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First published online 30 November 2005
doi: 10.1242/dev.02178

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Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

¹ Division of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
² The 21st Century COE program "Cell Fate Regulation Research and Education Unit", Kumamoto University, Kumamoto 860-0811, Japan.
³ Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
⁴ PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
⁵ Department of Oncology, The Hutchison/MRC Research Centre, University of Cambridge, Hills Road, Cambridge CB2 2XZ, UK.

View larger version (123K): [in a new window]   Fig. 1. Expression patterns of X-TSK and marker genes during development. Results of whole-mount in situ hybridization. (A,B) Stage13, anterior views; dorsal is upwards. (A) X-TSK and (B) Xdlx3. Both genes are expressed in the presumptive epidermal region (arrows). (C,D) Stage 14, anterior views. (C) X-TSK and (D) Xbmp4. Both genes are expressed in the anterior neural plate fold (arrowheads). (E,F) Stage 14, dorsal views; anterior is upwards. (E) Bilateral expression of X-TSK outside the neural plate (arrowheads). (F) Xslug expressed in premigratory neural crest cells (arrowheads). (G,H) Stage 23, anterior views. (G) Expression of X-TSK in the hindbrain (hb), lens placode (lp) and cranial neural crest derivative (arrow), but not in the epidermis or cement gland (cg). (H) Expression of Xbmp4 in the roof plate (rp), dorsal retina (dr), cement gland and epidermis (arrow). (I,J) Sections of hybridized embryos. Expression of X-TSK (I) and Xslug (J) in the anterior neural plate region in stage 14 embryos. X-TSK is expressed in the anterior neural fold (nf) and epidermal region (ep) (I), while Xslug is expressed in a narrow area of the neural crest (nc; J). (K,L) Section at the cranial neural crest level at stage 16. Lines indicate the border of each area. (K) X-TSK protein localization. (L) The merged image of K and the image of Xslug hybridized embryo. X-TSK is localized on the surface ectoderm and in the proximal edge of neural crest cells (arrowheads). (M) X-TSK expression at stage 26 was found in mandibular neural crest (m), anterior branchial crest (ab) and posterior branchial crest (pb), and very weakly in hyoid neural crest segments (hy). (N) Sox9, (O) ADAM 13 and (P) Xslug expression at stage 26.

View larger version (96K): [in a new window]   Fig. 2. X-TSK expression requires BMP signaling. Results of the animal cap (AC) assays. (A-H) Animal caps were prepared from stage 8-9 embryos injected with 0.5 ng Chordin (B,E), 0.5 ng Chordin + 1 ng BMP4 (C,F), 1 ng truncated BMP-receptor (tBR) (H), or none of the above (controls; A,D,G). ACs were harvested at the time equivalent to stage 14 and hybridized with XK81 (A-C) or X-TSK (D-H) probes. Chordin downregulated XK81 expression (B), which was rescued by BMP4 (C). The expression of X-TSK was repressed by Chordin (E), but rescued by BMP4 (F). (H) Truncated BMP receptors repressed the expression of X-TSK. (I) X-TSK expression in a normal embryo at stage 17. (J) RT-PCR analysis with ACs harvested at stages 14 (lanes 1-8) and 17 (lanes 9-16). ODC is used as an internal control. X-TSK expression in the ACs was recovered by increasing amounts of BMP4. BMP4 overexpression induced X-TSK expression at both stages. WE, whole embryo; NI, non-injected ACs.

View larger version (23K): [in a new window]   Fig. 3. X-TSK functions as a BMP antagonist in Xenopus. (A) Western blot analysis of X-TSK-Myc-His, C-TSK-Myc-His and XBMP4-Flag protein. Each protein was detected by anti-tag antibody. Co-immunoprecipitation of X-TSK-Myc-His or C-TSK-Myc-His and XBMP4-Flag. After immunoprecipitation, bound BMP4 was detected by immunoblotting with anti-Flag antibody. IP, immunoprecipitation. (B) RT-PCR analysis of X-TSK, Noggin or a mixture of both. The numbers above the columns show the amount of X-TSK mRNA in each cap (ng). Plus or minus indicates co-injection or not of Noggin (0.5 ng).

View larger version (61K): [in a new window]   Fig. 4. Effects of X-TSK depletion using siRNA on ectodermal patterning. (A) A normal Xenopus embryo at stage 17; the arrowhead shows the neural fold. (B) Stage 17; an embryo injected with X-TSK siRNA (X-TSK-si). The arrowhead shows a flattened neural fold and enhanced pigmentation. (C) RT-PCR analysis of embryos injected with siRNA radially into all blastomeres at the four-cell stage. The embryos were harvested at the appropriate stages. ODC is used as an internal control. Co, Control embryo; Si, X-TSK-si-injected embryo. (D-S) Whole-mount in situ hybridization of injected samples at stage 15. The injected samples are indicated in the upper right-hand corner, and the probes are in the lower right-hand corner. All pictures show the injected side on the right. The results are described in Table 1, except M-O,R,S. (D,F) An embryo injected with C-TSK siRNA (C-TSK-si). The expression of Sox9 and Zic5 was unchanged (arrowheads). (E,G) An embryo injected with X-TSK siRNA (X-TSK-si). The expression Sox9 or Zic5 was not observed (arrowheads). (H) Sox2 expression was not disturbed in the X-TSK-si-injected embryo. The bars indicate the width of neural plates. (I) Epidermal keratin was activated in the X-TSK-si-injected side (arrowheads). (J) Xrx1 expression was not disturbed in the X-TSK-si-injected side (arrowhead). (K) Xbmp4 expression was not activated (arrowhead). (L,M) Results of the rescue experiment with co-injection of X-TSK-si and mRNAs. (L) X-TSK-si (0.5 pmol) injection diminished Xslug expression (arrowhead). (M) X-TSK-si (0.5 pmol) and C-TSK mRNA (0.5 ng) were injected into one blastomere of a stage 2 embryo. Xslug expression was weak, but regionally rescued (arrowhead). (N,O) X-TSK-si (0.5 pmol) and X-TSK mRNA (250 pg) were injected. (N) Sox9 expression was slightly extended alongside ß-gal staining (arrowhead). (O) Xslug expression was restored to the same level as control side (arrowhead). (P,Q) An embryo injected with a morpholino oligo of X-TSK (X-TSK MO) (5 ng). Neural crest marker expression, Sox9 (P) and Xslug (Q) levels were decreased. (R,S) X-TSK MO phenotype is restored by X-TSK in which the signal peptide is replaced with N-cadherin signal peptide. The sequence around the initiation codon, which is a MO target, is swapped for the N-cadherin sequence. (R) Sox9 expression was observed alongside ß-gal-positive cells (arrowhead). (S) Xslug expression was restored (arrowhead). (T-W) The pre-neural crest genes in injected embryos. (T,V) C-TSK-si did not change the expression of Hairy2A or Msx-1 (arrowheads). (U,W) X-TSK-si diminished Hairy2A and Msx-1 expression in the cranial to trunk lateral neural plate (open arrowheads). (X) Triple staining sections. Sox9 and XK81 phenotypic embryos are sectioned, and stained using anti-X-TSK antibody. (Top) Sox9 expression is missing in the ß-gal-positive region (left, open arrowhead) and neural crest expression of X-TSK has also disappeared in the injected side (left). Normal expression of X-TSK protein was observed as red fluorescence (right, triangle). (Middle) The border of epidermis is indistinguishable in anterior neural plate area; X-TSK protein levels are diminished in ß-gal-positive region. (Bottom) The trunk region of epidermis is clearly enhanced on the injected side (arrowheads). Arrowheads indicate the epidermal borders. X-TSK protein was seen only in the non-injected side (right, triangle).

View larger version (85K): [in a new window]   Fig. 5. X-TSK induces the neural crest when in combination with Wnt. (A-E) Conjugate assay of ACs (stage 8-9) and the dorsolateral marginal zone (DLMZ) (stage10.5) analyzed by whole-mount in situ hybridization with a Sox9 probe. The numbers of phenotypic explants are described in the lower right-hand corners. (A) Sox9 expression in the conjugate of a non-injected animal cap and non-injected DLMZ. (B) The conjugate of X-TSK-si-injected AC and DLMZ; Sox9 expression is decreased. (C) The conjugate of AC and X-TSK-si-injected DLMZ. (D) The conjugate of X-TSK overexpressing AC and DLMZ; Sox9 is expressed in all explants. (E) The conjugate of AC and X-TSK expressing DLMZ. (F) The expression of Sox9 at the same stage as ACs. (G) RT-PCR analysis of the AC/DLMZ conjugate assay. X-TSK-si (0-4 pmol) was injected into the animal blastomeres evenly. (H-M) Whole-mount in situ hybridization analysis of the animal cap assay. ACs were prepared from stage 8-9 embryos injected with 0.5 ng X-TSK + 0.5 ng XWnt-8 (H,K), 0.5 ng X-TSK (I,L), or 0.5 ng XWnt-8 (J,M). ACs were harvested at the time equivalent to stage 21/22 and hybridized with XAG-1 (H-J) and Xslug (K-M) probes. (N) RT-PCR analysis of the animal cap assay. ODC was used as an the internal marker. The amount of injected X-TSK mRNA was 1 ng/embryo, which is enough to induce anterior markers. XWnt-8 mRNA (0.5 ng) was co-injected with X-TSK. X-TSK induced neural crest marker expression in combination with XWnt-8. (O-R) The embryos are transfected by the mRNA electroporation at stage 11.5 after gastrulation. The distribution of GFP fluorescence is indicated in the inset of each figure. (O) Sox9 expression in GFP mRNA transfected embryo. (P) A variation of X-TSK transfected embryo. Sox9 expression is extended to anterior side (arrowhead). (Q) A variation of X-TSK transfected embryo. Sox9 expression is ectopically observed (arrowhead), but endogenous expression was interfered by ectopic expression of X-TSK. (R) Xslug expression is also affected.

View larger version (74K): [in a new window]   Fig. 6. X-TSK interacts with the Notch signaling pathway. Whole-mount in situ hybridization analysis of mRNA-injected embryos. Injected samples are indicated in the upper right-hand corners; probes are indicated in the lower right-hand corner. (A,C) Neural crest markers Xslug (A) or Sox9 (C) disappeared or diminished (arrowheads) with X-TSK (1 ng) overexpression. (B) The ß-gal mRNA (0.5 ng) injection had no effect. (D) X-TSK-CD2 mRNA (1 ng) showed a similar effect to that in C. (E) Sox2 expression expanded laterally (right bar) in the X-TSK (1 ng)-injected side. (F) Epidermal keratin levels diminished. The anterior border disappeared on the injected side (open arrowhead). (G) An embryo injected with Chordin (100 pg). The Sox2-expressing neural plate is expanded (right bar). (H) An embryo injected with Chordin (100 pg). Expression of the neural crest marker Sox9 diminished (arrowhead). (I,J) X-TSK-si (0.5 pmol) and Chordin mRNA (100 pg). Faint expression of Sox9 (I) or Xslug (J) was observed in the far lateral side (arrowheads). (K) Hairy2A expression is downregulated on X-TSK-injected side, though it appears to spread over a broader domain (arrowhead and open arrowhead). (L) X-TSK mRNA (1 ng) injection resulted in decreased expression of Msx-1 (arrowhead) though the width of its expression area (right bracket) becomes wider than in the control side. (M) An embryo injected with X-TSK (1 ng). Xbmp4 expression increased outside the injected side (arrowhead). (N) An embryo injected with Chordin (100 pg). Xbmp4 expression disappeared around the ß-gal-positive cells (arrowhead). (O) BMP4 (0.5 ng) mRNA and (P) Notch ICD (0.5 ng) injections caused reduction of Xslug expression (arrowheads). (Q) ß-Gal mRNA injection did not affect Xbmp4 expression. (R) X-delta-1Stu injected areas are marked by ß-gal staining (light blue), the arrowhead indicates ectopic Xbmp4 expression (dark purple). (S,T) An embryo injected with X-TSK. (S) Two out of the three strips of N-tubulin expression disappeared (arrowheads). (T) X-delta-1 expression disappeared on the outer border of the neural crest (arrowhead). (U) ß-gal did not affect X-ESR-1 expression. (V) X-TSK mRNA (1 ng) injection resulted in expanded peripheral expression of X-ESR-1 on the injected side (arrowheads). (W,X) Co-injection of 1 ng X-TSK and 0.5 ng Su(H)DBM mRNAs. (W) Diffused signals of Sox9 were observed around the arrowhead. (X) Xbmp4 expression was extended towards the ß-gal-positive side (arrowhead). (Y) Sox9 expression was reduced in Su(H)/Ank 500 pg injected side (arrowhead). (Z) Sox9 expression was completely missing in X-TSK 1 ng+ Su(H)/Ank 500 pg injected side (open arrowhead). (AA) Xbmp4 expression was very weakly inhibited in Su(H)/Ank 500 pg injected side (open arrowhead). (BB) The patchy expression of Xbmp4 was observed in X-TSK 1 ng+ Su(H)/Ank 500 pg injected side (arrows). The expansion of Xbmp4-expressing regions was not observed (open arrowhead).

View larger version (30K): [in a new window]   Fig. 7. A molecular interaction between X-TSK and X-delta-1. (A) Immunoprecipitation of X-delta-1 extracellular domain with X-TSK. (B) RT-PCR analysis of acute XESR-1 activation by Notch canonical pathway with or without X-TSK. Animal caps are excised at stage 8.5 and analyzed at stage 9.5. As a positive control, the normal neurula was used (lane 1). Notch ICD (NICD, 1 ng), 1 ng X-delta-1 mRNA, 2 ng X-TSK mRNA, 10 ng X-TSK MO and mixtures thereof were injected into all blastomere at the four-cell stage.

View larger version (31K): [in a new window]   Fig. 8. A model of consecutive step of the neural crest specification. (A) A proposed model of sequential steps of neural crest specification. (Phase I) During gastrulation, midline BMP antagonists inhibit BMP4 expression and specify the neural plate (orange). After this, X-TSK expression is repressed in the presumptive neural plate (yellow), while it is maintained in the lateral ectoderm by BMP4 signaling. (Phase II) After stage 12.5, X-TSK expression is upregulated in the presumptive neural crest region, which has intermediate levels of BMP signaling. (siRNA) In X-TSK depleted embryo, BMP signaling levels (green) are increased in the TSK expressing-region. Under these conditions, epidermal fates are expanded up to the neural plate margin (blue), while the neural crest region (yellow) is repressed. (B) Molecular network of neural crest specification. X-TSK inhibits BMP4 (red asterisk) and modulates Notch signaling via direct binding to X-delta-1 (purple asterisk). Notch signaling can regulate the expression of BMP4, and BMP and Notch signaling interact to control the expression of Hairy2A and Msx1. Timing of Notch activation alters its effect on BMP4 expression (see Glavic et al., 2004) (black asterisk). Finally, caudalizing signals such as XWnt-8 converge on this network downstream of Msx1 to control the activation of Pax3 expression and neural crest cell specification within the Msx1-expressing region. The interactions indicated by `1' and `2' have been reported previously (Glavic et al., 2004; Monsoro-Burq et al., 2005).