XB-IMG-24070
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Xenbase Image ID: 24070
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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). Image published in: Kuriyama S et al. (2006) Copyright © 2006. Image reproduced with permission of the Publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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