Comparative Regenerative Biology

We use animals that demonstrate a high ability in regenerating body parts, such as planarians and newts, to understand the principle of regeneration. In particular, we investigate the difference between regenerative and non-regenerative animals to evoke said abilities from non-regenerative animals. We have already succeeded in achieving this with planarians, which were able to regenerate their heads through RNAi (Umesono et al., 2013 Nature), and accomplishing functional joint regeneration in frogs through the activation of reintegration systems (Tsutsumi et al., 2016 Regeneration).    
 
We are currently trying to induce limb regeneration abilities in frogs, as they lose the capability to achieve complete limb regeneration after metamorphosis. We are now focusing on the Sonic hedgehog (Shh) super-enhancer MFCS1 (mammals-fishes conserved sequence 1), since it has been suggested that the loss of MFCS1 activity after metamorphosis might cause a failure in achieving the aforementioned limb regeneration in adult frogs (Yakushiji et al., 2009). When we compared the MFCS1 sequences between newts and frogs, newts were found to possess several specific sequences (Figure 1), suggesting that sequence differences might affect super-enhancer formation between newts and frogs after metamorphosis.
 

Figure.1 Comparison of the MFCS1 sequences between newts and frogs.

 
We subsequently tried to detect enhancer RNA (eRNA) which might be transcribed in the MFCS1 region. An interesting aspect of this was that eRNA was detected in regenerating blastema of adult newt from st. 2.0 (Pleurodeles waltl: Figure 2A).
Conversely, expression of eRNA was suppressed in the frog’s blastema (Xenopus laevis) after metamorphosis (Figure 2B).
 

Figure.2 Expression of MFCS1-eRNA and Shh mRNA in the regenerating blastema of newt (P.w.) and frog (X.l.)
(A) The left and right panels show expression levels of MFCS1-eRNA and Shh mRNA, respectively. The left and right graphs in each panel were obtained from regenerating blastema at St.2.0 and St.2.5 of adult newts after amputation.
(B) The left and right panels shows expression levels of MFCS1-eRNA and Shh mRNA in frogs before and after metamorphosis.

Trials in isolating viable adult pluripotent stem cells derived from planarian using FACS

We have tried to develop an isolation method for viable adult pluripotent stem cells (aPSC) from planarians using FACS and succeeded in conditioning the low toxic staining method with both nuclear and cytoplasimic fluorescence dyes. 1µM Hoechst 33342 or 0.05 µg/ml Calsein AM could be used for isolating viable aPSC of the planarian, Dugesis japonica. We are presently investigating the effects of FGF- and Wnt-morphogens to cultured aPSC to demonstrate “the double gradient hypothesis”, which was proposed by Thomas Hunt Morgan (Morgan, 1901) and our group (Umesono et al., Nature, 2013).

• Annual Report 2021（PDF）

Satoh, A., Kashimoto, R., Ohashi, A., Furukawa, S., Yamamoto, S., Inoue, T., Hayashi, T., Agata, K. (2022). An approach for elucidating dermal fibroblast dedifferentiation in amphibian limb regeneration. Zoological Lett. 8, 6. doi: 10.1186/s40851-022-00190-6.
 
Sato, Y., Shibata, N., Hashimoto, C., Agata, K. (2022). Migratory regulation by MTA homologous genes is essential for the uniform distribution of planarian adult pluripotent stem cells. Dev. Growth Differ. 64, 150-162. doi: 10.1111/dgd.12773. Epub 2022 Feb 28.
 
Takeuchi, T., Matsubara, H., Minamitani, F., Satoh, Y., Tozawa, S., Moriyama, T., Maruyama, K., Suzuki, K.T., Shigenobu, S., Inoue, T., Tamura, K., Agata, K., Hayashi, T. (2022). Newt Hoxa13 has an essential and predominant role in digit formation during development and regeneration. Development 149, dev200282. doi: 10.1242/dev.200282. Epub 2022 Mar 11.
 
Lee, H., Hikasa, K., Umesono, Y., Hayashi, T., Agata, K., Shibata, N. (2022). Loss of plac8 expression rapidly leads pluripotent stem cells to enter active state during planarian regeneration. Development 149, dev199449. doi: 10.1242/dev.199449. Epub 2022 Feb 3.