Larger Image Figure 3. Clk2 promotes neural induction and suppresses epidermis differentiation via inhibition of BMP signal. (a) Clk2 induced neural tissues in X. laevis ectodermal explants. clk2‐GR and clk2 SYA‐GR mRNAs (500 pg, 1,000 pg and 1,500 pg) were injected into the animal hemisphere of 4‐cell stage embryos. At the blastula stage, embryos were transferred to a medium containing DEX and the ectodermal explants were isolated. At the neurula stage, the explants were subjected to an RT‐PCR analysis to examine the expression of marker genes. Expression of early neural markers (sip1, sox2 and sox3), an anterior neural marker (otx2) and a posterior neural marker (hoxb9) was upregulated by Clk2 overexpression, whereas the differentiated neural marker (ncam) was only slightly increased. The expression of epidermal keratin was reduced in the presence of Clk2. “Embryo” and “−RT” indicate whole embryos examined in the presence or absence of reverse transcriptase, respectively. histone h4 was used as the loading control. (b) Clk2 inhibits BMP signal downstream of receptor activation. noggin (100 pg), CA‐alk2 (200 pg) and clk2 SYA‐GR (500 pg, 750 pg and 1,000 pg) mRNAs were injected into the animal hemisphere of 4‐cell stage embryos. At the blastula stage, embryos were transferred to a medium containing DEX and the ectodermal explants were isolated. At the neurula stage, the explants were subjected to an RT‐PCR analysis to examine the expression of marker genes. CA‐Alk2 induced the expression of epidermal keratin, even when the endogenous BMP signal was blocked by Noggin (lanes 4 and 5). Overexpression of Clk2 reversed the CA‐Alk2‐induced epidermal keratin expression and promoted the expression of neural marker genes (sip1 and sox3) in the ectodermal explants (lanes 6–8). (c–n’) Clk2 expanded the neural plate and reduced the epidermis region. clk2 SYA‐GR (250 pg and 500 pg) mRNA together with β‐galactosidase mRNA were injected unilaterally into a blastomere of 2‐cell stage embryos. The injected side of the embryos is marked by brackets. The expansion of sox2 gene expression (c–h’) and reduction of epidermal keratin expression (i–n’) are indicated by arrowheads. (o) Overexpression of Clk2 expanded the expression region of sox2. Expression of sox2 was classified as strongly or weakly expanded, or normal (■, Strongly; image, Weakly; □, Normal). (p) Clk2 reduced the expression region of epidermal keratin in Xenopus embryos. Expression of epidermal keratin was classified as strongly or weakly reduced, or normal (■, Strongly; image, Weakly; □, Normal). c–e and i–k: dorsal view with the anterior to the bottom. f–h and l–n: dorso‐lateral view with the anterior to the left. f’–h’ and l’–n’: magnification of the dorso‐lateral side shown in f–h and l–n, respectively. Image published in: Virgirinia RP et al. (2019) Copyright © 2019. Image reproduced with permission of the Publisher, John Wiley & Sons. Permanent Image Page Printer Friendly View XB-IMG-176155