餅井 真

Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.

Planarians have established a unique body pattern along the anterior-posterior (AP) axis, which consists of at least four distinct body regions arranged in an anterior to posterior sequence: head, prepharyngeal, pharyngeal (containing a pharynx), and tail regions, and possess high regenerative ability. How they reconstruct the regional continuity in a head-to-tail sequence after amputation still remains unknown. We use as a model planarian Dugesia japonica head regeneration from tail fragments, which involves dynamic rearrangement of the body regionality of preexisting tail tissues along the AP axis, and show here that RNA interference of the gene D. japonica mek kinase 1 (Djmekk1) caused a significant anterior shift in the position of pharynx regeneration at the expense of the prepharyngeal region, while keeping the head region relatively constant in size, and accordingly led to development of a relatively longer tail region. Our data suggest that DjMEKK1 regulates anterior extracellular signal-regulated kinase (ERK) and posterior β-catenin signaling pathways in a positive and negative manner, respectively, to establish a proper balance resulting in the regeneration of planarian's scale-invariant trunk-to-tail patterns across individuals. Furthermore, we demonstrated that DjMEKK1 negatively modulates planarian β-catenin activity via its serine/threonine kinase domain, but not its PHD/RING finger domain, by testing secondary axis formation in Xenopus embryos. The data suggest that Djmekk1 plays an instructive role in the coordination between the establishment of the prepharyngeal region and posteriorizing of pharynx formation by balancing the two opposing morphogenetic signals along the AP axis during planarian regeneration.