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Gene/Clone	Species	Stage	Anatomy Item	Experimenter
msx1	xenopus

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Experiment details for msx1

Kuriyama S et al. (2006) Assay

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

Gene	Clone	Species	Stages	Anatomy
msx1.L		laevis	NF stage 15	ectoderm , neural crest , dorso-lateral

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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 β-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 β-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 β-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 β-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).

Gene	Clone	Species	Stages	Anatomy
msx1.L		laevis	NF stage 17	neural crest , trunk region

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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 β-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 β-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) β-Gal mRNA injection did not affect Xbmp4 expression. (R) X-delta-1Stu injected areas are marked by β-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) β-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 theβ -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).