PROGRAMA DE DOUTORAMENTO INTER-UNIVERSITÁRIO EM ENVELHECIMENTO E DEGENERESCÊNCIA DE SISTEMAS BIOLÓGICOS COMPLEXOS

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Mechanisms of osteoblast reprogramming and differentiation during zebrafish caudal fin regeneration

Publication . Brandão, Ana Sofia da Silva Pereira; Jacinto, António

Regeneration is an impressive biological process that allows the replacement of lost body parts due to damage or injury, restoring both tissue architecture and function. It is well documented that while mammals have a limited capacity to regenerate lost tissues, other vertebrates, such as amphibians and teleost fish, exhibit a remarkable capacity to regenerate organs, like the heart and the retina, and large sections of the body, such as the limb and the fin. Zebrafish has become an important model system to study vertebrate regeneration and the adult caudal fin is one of the most used tissues to comprehend how tissues are restored. This structure is easily accessible to surgery, amenable to live imaging, its amputation does not compromise survival and regeneration is particularly fast, occurring over the course of two weeks. Fin regeneration is an epimorphic process since it relies on a specialized structure called blastema, which is composed of a proliferative heterogeneous population of dedifferentiated cells with restrictive lineage potential. After caudal fin amputation, a regenerative program is activated and occurs in three sequential phases: wound healing, blastema formation, and regenerative outgrowth. These events comprise a tight coordination between proliferation, patterning and differentiation to reconstitute the architecture and the size of the original tissue. The adult caudal fin is composed of multiple tissues, including blood vessels, nerves, mesenchyme and the structural support, the bony-rays (skeletal elements). Each bony-ray is surrounded and maintained by an outer and inner monolayer of bone secreting cells, the osteoblasts. Many studies have focused on bone regeneration since the zebrafish caudal fin provides a unique model to understand bone formation and osteoblast dynamics upon tissue damage and regeneration. After caudal fin amputation, formation of the new bone elements depends greatly on tissue plasticity (changes in cellular identity). This is achieved through the activation of two complementary processes that enable the assembly of an osteoblast progenitor pool during blastema formation: dedifferentiation of resident mature osteoblasts and commitment of joint-associated osteoblast progenitors. Complex regulatory mechanisms subsequently maintain and expand the osteoblast progenitor pool and promote their redifferentiation into mature osteoblasts to restore the skeletal tissue. Therefore, both osteoblast progenitor assembly and redifferentiation are critical aspects of caudal fin bony-ray regeneration. Interestingly, ablation of mature osteoblasts prior to caudal fin amputation does not affect normal bone regeneration, suggesting that de novo bone formation can rely solely on the commitment of joint-associated osteoblast precursors or on new osteoblast progenitors arising from alternative sources.In this PhD thesis, I aimed to unravel key aspects of caudal fin bone regeneration, focusing on new regulators of osteoblast dedifferentiation and redifferentiation and alternative sources for de novo osteoblast formation in osteoblast-depleted fins. To investigate novel regulators of osteoblast dedifferentiation, we performed a genome-wide gene expression analysis of osteoblasts undergoing dedifferentiation. With this analysis, we concluded that this process occurs much earlier in the regenerative process than what was previously thought. Furthermore, we characterized the molecular basis of osteoblast dedifferentiation regarding epigenetic modulation, signal transduction, cell adhesion reorganization, epithelial to mesenchymal transition and acquisition of migratory behaviour and proliferation. Particularly, we observed that osteoblasts change their metabolic signature upon injury. This was predicted based on the upregulation of several glycolytic and lactate producing enzymes, which is followed by an increase in the expression of oxidative phosphorylation electron transport chain components. We hypothesize that osteoblast dedifferentiation relies on a bivalent metabolism that uses both glycolysis and oxidative phosphorylation, which may reflect an adaption to the energetic demands of regeneration. Since the link between metabolic adaptation and regeneration remains poorly understood, we decided to address it by inhibiting the glycolytic influx. We observed major defects in the regenerative process, including impaired assembly of the wound epidermis, a major signalling centre during regeneration, and fewer cells re-entering the cell cycle. In addition, we showed that several osteoblast markers were downregulated and that osteoblast populations became disorganized. This suggests that metabolic adaptation plays an important role in regeneration, in particular during osteoblast dedifferentiation. In addition to the transcriptional analysis, we followed a targeted approach. We examined the role of the Hippo signalling pathway as a potential regulator of osteoblast dedifferentiation, by inhibiting Yap (Hippo pathway effector). This prevented mature osteoblasts to migrate, re-enter the cell cycle and to assemble the osteoblast progenitor pool. In parallel, we evaluated the role of this pathway in mediating osteoblast redifferentiation during regenerative outgrowth. We noticed that Yap inhibition leads to a decrease in the number of differentiating osteoblasts and to the misregulation of key signalling pathways, such as Bmp and Wnt signalling. We provide evidence that Yap not only promotes osteoblast differentiation through activation of Bmp signalling via bmp2a expression but also restricts the osteoblast progenitor pool by inhibiting Wnt signalling to the differentiation front by regulating dkk1a. Altogether, these results lead us to propose that the Hippo/Yap signalling pathway regulates osteoblast dedifferentiation as well as redifferentiation. This reveals a previously unknown duality if the Hippo/Yap pathway in controlling two different aspects of osteoblast biology during caudal fin regeneration. Lastly, we provide evidence into the cellular and molecular mechanisms that regulate de novo osteoblast formation in osteoblast-depleted caudal fins. We identified an additional osteoblast progenitor population that arises at the outer and inner bone surfaces adjacent to the epidermal and mesenchymal compartments, respectively. These cells are not part of a uniform population but seem to form two distinct osteoblast progenitor populations with different origins or expression profiles. Lineage tracing experiments revealed that mesenchymal cells within the intraray compartment, but not epidermal cells, contribute to generate new osteoblasts in osteoblast-depleted caudal fins. This provides new evidence of an additional source of osteoblasts for regeneration. Moreover, we showed that both Retinoic Acid and Bmp signalling pathways are activated in this osteoblast progenitor population and are important to induce their commitment and recruitment during caudal fin regeneration. Thus, we elucidate potentially dormant regenerative mechanisms that emerge to ensure correct bone formation in caudal fins lacking mature osteoblasts. Taken together, this PhD thesis provides novel insights into new regulators of bone formation and alternative cells that can contribute to correct bone regeneration upon injury. We expect that defining the mechanisms regulating tissue plasticity, reprogramming and fate specification during bone reconstitution have major implications not only to understand the basic mechanisms that regulate tissue regeneration but also to the field of regenerative medicine and bone cancer biology.

2019-11-12Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

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SFRH/BD/51990/2012