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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 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).
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