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Xenopus LIM motif-containing protein kinase, Xlimk1, is expressed in the developing head structure of the embryo.
The LIM double zinc finger motif locates in several developmentally functioning and cytoskeletal proteins, and is considered to act as a specific motif for protein-protein interactions. LIM kinase (LIMK) is a novel protein kinase containing two LIM motifs at the N-terminal, the function of which has yet to be clearly defined. In this study, we cloned a cDNA encoding Xenopus counterpart of human LIMK1 gene by RT-PCR mediated cloning, and designated in Xlimk1. Xlimk1 is highly homologous to mammalian LIMK1 in each structural domain, particularly in LIM and protein kinase domains. In Northern blot analysis, two distinct Xlimk1 transcripts of 9.0 Kb and 3.7 Kb were present in early cleavage stages of the embryo. Both mRNA species were subsequently decreased at the gastrula stages. The 9.0 Kb of Xlimk1 mRNA again appeared in late neurula stage, then the expression level gradually increased in later stages of the embryo. Whole-mount in situ hybridization analysis showed the localization of Xlimk1 transcripts in the animal half of the blastulaembryo. In post-neurula stages, specific signals for Xlimk1 were predominant in the anterior (head) region of the embryo, including developing brain, hyoid and branchial arches, and anlagen of sensory organs. These results indicate that Xlimk1 may play an important role in neural development and formation of anterior (head) structures in the Xenopus embryo.
Fig. 3. Expression of Xlimk1 mRNA during Xenopus embryogenesis. A: RT-PCR analysis. Random primed cDNA from indicated stages of embryo mRNA were used as templates. PCR conditions were as follows: denaturation at 96�C for 30 sec, followed by annealing at 55�C for 60 sec, and extension at 72�C for 90 sec. As a control, the amplifications of histone H4 fragment are shown. B: Northern blot analysis. Total (10 μg)RNA isolated from indicated stages of embryos are used. Positions of Xlimk1 transcripts are indicated by arrowheads. The relative migration of RNA molecular markers is shown on the left. The lower panel shows the ethidium bromide-stained 18S and 28S ribosomal RNA, as a loading control.
Fig. 4. Whole-mount in situ hybridization analysis of Xlimk1 mRNA expression. A, B: Stage 9. Dorsal (left) and ventral (right) view. The ventral pole of embryos are indicated by stars. C, D: Lateral view of stage 22 embryo. E�H: Lateral view of stage 35 embryo. Each embryo is hybridized with sense (A, C, E, and G) or antisense (B, D, F, and H)
riboprobe specific for Xlimk1. ev, Eye vesicle; op, olfactory placode; ov, otic vesicle; h, hyoid arch; ab, anterior branchial arch; pb, posterior branchial arch; fb, forebrain; mb, midbrain; hb, hindbrain. Bars represent 1.0 mm.
Fig. 5. Expression of Xlimk1 mRNA in head region of stage 35 embryos. After whole-mount in situ hybridization, horizontal cross sec- tions (A�C, corresponding positions are schematically drawn in the bottom figure) were prepared. nt, Neural tube; g, foregut; ev, eye vesicle; ov, otic vesicle; h, hyoid arch; ab, anterior branchial arch; pb, posterior branchial arch. Bars represent 0.25 mm.
limk1 (LIM domain kinase 1) st 9
limk1 (LIM domain kinase 1)st 23
limk1 (LIM domain kinase 1) st 35
limk1 (LIM domain kinase 1) st 35 head region only