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bmp4xenopus   

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Experiment details for bmp4

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
bmp4.L laevis NF stage 12 to NF stage 17 neuroectoderm , cement gland primordium , anterior neural fold

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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).

Gene Clone Species Stages Anatomy
bmp4.L laevis NF stage 14 ectoderm , neuroectoderm , cement gland primordium , anterior neural fold
bmp4.L laevis NF stage 23 ectoderm , retina , roof plate , epidermis , neuroectoderm , [+]

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  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.

Gene Clone Species Stages Anatomy
bmp4.L laevis NF stage 15 neuroectoderm , anterior neural fold

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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).