Ectopic pharynxes arise by regional reorganization after anterior/posterior chimera in planarians

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.