Movement, secretion and blood flow in the intestinal tract are controlled by the enteric nervous system (Auerbach's plexus and Meissner's plexus). This enteric nervous system is formed during development when progenitor cells migrate up the intestinal wall, covering the intestinal tract in a network and differentiating. Disruption of this migratory process leads to Hirschsprung disease in humans, in which the nervous system does not form at the end of the large intestine. Therefore, we aimed to identify the molecular mechanisms underlying this migratory process.

To measure the molecular activity of enteric progenitor cells migrating on the intestinal wall, mice expressing the FRET biosensor were needed. At the time, genetically engineered mice expressing PKA and ERK had already been developed in the Matsuda lab (Kamioka et al., 2013CSF). In addition to this, in order to further investigate the molecular mechanisms of the cytoskeleton, genetically engineered mice expressing Rac1, Cdc42 and the FRET biosensor for JNK were newly generated. The developing intestinal tracts of these transgenic mice were organ-cultured and FRET imaging of enteric neural progenitors was performed using two-photon microscopy (Fig. 4 top).

Fig. 4. (Top) Live two-photon microscopy imaging of organ culture of the embryonic intestine of transgenic mice expressing the FRET biosensor. (Bottom) FRET images of Rac1, Cdc42, PKA, JNK and ERK in enteric neural progenitors, respectively; PKA-NC is a negative control lacking phosphorylated substrate. The low PKA activity and high Rac1 and Cdc42 activity in cells migrating in chains suggest a pathway in which PKA suppresses Rac1 and Cdc42. On the other hand, ERK and JNK showed random activity and did not correlate with migration speed, but experiments with inhibitors showed that JNK is required for cell migration.

GDNF and Endothelin 3, which are secreted by mesenchymal cells, are known to be important for progenitor cell migration. Therefore, when GDNF was administered to progenitor cells, PKA activity was reduced and, conversely, Rac1 was activated; when Endothelin 3 was administered, PKA was activated and Rac1 was reduced, in contrast to GDNF. In short, the results suggest a signalling pathway in which GDNF and Endothelin 3 secreted by mesenchymal cells positively and negatively regulate enteric neural progenitor cell migration via PKA.

This finding is expected to make a significant contribution to the elucidation of the pathogenesis of Hirschsprung disease.