistently expanded under conditions where neural crest precursors are lost (Bellmeyer using this mutant leads to the formation of a dramatically et al., 2003; Light et al., 2005). Moreover, we have found that expanded neural plate. experimental manipulations that lead to an increase Opl/Zic1 expression are frequently accompanied by expanded expression ΔXTcf3 inhibits placode and anterior neural plate border of neural plate markers such as Sox3 (Bellmeyer et al., 2003; markers Light et al., 2005). We therefore examined Sox3 expression in embryos injected with ΔXLef1 or ΔXTcf3. A modest increase While Sox2 and Sox3 are predominantly markers of the in Sox3 expression was noted in many embryos injected with multipotent CNS progenitor pool, expression of each of these ΔXLef1 (Fig. 2Ai). Unexpectedly, and in marked contrast to factors is also found in the pan-placodal region of the transverse what we observed in ΔXLef1-injected embryos, we found a neural fold. This is significant, as a recent study using avian dramatic upregulation of Sox3 in ΔXTcf3-injected embryos embryos suggested that inhibition of Wnt signaling is required (Figs. 2Aii, iv). This expansion was noted along the full length to position the placodal region in this system (Litsiou et al., of the A–P axis, and at high doses of injected mRNA (100 pg) 2005). Thus, the expanded expression of Sox2 and Sox3 seen in the entire expanse of the lateral ectoderm/flank expressed Sox3 ΔXTcf3-injected embryos could theoretically reflect an in the majority of the embryos (N=100, Figs. 2Aii, iv). To increase in cells adopting placodal fates. Arguing against such determine if this dramatic expansion of Sox3 expression in an interpretation is the expanded expression of Nrp1 in these ΔXTcf3-injected embryos was specific to this gene or instead embryos (Fig. 2Bii). Nrp1, an RNA binding protein implicated reflected a more general expansion of the neural plate, we also in neuronal maintenance (Good et al., 1998; Okano examined expression of the neural plate markers Sox2 and et al., 2002; Richter et al., 1990; Sakakibara et al., 1996), is Nrp1. We found that similar to Sox3, the expression of both expressed throughout the newly formed neural plate (Richter Sox2 and Nrp1 was greatly expanded in ΔXTcf3-injected et al., 1990) but, unlike Sox2 and Sox3, it does not mark the embryos (Fig. 2B), indicating that inhibition of Wnt signals placodes. Moreover, we had observed that ΔXTcf3 inhibited E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 75 the expression of c-myc not only in neural crest forming crest forming regions of the neural fold, ΔXLef1 did not and regions, but also in adjacent placodal regions (Figs. 3iii, iv). indeed frequently led to expanded expression of c-myc in this In addition to being expressed at the neural plate border in a region. region encompassing both neural crest and placodal precursors, The loss of anterior c-myc expression in ΔXLef1- and c-myc is also expressed in regions of the anterior/transverse ΔXTcf3-injected embryos was quite unexpected, as current neural fold. These c-myc expressing cells will not contribute to models for the role of canonical Wnt signaling in anterioposter- neural crest but instead give rise to sensory placodes and ior neural patterning suggest that these signals should not be portions of the forebrain (Bellmeyer et al., 2003). We found that active in anterior regions of the neurula, and that inhibiting Wnt both ΔXLef1 and ΔXTcf3 inhibited c-myc expression in the signals should increase rather than decrease the expression of transverse neural fold (Figs. 3i–iv), further supporting the anterior markers. To further examine the fate of transverse hypothesis that the expanded Sox2/Sox3 expression domain in neural fold-derived cell types in ΔXLef1 and ΔXTcf3-injected ΔXTcf3-injected embryos is not placodal in nature. Interest- embryos, we targeted these mutants to anterior-most regions of ingly, whereas ΔXTcf3 inhibited expression of c-myc in neural the ectoderm, and then examined the expression of the pan-

Fig. 3. ΔXTcf3 inhibits placode and anterior neural plate border markers. Embryos injected with mRNA encoding ΔXLef1 or ΔXTcf3 and the lineage tracer β-gal were examined by in situ hybridization (Stage 14) for cement gland and placodal markers with “p.” denoting probe used. Inhibition of c-myc expression in ΔXLef1- and ΔXTcf3-injected embryos is not restricted to the neural crest forming regions; expression in the anterior/transverse neural fold is also inhibited (i–iv). The placodal component of the Sox3 expression domain is inhibited by ΔXLef1 (v, vi) and ΔXTcf3 (vii, viii). Six1 expression was largely unaffected by ΔXLef1 (ix, x), while FoxL1C expression was shifted more posterior in some (xii) but not all (xiv) ΔXLef1-injected embryos. ΔXTcf3 inhibited both Six1 (xi, xii) and FoxL1C (xv, xvi) expression. Both ΔXLef1 and ΔXTcf3 inhibited the cement gland marker, CG-1 (xvii–xx), although ΔXTcf3 did so more potently. Arrow indicates injected side. Red stain is lineage tracer β-gal. 76 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 placodal markers Six1 (Figs. 3ix–xii) and FoxL1c (Figs. 3xiii– restricted to forebrain fates. We also wished to ascertain whether xvi), the cement gland marker CG-1 (Figs. 3xvii–xx) and the the overall A–P pattern of the neural plate had been perturbed. placodal component of Sox3 expression (Figs. 3v–viii) at Accordingly, we first examined whether Otx2, a marker of neural plate stages. We found that each of these markers was forebrain and anterior midbrain, was expressed throughout the inhibited by ΔXTcf3, confirming that there is no increase in the expanded neural plates induced by ΔXTcf3. mRNA encoding formation of placode-fated cells in these embryos. Importantly, ΔXTcf3 was injected into one animal blastomere at the eight-cell the inhibition of both transverse neural fold and cement gland stage in order to target the ectoderm, and we injected a 5-fold markers in ΔXTcf3-injected embryos provides additional lower dose of mRNA (20 pg) to avoid distortions of neural plate evidence that Wnt signals are required for proper development morphology due to excessive ectopic neural tissue. As seen with of the anterior-most region of the neural plate, as opposed to injections at the two cell stage, enlargement of the neural plate only caudal CNS fates. ΔXLef1 proved to be a less potent was seen along the anterior–posterior axis as evidenced by a inhibitor of CG-1 than ΔXTcf3 (Figs. 3xvii–xx) and did not laterally expanded domain of Sox3 expression (Figs. 4Ai, ii). inhibit Six1 and FoxL1c (Figs. 3ix, x, xiii, xiv), further While some increase in Otx2 expression was frequently noted on supporting the hypothesis that these two transcription factors the injected side of these embryos (Figs. 4Aiii, iv), this regulate distinct targets in the ectoderm. Interestingly, ΔXLef1 expansion was limited to anterior most regions, even in embryos but not ΔXTcf3 sometimes mediated a caudal shift in the in which the lineage tracer β-gal was present along the full expression domain of FoxL1c (Fig. 3xiii), consistent with a role anterior–posterior extent of the enlarged neural plate (Figs. in A–P patterning of the neural plate. 4Aiii, iv). These findings indicate that the ectopic neural tissue induced by targeted expression of ΔXTcf3 in the ectoderm is not ΔXTcf3-mediated expansion of the neural plate can be obligatorily “anterior” in nature. uncoupled from effects on A–P patterning of the CNS To determine whether ΔXTcf3-mediated expansion of the neural plate could be uncoupled from effects on A–P patterning The dramatic increase in Sox3 expression throughout regions or convergence/extension, we used in situ hybridization to of the lateral ectoderm normally fated to become epidermis, examine a range of markers that mark spatially restricted together with the inhibition of placodal markers, indicates that domains along the A–P neural axis. The markers examined the overall size of the neural plate has been greatly expanded in included Krox20, a hindbrain marker (4Biii), En2, which marks ΔXTcf3-injected embryos. Moreover, the loss of anterior/ the midbrain/hindbrain junction (4Biv), Pax6, which marks the transverse neural fold markers suggests that this ectopic neural presumptive eye field as well as more posterior portions of the tissue is not obligatorily anterior in character. However, because CNS (4Bv), and XBF-1 (4Bvi), and Xanf (4Bvii) both of which during the establishment of the embryonic axes, antagonization mark the forebrain. In experiments in which ΔXTcf3 elicited of Wnt signals by factors such as Dickkopf has been linked to substantial increases in Sox3 expression along the anterior– head induction (Bouwmeester et al., 1996; Glinka et al., 1997, posterior axis (Fig. 4Bi) no substantial displacement of the 1998; Mukhopadhyay et al., 2001), we nevertheless wished to above noted A–P markers was noted on the injected side determine if the ectopic neural tissue induced by ΔXTcf3 was relative to the unmanipulated/control side of the embryo (Figs.

Fig. 4. ΔXTcf3-mediated expansion of the neural plate can be uncoupled from effects on A–P patterning. (A) Embryos injected into one cell at the eight-cell stage with mRNA encoding ΔXTcf3 show expanded expression of Sox3 along the entire A–P axis (i, ii). However, sibling embryos show little or no expansion of Otx expression (iii, iv); note the lineage tracer, β-gal (light blue stain) along the entire A–P axis and highlighted by red arrow. (B) At doses of ΔXTcf3 RNA that expand Sox3 (i), the mesoderm marker Muscle Actin (ii) appears relatively unaffected on the injected side (arrow). A–P neural markers show normal expression on the injected side (arrow) relative to contralateral control. Markers examined are (iii) Krox20; (iv) En-2; (v) Pax6; (vi) BF-1: (vii) Xanf (viii) Otx with “p.” denoting probe used. E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 77

4Biii–viii). Moreover, in these experiments, no effects on embryos (Figs. 5Avi, viii), indicating that despite the muscle actin were noted (Fig. 4Bii), indicating that the ΔXTcf3-mediated increase in the size of the multipotent CNS ectodermally targeted ΔXTcf3 had not affected the develop- progenitor pool, the ectopic Sox2/3 expressing cells were ment of the underlying mesoderm. unable to progress to form differentiated neurons. Moreover, we found that expression of ESR7, a Notch target gene that is The increased size of the multipotent CNS progenitor pool is induced in cells responding to lateral inhibition, and thus not accompanied by an increase in neuronal differentiation blocked from neuronal differentiation, was increased in ΔXTcf3-injected embryos (Fig. 5Aiv). In contrast to these We next wished to determine if the increased size of the findings, we found that ΔXLef1 had little or no effect on neural plate observed in ΔXTcf3-injected embryos was neuronal differentiation as assayed by Xash or Delta expression, accompanied by a corresponding increase in neuronal differ- and did not increase ESR7 expression (Figs. 5Aiii, v, vii). entiation. Accordingly, we examined the expression of markers Together, the above results suggested that Tcf3-mediated of differentiating neural cells such as Xash and Delta. Xash, a Wnt signals may play multiple temporally distinct roles during homolog of the Drosophila Acheate Scute-complex,isa CNS development—they must first be inhibited to allow transcription factor that has been shown to promote neural specification of the neuronal progenitor pool but may subse- differentiation (Ferreiro et al., 1994), while Delta is a Notch quently be required for neuronal differentiation to proceed. To ligand expressed in differentiating primary neurons (Chitnis et test this hypothesis, we wished to induce ectopic neuronal al., 1995). Somewhat surprisingly, we found that expression of differentiation at a point in the neurogenic cascade downstream both of these markers was inhibited in ΔXTcf3-injected of “neural induction,” and then ask whether ΔXTcf3 could

Fig. 5. ΔXTcf3 expands the neuronal precursor pool but inhibits neuronal differentiation. (A) mRNA encoding ΔXTcf3 or ΔXLef1 was coinjected with β-gal into one blastomere at the two-cell stage. Embryos were examined by in situ hybridization at stage 18 for expression of Sox3 (i, ii); ESR7 (iii, iv); Xash (v, vi); and Delta (vii, viii). All embryos are viewed dorsally with anterior down. Blue staining represents β-gal staining and arrows indicate injected side. ΔXTcf3 expands expression of Sox3 (ii), and ESR7 (iv), but inhibits markers of neuronal differentiation including Xash (vi) and Delta (viii). While ΔXLef1-injected embryos show a limited expansion of markers of neuronal precursors, there is no significant effect on markers of neuronal differentiation. (B) Embryos were injected with mRNA encoding β-gal and ΔXTcf3, Neurogenin, or both. Arrows indicate injected side. (i) ΔXTcf3 inhibits N-tubulin. Embryos injected with mRNA encoding Neurogenin induces ectopic neural differentiation marked by N-tubulin. (iii) ΔXTcf3 is able to block ectopic neural differentiation caused by Neurogenin. 78 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 block this neuronal differentiation. The proneural bHLH protein repression through interaction with Wnt target promoters. Neurogenin is a potent inducer of the neurogenic cascade in Instead, these proteins block canonical Wnt signals by binding early Xenopus embryos and can induce the expression of β-catenin and preventing it from interacting with endogenous neuronal differentiation markers throughout the ectoderm (Ma Lef/Tcf family transcription factors and activating transcription. et al., 1996; Talikka et al., 2002). Consistent with this, embryos When NLEF and NTCF were targeted to the ectoderm of Xe- injected with Neurogenin mRNA display widespread N-tubulin nopus embryos, we found that both factors were able to inhibit expression in the ectoderm at neural plate stages (Fig. 5Bii). Slug expression (Figs. 6Biii, iv) and expand Sox3 expression Coexpression of ΔXTcf3 significantly blocked neurogenin-me- (Figs. 6Bvii, ix). diated N-tubulin expression (Fig. 5Biii), supporting a role for The above results demonstrate that inhibition of active Wnt signaling in late events leading to neuronal differentiation. canonical Wnt signals, and not simply promoter-dependent repression of Wnt target genes, underlies the loss of neural crest Inhibition of Wnt/β-catenin signals induces neural tissue precursors and the expansion of the neural plate observed in the previous experiments. To confirm these findings, we wished to Lef/Tcf family transcription factors not only activate use an additional, cell autonomous, means of inhibiting transcription of Wnt target genes in response to canonical canonical Wnt signals. To this end, we took advantage of Wnt signals but also actively repress these target genes in the previously characterized morpholinos that are able to potently absence of ligand stimulation. While both Lef1 and Tcf3 can deplete β-catenin from early Xenopus embryos (Heasman et al., bind Groucho/TLE family transcriptional corepressors, the 2000). Consistent with our other findings, embryos injected in Tcf3 protein has binding sites for an additional corepressor, one animal blastomere at the eight-cell stage with β-catenin CtBP, in its extended C-terminus (Brannon et al., 1999; morpholinos showed inhibition of Slug and expanded expres- Brantjes et al., 2001; Daniels and Weis, 2005; Levanon et al., sion of Sox3 at neural plate stages (Figs. 6Bv, x). Together, 1998; Roose et al., 1998). Moreover, in addition to these data demonstrate that inhibition of canonical Wnt signals differences in corepressor binding, there is evidence that in the embryonic ectoderm leads to a loss of neural crest Lef/Tcf family proteins can differ with respect to the precursors and a greatly expanded CNS progenitor domain. transcriptional coactivators they recruit to target promoters Given our findings that ΔXLef1 and ΔXTcf3 differ in their (Hecht and Stemmler, 2003). To begin mapping the protein ability to inhibit Wnt/β-catenin signals, what underlies this domains that underlie the differential activities that these difference? As these proteins differ in their ability to recruit proteins display during ectodermal patterning, we first asked CtBP, we wished to determine if the functional differences they whether rendering XLef1 and XTcf3 equivalent with respect exhibit reside in their C-terminal tails. Accordingly, we replaced to their transcriptional repressor activity would make them the portion of ΔXLef1 C-terminal to the HMG domain with the equally potent in their ability to inhibit neural crest formation corresponding region of ΔXTcf3 and asked if this domain swap and expand the neuronal progenitor domain. To this end, the was sufficient to make the resultant chimeric protein behave like DNA binding domains of each of these factors were fused in ΔXTcf3 in ectodermal patterning assays. We found that the frame to the strong constitutive repression domain from ΔXLef1 Tailswitch protein was an efficient inhibitor of Slug Engrailed to generate XLef1HMGEnR and XTcf3HMGEnR. expression (Figs. 6Ciii, iv), behaving indistinguishably from We found that in embryos injected with either XLef1HM- ΔXTcf3 in this assay (Figs. 6Ci, ii). By contrast, although the GEnR or XTcf3HMGEnR formation of neural crest precur- chimeric protein proved a better inducer of ectopic Sox3 than sors was blocked, as evidenced by loss of Slug expression at ΔXLef1 (Figs. 6Cvii, viii, and not shown), it did not expand the the neural plate border (Figs. 6Bi, ii). In addition, both of neural plate as potently as ΔXTcf3 (Figs. 6Cv, vi). These data these repressor fusion proteins dramatically expanded Sox3 suggest that the molecular mechanisms that underlie neural crest expression to an extent equal to or greater than that achieved inhibition and neural plate expansion may be at least partially using ΔXTcf3 (Figs. 6Bvi, vii). These results demonstrate distinct. that the functional differences between ΔXLef1 and ΔXTcf3 lie outside their DNA binding domains. Upregulation of Wnt/β-catenin signals inhibits neural We next wished to determine if the ability of ΔXTcf3, induction XLef1HMGEnR, and XTcf3HMGEnR to expand the neuronal progenitor domain is due to their function as constitutive In the above-described loss of function experiments, four transcriptional repressors of Wnt target promoters, or alterna- different methods of inhibiting Wnt signaling all resulted in tively, if it is due to their ability to dominantly inhibit Wnt/β- ectopic neural induction. These findings suggest that during catenin signals. To distinguish between these possibilities, we normal development, canonical Wnt signaling plays an constructed XLef1 and XTcf3 deletion mutants consisting of important role in restricting the size of the neural plate by only the N-terminal β-catenin binding domains of these actively suppressing neural fates. If this model is correct, then transcription factors (NLEF and NTCF, modeled after pre- upregulation of Wnt signals within the prospective neural viously described constructs (Hamilton et al., 2001; Molenaar et progenitor domain might be expected to inhibit neural al., 1996). These constructs lack both the corepressor binding induction. To test this hypothesis, we used a stabilized form sites and DNA binding domain of the respective full-length Lef/ of β-catenin (Δβ-catenin) that functions as a constitutive Tcf factors, and therefore are unable to mediate transcriptional activator of canonical Wnt signaling (Yost et al., 1996). In E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 79

Fig. 6. Inhibition of Canonical Wnt signals induces neural tissue. (A) Schematic showing the XLef1 and XTcf3 constructs utilized in these experiments compared to full-length coding regions. (B) Embryos were injected in one blastomere at the two-cell stage with mRNA encoding constitutively repressing forms of XLef1 or XTcf3 (XLef1HMGEnR i, vi; XTcf3HMGEnR, ii, vii); the β-catenin binding domain of XLef1 or XTcf3 (NLEF, iii, viii; NTCF, iv, ix); or were injected in one blastomere at the eight-cell stage with morpholinos targeting β-catenin (v, x). For all means of inhibiting canonical Wnt signals, expression of the neural crest marker Slug was inhibited (i–v) and expression of the pan-neuronal marker Sox3 was dramatically expanded (vi–x). (C) When expressed at the same level, ΔXTcf3 and ΔXLef1 Tailswitch have equivalent ability to inhibit Slug (i–iv). However, ΔXTcf3 expands Sox3 expression (iii, iv) more potently than ΔXLef1 Tailswitch (vii, viii). 80 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 addition, we constructed fusion proteins in which the stabilized animal cap assays. Animal caps explanted from noggin- or β-catenin was fused in frame to our ΔXLef1 or ΔXTcf3 chordin-injected embryos strongly expressed Sox3 throughout proteins (β-cateninΔXLef1 and β-cateninΔXTcf3). mRNA the explants, in contrast to control explants or those explanted encoding either Δβ-catenin, β-cateninΔXLef1, or β-cate- from β-cateninΔXTcf3-injected embryos (Fig. 8A and not ninΔXTcf3 was coinjected with mRNA encoding β-gal into shown). Significantly, explants from embryos coinjected with one dorsal animal blastomere of eight-cell stage embryos. noggin and β-cateninΔXcf3 also failed to express Sox3 (Fig. Consistent with previous findings (LaBonne and Bronner- 8A and not shown). This result indicates that activation of Wnt/ Fraser, 1998; Wu et al., 2005), embryos expressing Δβ-catenin β-catenin signals is sufficient to block neural induction showed expanded Slug expression (Fig. 7Bi). Embryos mediated by BMP attenuation, providing further support for a expressing β-cateninΔXLef1 or β-cateninΔXTcf3 showed a model in which canonical Wnt signals restrict the size of the similar increase (Figs. 7Bii, iii). When the lineage tracer β-gal neuronal progenitor domain endogenously. Furthermore, the (and therefore β-cateninΔXLef1 or β-cateninΔXTcf3) was ability of β-cateninΔXTcf3 to inhibit neuronal progenitor fates localized within the prospective neural plate, expression of the is cell autonomous. When β-cateninΔXTcf3 was expressed in neural plate markers Sox3 and Nrp1 was inhibited (Figs. 7Biv– only a portion of the noggin-expressing explants, only the β- ix). Importantly, this inhibition was noted along the A–P axis, cateninΔXTcf3-expressing region of the explant (as marked by and in medial as well as lateral regions of the neural plate (Figs. the lineage tracer beta-gal) was prevented from expressing Sox3 7Bv–vi), indicating that Wnt/β-catenin signals inhibit neuronal (Fig. 8B and not shown). Importantly, the ability of Wnt/β- progenitor formation at all axial levels. Coexpression of catenin signals to inhibit neural induction could be distin- ΔXTcf3 with β-cateninΔXTcf3 was sufficient to rescue these guished experimentally from the neural crest inducing activity effects (Fig. 7C). of these signals. Neural crest induction by the combined action of Wnts and BMP antagonists is sensitive to activity levels of Neural induction following Wnt inhibition requires BMP both signaling pathways (LaBonne and Bronner-Fraser, 1998). attenuation We find that β-cateninΔXTcf3 is a potent inhibitor of Sox3 expression in animal cap assays under experimental conditions The above findings indicated that Wnt/β-catenin signals that are not permissive for neural crest induction (Supplemental play a previously unrecognized role in the process of neural Fig. 2). This indicates that the inhibition of neural progenitor induction and raised the question of whether the inhibition of fates in this assay is independent of, and not an indirect these signals might be sufficient for neural induction in the consequence of, neural crest induction. way that BMP inhibition has been proposed to be under the Consistent with the hypothesis that blocking both BMP and neural default model. To address this, and to more generally Wnt signals is necessary for neural induction, we found that the put our results in the context of previous work on neural expansion of the neural plate in ΔXTcf3-injected embryos induction, we asked if ΔXTcf3-mediated inhibition of Wnt could be significantly rescued by upregulation of BMP signals signaling was sufficient to induce neuronal precursor fates in using a constitutively active BMP receptor (Candia et al., 1997). animal caps. Embryos were injected at the two-cell stage with When caBMPR was targeted to the neural ectoderm a mRNA encoding ΔXTcf3, and animal pole ectoderm was significant reduction in neural plate size was noted, and explanted at blastula stage and cultured to stage 19 when it similarly, the expanded Sox3 expression characteristic of was examined by RT-PCR for the expression of Sox3.In ΔXTcf3-injected embryos was largely reversed following contrast to the BMP antagonist noggin, which potently coexpression of caBMPR (Fig. 8C). Given recent evidence induced expression of this neural marker, no significant implicating FGF in vertebrate neural induction, we also induction of Sox3 was observed in animal caps expressing examined what effect blocking FGF signaling would have on ΔXTcf3, suggesting that the neural induction mediated by this ΔXTcf3-mediated neural induction. We found that a dominant mutant in whole embryos requires additional signals not negative FGF receptor alone led to a minimal reduction in Sox3 present in explants (not shown). Since recent work has expression and primarily at the lateral margins of the neural implicated FGF signaling in neural induction in both Xenopus plate (Fig. 8C). By contrast, coexpression of this inhibitory and chick (Stern, 2005) we asked if FGF could cooperate receptor significantly reduced the extent of ectopic Sox3 with Wnt inhibition to induce neural fates in this assay, expression induced by ΔXTcf3. These results suggest that however, no induction of Sox3 was observed in these FGF signaling plays at least a permissive role in the induction of explants expressing FGF8 and ΔXTcf3 (not shown). ectopic neural tissue via Wnt inhibition, possibly through the We next asked if activation of Wnt/β-catenin signals would ability to decrease Smad1 function through Map kinase counteract the neural inducing activity of noggin or chordin in mediated linker phosphorylation (Pera et al., 2003). These

Fig. 7. Activation of Wnt/β-catenin signals inhibits neuronal precursor formation. (A) Schematic showing the XLef1 and XTcf3 constructs utilized in these experiments. (B) Upregulation of canonical Wnt signals via expression of Δβ-catenin (i, iv, vii) or constitutively activating forms of XLef1 or XTcf3 (β-cateninΔXLef1, ii, v, viii; β-cateninΔXTcf3, iii, vi, ix) expands expression of the neural crest marker Slug (i–iii) and inhibits expression of the pan-neural markers Sox3 (iv–vi) and Nrp1 (viii–ix). The red arrow in panels v, vi, and ix point to cleared patches of staining where the Wnt pathway is ectopically activated. (C) The inhibition of Sox3 by β-cateninΔXTcf3 (ii, v) can be rescued by coexpression of ΔXTcf3 (iii, vi). E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 81 82 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86

Fig. 8. Neural induction requires the interplay of BMP, FGF and Wnt signals. (A) (i) Schematic of an animal cap assay. (ii) In situ hybridization examining Sox3 expression in animal caps injected as indicated and cultured to Stage 15. Sox3 expression is induced by BMP attenuation (Noggin expressing caps) but not by β-cateninΔXTcf3. β-CateninΔXTcf3 inhibits Noggin-mediated Sox3 expression when coexpressed. (B) β-CateninΔXTcf3 inhibits Sox3 expression cell autonomously. Embryos were injected with mRNA encoding Noggin in both cells at the two-cell stage (i–iii) and subsequently at the four-cell stage a single blastomere was injected with β-gal alone (i) or β-gal plus β-cateninΔXTcf3 (ii, iii). Note that cells expressing lineage tracer and β-cateninΔXTcf3 do not also express Sox3. (iii) Magnified view of upper explant pictured in ii. (C) Embryos were injected into one cell at the two-cell stage with the indicated mRNA (noted in boxes above the embryo) and β-gal RNA. Embryos were harvested at stage 15 and probed for Sox3. Blocking FGF signals with a dominant inhibitory FGF receptor (dnFGFR) partially rescues expansion of the neural plate seen with ΔXTcf3 alone. Upregulation of BMP signaling with a constitutively active type 1 BMP receptor (caBMPR) also rescues the ΔXTcf3-mediated expansion of the neural plate and also decreases expression of Sox3 within the normal neural plate forming region. These findings indicate that the ability of ΔXTcf3 to induce ectopic neural tissue is dependent on the status of BMP and FGF signals. Arrows indicate injected side. findings further underscore the complex interplay of signals that the predominant neural inducer in ascidians (Bertrand et al., underlies the process of neural induction in vertebrates. 2003; Delaune et al., 2005). In Xenopus, inhibition of FGF signaling has been shown to block neural induction by BMP Discussion antagonists such as Noggin, Chordin or Smad6 (Launay et al., 1996; Sasai et al., 1996). However, it has also been shown that Neural induction and the neural default model FGF-dependent MAP kinase activity downregulates Smad1 activity (Pera et al., 2003) and therefore the extent to which Until recently, the dominant model for neural induction in FGF-mediated neural induction is a consequence of inhibitory vertebrates has been “the default model” which posits that the cross-talk with BMP signaling remains a matter of debate. default state of cells in the embryonic ectoderm is to adopt neural fates (Hemmati-Brivanlou and Melton, 1997). The Inhibition of Wnt/β-catenin signals is required for neural origins of this model stem from experiments demonstrating induction that any experimental manipulation that attenuates the con- stitutive levels of BMP signaling found in explanted ectoderm In the course of studies initiated to examine whether XLef1 (“animal caps”) will cause these explants to differentiate as and XTcf3 play functionally distinct roles in ectodermal pat- neuronal tissue (Harland, 2000). If BMP signaling is not terning, we found ΔXTcf3-injected embryos had dramatically inhibited, these same cells will instead adopt epidermal fates. expanded neural plates. In these embryos, the domain of Sox3 The default model has proven valuable as a paradigm against expression sometimes encompassed the entire expanse of the which subsequent experimental findings could be contextua- lateral ectoderm along the length of the anterioposterior axis lized and evaluated. However, the sum of recent work from a (Figs. 2Aii, iv). Other markers of the multipotent CNS number of model organisms (reviewed in Stern, 2005) has progenitor pool, including Sox2 and Nrp1, were also greatly suggested that the process of neural induction may be more expanded (Fig. 2B and not shown). These findings strongly complex than envisioned by this model. One major challenge to suggested that blocking canonical Wnt signals with ΔXTcf3 is the default model has derived from work suggesting that FGF sufficient to direct presumptive non-neural ectoderm to adopt a signaling is important for neural induction, and that it may be neuronal precursor fate. Importantly, our experiments further E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 83 demonstrate that this dramatic expansion of the neuronal patterning. This is clearly seen in Fig. 4B by comparing the progenitor domain is not due to the misexpression of a manipulated and contralateral sides of ΔXTcf3-injected constitutive repressor of Wnt target promoters, but instead is embryos probed for a range of spatially restricted markers due to the active inhibition of canonical Wnt signals. This is including Krox20, engrailed, and BF-1. Thus, the massive shown by the ability of the β-catenin binding domains from ectopic neural tissue that is induced in these embryos cannot be either XLef1 or XTcf3 to expand the neural plate, as well as by readily explained in the context of alterations in anterioposter- the finding that morpholino-mediated depletion of β-catenin ior neural fates. Moreover, our finding that transverse neural has similar effects (Fig. 6Bviii–x). These findings are consistent fold markers are inhibited in ΔXTcf3-injected embryos with an inhibitory role for canonical Wnt signaling in regulating indicates that Wnt signaling plays an active patterning role neuronal precursor fates in the early ectoderm. Consistent with even in rostral-most regions of the ectoderm. Importantly, our such a role, we find that upregulation of canonical Wnt signals findings do not exclude a role for canonical Wnt signals in A–P in the ectoderm inhibits the expression of pan-neural markers neural patterning but instead indicate that their role in neural such as Sox3 and Nrp1 (Fig. 7B). induction can be experimentally distinguished from any role in While Wnts play a well-established role in dorsal axis A–P patterning, similar to what has been shown for neural crest formation in Xenopus, these signals have not been previously induction (Wu et al., 2005). implicated in the process of neural induction in this system, with Another suggested role for Wnt signaling that is relevant to the exception of one study by Baker et al. (1990). These authors our findings stems from studies examining the molecular proposed that stabilized β-catenin induces neural tissue in Xe- mechanisms of head formation in Xenopus (Bouwmeester et al., nopus ectodermal explants via a mechanism that involves 1996; Glinka et al., 1997). These authors proposed a “two inhibition of BMP4 expression (Baker et al., 1999). At the doses inhibitor model” for axis formation under which the embryonic of Δβ-catenin and β-cateninΔXTcf3 used in our study, we inducers of “head” and “trunk” fates are molecularly distinct, noted no induction of neural markers. The results of that study whereby “head” induction is thought to require inhibition of and the findings that we present here would thus appear to be in both Wnt and BMP signals. Consistent with such a role, the conflict. However, more recent work has shown that when Spemann “organizer” secretes a number of Wnt antagonists in expressed at high levels, stabilized β-catenin can induce addition to its better studied BMP antagonists, including expression of the BMP antagonists noggin and chordin in Cerberus (Bouwmeester et al., 1996), Fzb (Wang et al., 1997), animal caps (Wessely et al., 2001), and this is likely to explain and Dickkopf (Glinka et al., 1997). Indeed, Dickkopf1 was the induction of neural markers observed by Baker et al. (1999). initially isolated as a head inducing factor, and subsequently Moreover, a subsequent study by Domingos et al. has was shown to function by non-productively binding the Wnt demonstrated that the ability of stabilized β-catenin to induce coreceptor LRP5/6 (Bafico et al., 2001; Mao et al., 2001; neural fates in animal caps is always non-cell autonomous Semenov et al., 2001). In these studies of axis formation and (Domingos et al., 2001). This result is fully consistent with our head induction, Wnt antagonists were mainly targeted to the findings that Wnt/β-catenin inhibits neuronal precursor fates in mesoderm and effects on neuronal precursor fates in the a cell autonomous fashion (Fig. 8B). ectoderm were not examined. Nevertheless, it is important to consider our findings in the light of this role for Wnt Wnt signaling and A–P neural patterning antagonization. Significantly, we have shown that when Wnt signals are inhibited in the ectoderm, the massive induction of A number of previous studies have proposed a role for Wnt cells expressing pan neural markers is not accompanied by signaling in the posteriorization of anterior neural tissue in expression of “anterior neural” or forebrain markers, and Xenopus (Fredieu et al., 1997; Houart et al., 2002; Kiecker therefore cannot be explained as an increase in “head” and Niehrs, 2001; Kudoh et al., 2002; McGrew et al., 1995; formation. Nordstrom et al., 2002). These studies reported that embryos in which Wnt/β-catenin signals had been upregulated showed Distinct roles for Lef/Tcf factors in ectodermal patterning displaced expression of genes that mark specific anterioposter- ior domains along the neural axis on the injected side of the If canonical Wnt signals play essential roles in restricting the embryo relative to the control side, and that canonical Wnt size of the neural plate, as well as in neural crest and cranial signals can alter the anterioposterior character of neuralized placode induction, what mechanism is used to dictate the animal caps. However, none of these previous studies precise response to these signals? Several recent reports have examined the effects of canonical Wnt signaling on neural suggested one mechanism by which distinct outcomes might be plate markers. Furthermore, in light of recent evidence that achieved by demonstrating that individual Lef/Tcf family Wnt-mediated effects on neural crest induction can be members can mediate different transcriptional responses to experimentally uncoupled from posteriorization of anterior Wnt signals (Gradl et al., 2002; Labbe et al., 2000; Letamendia neural fates (Wu et al., 2005), we wished to examine if this et al., 2001; Liu et al., 2005; Pukrop et al., 2001; Roel et al., would also be true for effects on neuronal progenitor fates. 2002; Sasaki et al., 2003). Because both Lef1 and Tcf3 are Importantly, we found that we could substantially expand the broadly expressed in the neural plate and the neural crest in size of the neural plate along the entire length of the Xenopus (Molenaar et al., 1998), it seemed possible that these anterioposterior neural axis without dramatic effects on neural factors might also play distinct roles in mediating Wnt 84 E. Heeg-Truesdell, C. LaBonne / Developmental Biology 298 (2006) 71–86 responses in neural crest and/or CNS progenitors. Consistent these same cells from adopting CNS progenitor fates. When with this, we find that ΔXLef1 and ΔXTcf3 differ dramatically Wnt/β-catenin signals are antagonized, not only do neural crest in their ability to influence neural crest, neural plate, and cranial precursors fail to form, but a massive induction of neuronal placode formation in Xenopus. progenitors also results. The direct molecular targets of Wnt Moreover, we found that the functional differences between signals during this process remain to be determined, but seem these factors resides outside of the DNA binding domain, likely to include c-myc, Slug, and at least one SoxD family because when the HMG domain of either XLef1 or XTcf3 is member. Future challenges will include understanding the fused in frame to a constitutive repressor domain, both factors mechanisms by which the combined output of multiple are equally potent at inducing ectopic neural tissue (Figs. 6Bvi, signaling pathways, including Wnts, FGFs, and BMPs is vii). Interestingly, replacing the C-terminus of ΔXLef1 with the integrated and interpreted on target promoters during ectoder- CtBP binding C-terminus of ΔXTcf3 conferred the ability to mal patterning. potently inhibit neural crest formation on this factor but had a less dramatic effect on its ability to expand Sox3 expression, Acknowledgments indicating that these two activities are mechanistically distinct. Beyond differences in their ability to recruit CtBP, Lef/Tcf The authors gratefully acknowledge T. Lallier, R. Harland, family members have other structural differences that could C. Kintner, and T. Moreno for reagents, A. Vernon and members prove to be of functional importance. These include the presence of the laboratory for their valuable discussions. E.H.-T. was or absence of several small domains that have been proposed to supported by the Cellular and Molecular Basis of Disease modulate repressor activity (Pukrop et al., 2001) and which training program (NIGMS). C.L. is a Scholar of the GM Cancer reportedly underlie the distinct activities displayed by Lef/Tcf Research Foundation. This work was also supported by grants factors during mesodermal patterning (Liu et al., 2005). It will from the American Cancer Society and the NIH to C.L. be important to determine the contribution these additional domains make to Lef/Tcf-mediated ectodermal patterning. Appendix A. Supplementary data While no previous work has examined the distinct abilities of different Lef/Tcf family factors to modulate the formation of the Supplementary data associated with this article can be found, neural plate and neural crest, two zebrafish Tcf3 genes have in the online version, at doi:10.1016/j.ydbio.2006.06.015. been shown to play essential roles in head formation and brain patterning (Kim et al., 2000; Dorsky et al., 2003). The zebrafish References Tcf3 mutant “Headless” displays defects in organizer gene expression as early as the onset of gastrulation resulting in Amaya, E., Musci, T.J., Kirschner, M.W., 1991. Expression of a dominant defects in head induction (Kim et al., 2000). These effects can negative mutant of the FGF receptor disrupts mesoderm formation in be rescued with forms of Tcf3 that have constitutive repressor Xenopus embryos. 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