In previous posts I have discussed the positive role that ROS species have during tail regeneration in Xenopus as well as the important role played by apoptosis to trigger head regeneration in Hydra. Now a recent paper from the laboratory of Sophie Vriz links both processes during adult caudal final regeneration in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/23803955).
First, the authors show how whereas wounding induced a transitory ROS production with a peak 30 minutes post-lesion that disappeared after 2 hours, fin amputation induced a much prolonged production of ROS peaking at about 12-16 hours post-amputation (hpa). ROS production was not detected after 24 hours of regeneration. Then, and to try to see the functional relevance of this regeneration-specific extended ROS production the authors used different inhibitors of ROS. Thus, inhibiting NOX (NADPH oxidase) activity from 12 to 24 hpa seemed sufficient to reduce the size of the regenerated fin at 72 hours. This reduced regeneration was accompanied by a decrease in the expression of klf4 (a progenitor cell fate marker) and dio3 (involved in progenitor cell proliferation). The expression of another progenitor cell fate marker as it is myc was not affected.
As oxidative stress has been related to MAP kinase activation and apoptosis, the authors wanted to search whether ROS was also involved in those processes during caudal fin regeneration. First, they observed that the normal activation of the JNK pathway at 6 hpa was reduced about at 50% after NOX inhibition, suggesting that ROS has a role in the early activation of the JNK pathway. Second, the inhibition of ROS production reduced also the number of apoptotic cells during the first 18 hpa. Then, the authors checked in more detail the cell death and apoptotic responses after amputation. Remarkably they found a bimodal response with two peaks of TUNEL positive cells (also active caspase-3 positive cells) at about 5 hpa and 15-18 hpa mainly concentrated in the stump epithelium and very few cells in the mesenchyme. In contrast, after wounding, and in agreement with the only early peak observed for ROS production, cell death increased and peaked around 6 hpa without a second peak later. To further characterize the function of apoptosis on regeneration the authors inhibited this process during fin regeneration. Remarkably, blocking the 2nd wave of apoptosis (from 12 to 72 hpa) was sufficient to impair regeneration. In fact, inhibiting the first apoptotic wave (from 4 to 10 hpa) neither blocked regeneration nor affected the 2nd apoptotic wave. These results suggest that the 2nd apoptotic wave was the one required for regeneration. However, these effects on regeneration affect only the size of the regenerate because those small blastemas appeared to be normally patterned.
Next, the authors looked how proliferation was affected after JNK and apoptosis inhibition. In control animals, at 24 hpa the mitotic cells were mainly localized in the inter-ray epidermis of the stump. The inhibition of ROS or apoptosis reduced the number of mitotic cells by 50%. In contrast to what it has been described in other models JNK pathway did not seem to be involved in the induction of apoptosis during zebrafish caudal fin regeneration. So it seems that apoptosis and JNK work in parallel downstream of ROS production to induce cell proliferation during regeneration.
Finally the authors checked whether the inhibition of apoptosis or JNK pathway impairs cellular reprogramming or signalling molecules required for blastema growth. Inhibiting the second wave of apoptosis reduced the expression of klf4 and dio3, as NOX inhibition does. However, JNK inhibition did not seem to affect these markers. At the level of signalling pathways, apoptosis inhibition reduced the expression of fgf20, sdf1 and enhanced the expression of igf2b and wnt10a. On the other hand, inhibiting the JNK pathway reduced the expression of sdf1, wnt5b and igf2b. In summary, the authors found out that that the production of ROS during regeneration is important to induce apoptosis and JNK pathway that would work in parallel to promote epidermal cell proliferation and blastema formation during zebrafish caudal fin regeneration.