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Title: Zebrafish caudal fin regeneration : the effect of repeated amputations and ageing
Other Titles: Regeneração da barbatana caudal do peixe zebra : o efeito de amputações repetidas e envelhecimento
Authors: Azevedo, Ana Sofia Rodrigues dos Santos 
Orientador: Duarte, Carlos
Keywords: Peixe-zebra; RNA
Issue Date: 2012
Abstract: Whilst all organisms developed schemes to respond to injury and illness, their capacity to recover from severe loss or damage of organs and appendages diverge quite a lot. A vertebrate organism that retained regenerative capacity is the zebrafish (Danio rerio). Its amenability to molecular and genetic manipulation turned it into a powerful regeneration model. In particular, zebrafish caudal fin regeneration has emerged as an ideal model to further study vertebrate regeneration due its accessibility and simple anatomical structure. The caudal fin is composed of several segmented bony rays. Each bony ray, with the exception of the most lateral, is bifurcated in the distal region of the fin. Regarding the caudal fin regeneration process, it is commonly believed that regeneration efficiency is lost upon repeated amputations. The aim of my thesis was to characterize in detail whether there is a decrease in regeneration efficiency and to identify the signalling pathways that are altered, in response to repeated injuries. To this end, we designed a protocol of consecutive repeated amputations in which the same caudal fins were subjected to three consecutive amputations every month. This protocol was repeated 10 times and resulted in a total of 29 amputations in the end of the protocol. Our results show that the size of the blastema, which is a structure comprised of progenitor cells that direct regeneration, and of the fully regenerated fin remains unchanged. Thus, consecutive repeated amputations of the zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. The inhibition of Wnt/β-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 (Dkk1) completely blocks fin regeneration. We overexpressed dkk1-gfp twice daily starting shortly before fin amputation and until 4 days-post-amputation (dpa) to completely inhibit fin regeneration. ii Once these fish were relieved from the heat-shock treatment, spontaneous regeneration did not occur. However, when fins were re-amputated at the non-inhibitory temperature, the caudal fin regenerated and reached its original length. To further challenge the regenerative capacity we performed repeated cycles of amputation, inhibition of Wnt/β-catenin signalling, recovery and second amputation. Remarkably, repeated blockage of blastema formation and fin regeneration after inhibition of Wnt/β-catenin signalling, did not diminish the regenerative capacity after a new amputation stimulus. We conclude that, blastema formation and regenerative outgrowth do not depend on a biological process that is permanently disrupted or depleted by loss of Wnt/β-catenin signalling. In spite of this amazing capacity to regenerate, we observed that, while the bone distal to the amputation plane (new bone) regenerated with a normal morphology, the bone proximal to the amputation plane (old bone) became progressively thickened with the repeated cycles of amputations. We suggest that this progressive bone thickening can be due to an inappropriate activation of osteoblasts that secrete matrix far away from the amputation plane or, alternatively, an unbalanced ratio of bone-forming and bone-degrading cells. Moreover, we detected an alteration in the original pattern of pigment cells and a distal shift in the position of the bony ray bifurcations in the regenerated caudal fins. We wanted to further investigate how the positional information is established during fin regeneration and whether it is altered by repeated amputations at different proximo-distal (PD) places along the fin. Our results show that upon a first amputation at 4 segments of the bony ray from the base of the fin (proximal amputation), the bifurcation position was immediately distalized when compared to its previous position in the uncut fin. Following the second, third and fourth amputation, the bifurcation position was maintained in the regenerated fin. On the other hand, the bifurcation position was progressively distalized when the amputations were done at 1 iii segment proximal to the bifurcation (near bifurcation – distal amputation). Thus, we show that while amputations performed at a long distance from the bifurcation do not change its PD position in the regenerated fin (after a first amputation), consecutive distal amputations induce a positional reset and progressively shift its position distally. Therefore, it is possible that an amputation proximally near the bifurcation will inhibit the signal responsible to initiate the formation of a bifurcation and consequently delay this process. We aimed to determine the signals involved in the control of the bifurcation position by the amputation place. To this end, we analyzed in detail the role of Sonic hedgehog (Shh), since previous reports propose that, preceding the formation of a bony ray bifurcation, shh duplicates its single domain. However, in contrast, our analysis shows that the dynamics of shh expression does not change in response to different amputation places, being always two domains of expression throughout the regeneration process. Thus, Shh does not seem to be the factor that modulates the bifurcation position during fin regeneration. Given the fact that it has been proposed that Shh might play a role in the osteoblasts patterning and/or differentiation during fin regeneration we analyzed Zns5 expression, an osteoblast marker in a shh-gfp transgenic reporter line. We observed that soon after the detection of shh expression, the bone alters its growing tip, and the forming osteoblasts start to be aligned close to the basal layer of the epidermis next to shh expressing cells. This leads to the hypothesis that shh expression in two separate domains might be important to align and direct the growth of the regenerating bone. Finally, we analyzed the implication of Fibroblast growth factor (Fgf) signalling in the modulation of the bifurcation position by the amputation place, since it was previously reported that the levels of Fgf signalling activation vary according to the PD place of amputation. This reveals the existence of positional memory in the regenerating fin that can be mediated or act through Fgf signalling. In order to investigate whether Fgf signalling would determine the PD position of the bifurcation in the regenerated fin, we iv made use of the hsp70:dn-fgfr1 zebrafish transgenic. This transgenic contains a dominant-negative fgfr1-egfp fusion gene (dn-fgfr1) driven by a heat-inducible zebrafish hsp70 promoter and efficiently attenuates Fgf signalling during fin regeneration in a dose dependent manner. However, Fgf signalling attenuation did not alter the position of the bony ray bifurcation, when compared to the controls, indicating that Fgf signalling may not be the trigger signal for the formation of a bifurcation in zebrafish fin regeneration. The establishment of positional memory during vertebrate regeneration has been mainly investigated in the amphibian limb. Nevertheless, the signals involved in the maintenance of positional memory remain poorly understood. The better understanding of this process in model organisms will be of great importance in the regenerative medicine field, namely to achieve the proper tridimensional structure for a successful and functional integration of the in vitro generated organs into patients. Additionally, we believe that better understanding of the cellular mechanisms underlying the virtually unlimited regenerative capacity of fish caudal fin regeneration will be informative for efforts to improve repair in humans.
Description: Tese de doutoramento em Biologia apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Rights: openAccess
Appears in Collections:FCTUC Ciências da Vida - Teses de Doutoramento

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