Kinase activity assay of zcdkl1 in HEK 293 cells transfected with pEGFP or pEGFP-zcdkl1. Twenty-four h post-transfection, cells were lysed with lysis buffer and subjected to immunoprecipitation with anti-GFP antibody. (A) Immunoprecipitated complexes underwent in vitro kinase assay using myelin basic protein (MBP) and histone H1 as substrates. Phosphorylation of histone H1 (lane 1 and 3) and MPB (lanes 2 and 4) was detected by autoradiography; (B) Immunoprecipitated complexes (lane 1 for GFP alone; lane 2 for GFP-zcdkl1) were subjected to Western blot analysis using anti-GFP antibodies.

Temporal and spatial expression of zcdkl1 gene. (A) Total RNA derived from different stages of development (upper panel) and adult tissues (lower panel) were converted into cDNA and subjected to PCR amplification. PCR fragments were separated by electrophoresis on 3% agarose gel and visualized by ethidium bromide staining. Elongation factor 1α was included as internal control.

(3 cont.) Temporal and spatial expression of zcdkl1 gene. (B–H) Expression of zcdkl1 was analyzed in whole mount in situ hybridization. The developmental stage is indicated in the lower left (G′ and G′′), while the transverse section is indicated by line in panel (G). Sections were counterstained with eosin Y. (B–H) Lateral view with the anterior pole to the left. Insert in (F) represents the caudal region with higher magnification. The arrowhead indicates the dorsal neurons in the spinal cord. The arrow indicates the hypochord. The asterisk indicates the pronephric duct.

Knockdown of zcdkl1 produced defective phenotypes. Embryos at 1 to 4 cells stages received different dosages of morpholino. (A) Phenotype of zcdkl1 morphant at 24 HPF. Lateral view with the anterior pole to the left; (B) Percentages of defective phenotype produced by different dosages of zcdkl1 MO, 10 ng zcdkl1 MO plus 11 pg zcdkl1 mRNA, and 10 ng control MO. N value denotes the total injected embryos from three independent experiments.

Expression pattern of neurogenin 1 (ngn1) and sonic hedgehog (shh) in zcdkl1 morphants. Morphants that received 10 ng control MO (A, D, and G), 10 ng cdkl1 MO (B, E, and H), and 10ng cdkl1 MO + 11 pg cdkl1 mRNA (C, F, and I) were subjected to whole mount in situ hybridization using ngn1 (A–F) and shh (G–I) riboprobes. Lateral (A–C and G–I) and dorsal (D–F) views with the anterior pole to the left.