Makoto Mochii

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.

Wound epidermis (WE) and the apical epithelial cap (AEC) are believed to trigger regeneration of amputated appendages such as limb and tail in amphibians by producing certain secreted signaling molecules. To date, however, only limited information about the molecular signatures of these epidermal structures is available. Here we used a transgenic Xenopus laevis line harboring the enhanced green fluorescent protein (egfp) gene under control of an es1 gene regulatory sequence to isolate WE/AEC cells by performing fluorescence-activated cell sorting during the time course of tail regeneration (day 1, day 2, day 3 and day 4 after amputation). Time-course transcriptome analysis of these isolated WE/AEC cells revealed that more than 8,000 genes, including genes involved in signaling pathways such as those of reactive oxygen species, fibroblast growth factor (FGF), canonical and non-canonical Wnt, transforming growth factor β (TGF β) and Notch, displayed dynamic changes of their expression during tail regeneration. Notably, this approach enabled us to newly identify seven secreted signaling molecule genes (mdk, fstl, slit1, tgfβ1, bmp7.1, angptl2 and egfl6) that are highly expressed in tail AEC cells. Among these genes, five (mdk, fstl, slit1, tgfβ1 and bmp7.1) were also highly expressed in limb AEC cells but the other two (angptl2 and egfl6) are specifically expressed in tail AEC cells. Interestingly, there was no expression of fgf8 in tail WE/AEC cells, whose expression and pivotal role in limb AEC cells have been reported previously. Thus, we identified common and different properties between tail and limb AEC cells.

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.