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Canonical Wnt signals have been implicated in multiple events during early embryogenesis, including primary axis formation, neural crest 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.
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 .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.
δXTcf3 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 Opl/Zic1 (iii). The lateral view of the embryo in ii is shown in iv. δXLef1 injected embryos show a modest increase in Sox3 and Opl/Zic1, whereas δXTcf3-injected embryos show dramatic increase in Sox3 expression. (B) DXTcf3 expands expression of neuronal progenitor markers Sox2 (i) and Nrp1 (ii). Red staining is from lineage tracer β-gal. Arrowheads indicate injected side.
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 .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 (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 (xviix), although δXTcf3 did so more potently. Arrow indicates injected side. Red stain is lineage tracer β-gal.
Fig. 4. δXTcf3-mediated expansion of the neural plate can be uncoupled from effects on A 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 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 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 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.
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.
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) and expression of the pan-neuronal marker Sox3 was dramatically expanded (vi). (C) When expressed at the same level, δXTcf3 and δXLef1 Tailswitch have equivalent ability to inhibit Slug (iv). However, δXTcf3 expands Sox3 expression (iii, iv) more potently than δXLef1 Tailswitch (vii, viii).
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 (iii) and inhibits expression of the pan-neural markers Sox3 (ivi) and Nrp1 (viiix). 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).
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 (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.
Supplemental Fig. 1. Embryos injected with mRNA encoding β-gal and untagged δLef1 (i, iii, v) or untagged δXTcf3 (ii, iv, vi) were examined by in situ hybridization for expression of Sox3 (i, ii); Slug (iii, iv); and C-myc (v, vi). All embryos are positioned showing anterior/dorsal side, with their injected side to the right (arrow). Red staining is lineage tracer β-gal. δXTcf3 potently expands the neural plate marker, Sox3, and strongly inhibits markers of the neural crest (Slug and C-myc) and placodal regions (C-myc). At equivalent levels of injected mRNA, δLef1 has little or no effect on expression of these markers. These findings are fully consistent with results using epitope tagged forms of these proteins.
Supplemental Fig. 2. Embryos were injected with mRNA encoding Noggin, β-cateninδXTcf3 or both along with the lineage trace β-gal into two cells at the two-cell stage. Explants were cultured to stage 15 and probed for either Sox3 or Slug. Note that the level of β-cateninδXTcf3 that can inhibit Sox3 in ectodermal explants does not induce Slug when coinjected with Noggin.