There are several cases in which regenerative capabilities vary depending on the regenerative stage of the organisms. Some examples of this loss of regeneration include that of limbs in post-metamorphic amphibians and the heart in one week-old mice. Now a recent paper from the laboratory of Kenneth Poss describes how the pectoral fins in zebrafish show a sexually dimorphic response to amputation and links that to the maintenance of male-specific structures and the regulation of the Wnt signalling pathway (http://www.ncbi.nlm.nih.gov/pubmed/24135229).
In a previous paper the same laboratory had found that pectoral fin regeneration is often impaired in males whereas it proceeds normally in females (http://www.ncbi.nlm.nih.gov/pubmed/22079110), being such impairment partially rescued by overactivating the Wnt/b-catenin pathway. Regeneration failure correlates with sexual maturity indicating an age- and sex-dependent loss of regenerative abilities for the male zebrafish pectoral fins. In this new paper the authors characterize in more detail the molecular basis of such sexual dimorphism and focus on the study of Dkk1, an inhibitor of the Wnt/b-catenin signalling pathway, which is well-known for being required for a successful regeneration in many contexts and species, including zebrafish fins. dkk1 was found to be expressed at higher levels in male pectoral fins that in females. By using a transgenic line the authors found that dkk1 displayed a sexual dimorphic expression being detected in what they called epidermal tubercles (ETs) in the anteromedial rays of the pectoral fins of males. Because similar structures had been described in other fishes and associated to mating and spawning, the authors first addressed the role of ETs in spawning. What they found was that zebrafish males used the pectoral fin to grasp the female abdomen to stimulate egg laying. In fact, males with amputated pectoral fins appeared mostly incapable of stimulating an efficient spawning.
Next, they analysed the role of androgens in the development of ETs. Remarkably, an androgenic factor was able to induce the development of ETs in the pectoral fins of females; conversely, the application of an androgen inhibitor in males decreased the number and definition of ETs. These changes were accompanied with an increase or decrease of dkk1 expression, respectively. In order to characterize the role of the Wnt/b-catenin pathway in the formation of ETs they crossed a transgenic line bearing a Wnt signalling reporter with the line with labelled dkk1-expressing cells. Following the maturation of ETs they found 3 distinct types of ETs: immature structures where Wnt signalling is active and dkk1 is not expressed, intermediate cells expressing dkk1 and active Wnt signalling and mature ETs expressing dkk1 and no active Wnt signalling. Because the balance of activated/inhibited Wnt signalling is important for the induction and/or patterning of epidermal appendages the authors further analysed the role of Wnt/b-catenin on ETs formation. Experiments inhibiting or overactivating Wnt signalling suggested that that this pathway has a positive role in the formation of new ETs. In fact, ETs go through a continuous cell turnover from a basal layer cell population(s) between adjacent ET units, being this renewal dependent on local dkk1 regulation and Wnt activation.
During fin regeneration, at 2 days post amputation (dpa) Wnt signalling is activated in early blastema cells. As regeneration proceeds Wnt pathway keeps active in the proliferative distal blastema, whereas dkk1 is expressed in more proximal differentiating cells. This dynamics was observed in different fins in both females and males, including female pectoral fins and posterior regions of male pectoral fins. Defects in male pectoral fin regeneration mostly appeared when amputation was performed across a region containing ETs. Further analyses on male pectoral fin regeneration revealed that the wound was capped by epidermal cells expressing high levels of dkk1 with little proliferative activity underneath. This was in contrast to females in which dkk1 was not expressed in the blastema cells at this stage (2 dpa). Also, whereas in females axin2, a known target of active Wnt signalling was detected in blastema cells, no expression was found in males clearly indicating a failure in Wnt activation in those regenerating males. As regeneration proceeded males appeared to achieve different degrees of recovery. After several weeks of regeneration males were then classified in three groups depending on whether the defects in their regenerated pectoral fins were mild or severe. Remarkably, males with severe defects were incapable of stimulating spawning during mating.
In summary, this paper describes the molecular basis of the sexual dimorphism shown by regenerating pectoral fins in female and male zebrafish. Thus, a fine balance between active and inactive Wnt signalling is pivotal for proper regeneration. In males, the high level of dkk1 required in ETs to maintain these basic structures for a successful mating appears to come at the expense of reducing their regenerative potential.