A general property of animal development is that all organs and appendixes grow up to specific proportions relative to the body size of the organism. Remarkably, in those species capable of regenerating, for example a complete limb de novo, the regenerated structure grows up to its original size restoring the normal body proportions. How this coordinated growth is regulated is not much known. Now a recent paper from the laboratory of Christopher Antos (http://www.ncbi.nlm.nih.gov/pubmed/24561038) describes the role of calcineurin on the coordination of growth during zebrafish fin regeneration.
Isometric growth refers to the situation where growth occurs at the same rate for all parts of an organism. Any deviation from this situation for a specific organ or structure is refereed as an allometry. During the development of the zebrafish fin, this appendix grows initially allometrically respect to the rest of the body until it approaches its final size when it switches to an isometrical growth. Similarly, during fin regeneration, the initial stages are characterized by an allometric growth that ends up to an isometric growth when the fin is regenerated. This paper describes the role of calcineurin on regulating this fin growth rate.
Calcineurin is a protein phosphatase that activates the T cells of the immune system. Because the immune response after an injury can modulate the regenerative output the authors wanted to check whether using the immunosuppressant Tacrolimus (FK506) had any effect on zebrafish fin regeneration. By treating regenerating fins with this drug they observed how those amputated fins grew much larger than the controls, giving rise to large fins disproportionate to the body size. The growth rate of the FK506-treated fins started to differ from the control rate around day 7 post-amputation and was kept higher beyond 4 weeks. In controls, the growth rate of the regenerated fin initially increased respect to the body growth rate but then decreased around 3 weeks post-amputation as the regenerated fin reached its original size. The authors show that after FK506 treatment the increased growth rate in the regenerating fin occurred in the distal regenerate and not in the stump region. In fact, FK506 binds to the so-called FK506-binding proteins (FKBPs) and fkbp1Aa, fkbp10 and fkbp14 genes were expressed in the blastema, further suggesting that FK506 acted on blastema cells. At the histological level this enlarged fins showed normal organization, normal tissue morphometry and there were no evidences of tumorous growth. The authors also checked that the expression of several genes that are normally reactivated during regeneration was maintained further after FK506 treatment, suggesting that the enlarged outgrowths were due to the maintenance of this proregenerative transcriptional program.
Remarkably, FK506 treatment of uninjured adult limbs also induced their allometric growth respect to the body resulting in enlarged fins. Here, again several proregenerative genes were upregulated in those uninjured fins. In juvenile zebrafish the development of the fins involves also an initial allometric growth. FK506 treatment of those juveniles also resulted in the maintenance of an allometric growth and enlarged fins. In all these experiments it should be noticed that the effects of FK506 were restricted to the growth rate of fins and not the rest of the body. Overall, these experiments suggested that FK506 affected a mechanism that regulates a switch between isometric and allometric growth. Not all immunosuppressant yielded the same effects pointing out to a FK506-specific target. It is known that the FK506-FKBP complex binds calcineurin inhibiting its activity. Using another drug that also inhibits calcineurin the authors found the same effects on fin outgrowth, suggesting that calcineurin could be regulating this allometric growth. Accordingly to this hypothesis the authors found that during early stages of regeneration when the regenerating fin is growing allometrically the activity of calcineurin was decreased compared to an uninjured fin. Conversely, this activity increased as the regenerating fin was achieving its final size and switching to an isometric growth. Therefore, calcineurin appeared to be involved in regulating allometric growth.
It is also known that amputated fins at more proximal regions regenerate at higher rates that those fins amputated more distally. Thus, for example if one lobe of the bilobed fin is amputated at a proximal region and the other lobe is amputated more distally, both lobes reaches their final size at the same time because the lobe amputated proximally regenerates faster. This suggests that the positional information along the proximodistal axis can determine the growth rate. If repeating this experiment of amputating the two lobes of the fin at different levels along the proximodistal axis and allowing them to regenerate in the presence of FK506 the authors found that both lobes grew at a high rate characteristic of the proximal regenerate of controls. The growth rate in the FK506-treated fins never converged so they regenerated enlarged and disproportionate lobes with the one amputated more distally larger than the one amputated more proximal (and both larger than controls). The authors concluded then that this enhanced growth rate was due to a proximalization either of the regenerating tissue or the growth program. This was further supported by the observation that the FK506-treated fins showed a distal shift of the bifurcation point of each fin ray.
Finally, as the retinoic acid (RA) pathway has been shown to regulate positional growth by proximalizing the outgrowth the authors analysed this pathway after FK506 treatment. FK506 induced the expression of raldh2 and crabp2b (proteins that increase RA signalling) as well as induced the downregulation of enzymes that degrade RA, suggesting that calcineurin inhibition promoted the expression of RA signalling during fin regeneration that lead to the proximalized allometric growth rate.
In summary, the authors describe the role of calcineurin as a molecular regulator of the switch between isometric and allometric growth during fin development and regeneration.