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Fig. 1. Comparison of X. tropicalis Clk family protein sequences.
Putative nuclear localization signals, N-terminal phosphodegron sites, kinase domain, and activation loop are indicated by green, yellow, red, and blue boxes, respectively. Amino acid residues that are conserved between at least two of the sequences are shaded in black. Sequence alignment was performed using the Clustal W method. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2. Expression of clk1 and clk3 during early development of X. tropicalis.
(A) RT-PCR analysis of temporal expression of clk1 and clk3. “-RT†indicates RNA of whole embryos (stage 20) examined in the absence of reverse transcriptase. (B–U′) Spatial expression of clk1 and clk3 analyzed by WISH in developing embryos at stages 9, 10.5, 15, 20, 23, and 30. The expression of clk1 and clk3 are shown in blue/purple. (B, D, L, and N) Animal view. (C, E, M, and O) Vegetal view. (F, H, J, P, R, and T) Dorsal view with the anterior to the left. (G, I, Q, and S) Posterior view with the dorsal to the left. (K and U) Lateral side view with the anterior to the left. (K′ and U′) Magnified view of K and U, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 3. clk family genes promote neural development in ectodermal explants.
clk1-GR, clk2-GR, and clk3-GR mRNAs (1500 pg and 2000 pg) were injected into the animal hemisphere of 4-cell stage embryos. Ectodermal explants were isolated at the blastula stage, cultured until the neurula stage (stage 21), and subjected to RT-PCR analysis. “Embryo†and “-RT†indicate whole embryos examined in the presence or absence of reverse transcriptase, respectively.
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Fig. 4. Knockdown of Clk3 inhibits early neural development.
(A–D) Knockdown of Clk family proteins caused phenotypic changes in Xenopus embryos. Control MO, Clk1 MO, Clk2 MO, and Clk3 MO (25 ng, 25 ng, 25 ng and 10 ng, respectively) were injected into the animal hemisphere of 2-cell stage X. tropicalis embryos. The embryos were raised until the late tailbud stage (stage 37/38) for phenotypic analysis. In this experiment, a lower level of Clk3 MO (10 ng) was used to avoid embryonic lethality caused by the higher dose of Clk3 MO. The phenotypic changes were classified into three types: I (normal head size, bent axis); II (small head and eyes, bent axis); and III (severely reduced head and eye size, bent axis). (E) Summary of phenotype observed in the experiment shown in A–D. The numbers of embryos analyzed are shown on the top of the graph. (F–H) Clk3 is required for early neural development. Clk3 MO (10 ng) was injected into the animal hemisphere of 2-cell stage X. tropicalis embryos. At the late tailbud stage (stage 37/38), the embryos were subjected to qPCR analysis to examine the expression of neural and mesodermal marker genes. ∗P < 0.05, t-test.
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Supplementary Figure 1
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clk1 (CDC like kinase 1) gene expression In a Xenopus laevis embryo, NF stage 9, assayed via in situ hybridization, animal view (B) and vegetal view (C).
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clk1 (CDC like kinase 1) gene expression In a Xenopus laevis embryo, NF stage 10.5, assayed via in situ hybridization.
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clk1 (CDC like kinase 1) gene expression In a Xenopus laevis embryo, NF stage 15, assayed via insitu hybridization, dorsal (F) and posterior-dorsal view (G), anterior left.
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clk1 (CDC like kinase 1) gene expression In a Xenopus laevis embryo, NF stage 20, assayed via insitu hybridization, dorsal (H) and posterior-dorsal (I) view, anterior left.
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clk1 (CDC like kinase 1) gene expression In a Xenopus laevis embryo, NF stage 23, assayed via in situ hybridization, dorsal view, anterior left.
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 9, assayed via in situ hybridization, animal view (L) and vegetal view (M).
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 10.5, assayed via in situ hybridization, dorsal view (N), vegetal/blastoporal view, with dorsal left (O).
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 15, assayed via inbsitu hybridization, dorsal (P) and posterior dorsal (Q) view
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 20, assayed via in bsitu hybridization, dorsal (R) and posterior-dorsal (S) view, anterior left.
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 23, assayed via in situ hybridization, dorsal view, anterior left.
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clk3 (CDC like kinase 3) gene expression In a Xenopus laevis embryo, NF stage 30, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
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