Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R ou S do gene Slc11a1 apresentam distinta capacidade regenerativa à perfuração de suas orelhas. Animais AIRmaxSS exibiram regeneração tecidual precoce em comparação aos animais AIRmaxRR, sugerindo que o alelo S favorece a regeneração nestes animais. Camundongos das sublinhagens AIRmin não apresentaram regeneração após perfuração de suas orelhas. Em resposta ao estímulo, animais AIRmaxSS exibiram inflamação local mais intensa e tardia do que animais AIRmaxRR, demonstrando elevados níveis de MPO e edema, e influxo celular predominantemente de neutrófilos. Ensaios de expressão gênica global demonstraram genes diferencialmente expressos entre as sublinhagens, evidenciando genes sobre-representados no tema biológico proliferação celular em ambas sublinhagens, enquanto somente nos animais AIRmaxSS ocorreu sobre-representação para resposta inflamatória nos genes ativados e para contração muscular nos genes reprimidos. Os resultados de microarray foram validados por qPCR. / Homozygous AIRmax and AIRmin sublines for Slc11a1 R and S alleles present distinct regenerative capacity to the ear hole. AIRmaxSS mice exhibited early tissue regeneration compared to AIRmaxRR animals, suggesting that the Slc11a1 S allele promotes regeneration in these animals. AIRmin sublines didnt show regeneration after ear punch. In response to the stimulus, AIRmaxSS animals exhibited more intense and later local inflammation than AIRmaxRR animals, presenting elevated levels of MPO, edema and cellular influx predominantly of neutrophils. Global gene expression analysis showed differentially-expressed genes between the sublines, in which over-represented biological theme is cell proliferation in both sublines. AIRmaxSS animals displayed over-representation of inflammatory response in up-regulated genes and of muscle contraction in down-regulated genes. Microarray results were validated by using quantitative PCR.

Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R ou S do gene Slc11a1 apresentam distinta capacidade regenerativa à perfuração de suas orelhas. Animais AIRmaxSS exibiram regeneração tecidual precoce em comparação aos animais AIRmaxRR, sugerindo que o alelo S favorece a regeneração nestes animais. Camundongos das sublinhagens AIRmin não apresentaram regeneração após perfuração de suas orelhas. Em resposta ao estímulo, animais AIRmaxSS exibiram inflamação local mais intensa e tardia do que animais AIRmaxRR, demonstrando elevados níveis de MPO e edema, e influxo celular predominantemente de neutrófilos. Ensaios de expressão gênica global demonstraram genes diferencialmente expressos entre as sublinhagens, evidenciando genes sobre-representados no tema biológico proliferação celular em ambas sublinhagens, enquanto somente nos animais AIRmaxSS ocorreu sobre-representação para resposta inflamatória nos genes ativados e para contração muscular nos genes reprimidos. Os resultados de microarray foram validados por qPCR. / Homozygous AIRmax and AIRmin sublines for Slc11a1 R and S alleles present distinct regenerative capacity to the ear hole. AIRmaxSS mice exhibited early tissue regeneration compared to AIRmaxRR animals, suggesting that the Slc11a1 S allele promotes regeneration in these animals. AIRmin sublines didnt show regeneration after ear punch. In response to the stimulus, AIRmaxSS animals exhibited more intense and later local inflammation than AIRmaxRR animals, presenting elevated levels of MPO, edema and cellular influx predominantly of neutrophils. Global gene expression analysis showed differentially-expressed genes between the sublines, in which over-represented biological theme is cell proliferation in both sublines. AIRmaxSS animals displayed over-representation of inflammatory response in up-regulated genes and of muscle contraction in down-regulated genes. Microarray results were validated by using quantitative PCR.

Les amphibiens urodèles (e.g. les axolotls) possèdent la remarquable capacité de régénérer plusieurs parties de leur corps. Ils peuvent, entre autres, régénérer parfaitement un membre amputé par épimorphose, un processus biphasique comprenant une phase de préparation, spécifique à la régénération, et une phase de redéveloppement, commune à l’épimorphose et au développement embryonnaire. Durant la phase de préparation, les cellules du moignon se dédifférencient en cellules pseudo-embryonnaires, prolifèrent et migrent distalement au plan d’amputation pour former un blastème de régénération. Parmi les vertébrés, la dédifférenciation est unique aux urodèles. Afin de mieux comprendre le contrôle moléculaire de la régénération chez les urodèles, nous avons choisi d’étudier BMP-2, un facteur de croissance, en raison de son implication dans la régénération des phalanges distales chez les mammifères. Le facteur de transcription MSX-1 a également été sélectionné en raison de sa capacité à induire la dédifférenciation cellulaire in vitro et de son interaction potentielle avec la signalisation des BMPs. Les résultats présentés dans cette thèse démontrent que BMP-2 et MSX-1 sont exprimés lors des phases de préparation et de redéveloppement de l’épimorphose, et que leur profil d'expression spatio-temporel est très semblable, ce qui suggère une interaction de leurs signaux. En outre, chez les tétrapodes amniotes, l’expression de Shh est restreinte au mésenchyme postérieur des membres en développement et chevauche l’expression de BMP-2. Toutefois, l’expression de BMP-2 n’est pas restreinte à la région postérieure mais forme un gradient postéro-antérieur. Shh est le principal régulateur de la formation du patron de développement antéro-postérieur du ii membre. Étant donné les domaines d’expression chevauchants de BMP-2 et Shh et la restriction postérieure d’expression de Shh, on croit que Shh régule la formation du patron de développement de postérieur à antérieur par l’activation de l’expression de BMP-2. Fait intéressant, l’axolotl exprime également Shh dans la région postérieure, mais le développement des pattes se fait de la région antérieure à la région postérieure au lieu de postérieur à antérieur comme chez les autres tétrapodes, et ceci durant le développement et la régénération. Nous avons utilisé cette caractéristique de l’axolotl pour démontrer que la signalisation Shh ne structure pas l’autopode via BMP-2. En effet, l’expression de BMP-2 n'est pas régulée par l'inhibition de la signalisation Shh, et son expression est du côté opposé à celle de Shh durant le développement et la régénération des pattes de l’axolotl. Il a été observé durant le développement du membre chez la souris que MSX-1 est régulé par la signalisation Shh. Nos résultats ont démontrés que chez l’axolotl, MSX-1 ne semble pas régulé par l'inhibition de la signalisation Shh au cours de la régénération du membre. De plus, nous avons démontré que contrairement à l’expression de Shh, l’expression de BMP-2 est corrélée avec l’ordre de formation des phalanges, est impliquée dans la condensation cellulaire et dans l'apoptose précédant la chondrogenèse. L’ensemble de ces résultats suggère un rôle de BMP-2 dans l’initiation de l’ossification endochondrale. Enfin, nous avons démontré que la signalisation BMP est indispensable pour l’épimorphose du membre durant la phase de redéveloppement. / Urodele amphibians (e.g. the axolotls) have a remarkable ability to regenerate parts of their body. They will, among other things, fully regenerate an amputated limb by epimorphosis, a biphasic process comprising a preparation phase, specific to the regeneration, and a redevelopment phase, common to epimorphosis and embryonic development. During the preparation phase, the cells of the stump dedifferentiate into embryonic-like cells, proliferate and migrate distally from the level of amputation to form a regeneration blastema. Among vertebrates, the process of dedifferentiation is unique to urodeles. To better understand the molecular control of regeneration in urodeles, we chose to study BMP-2, a growth factor, because of its involvement in mammalian digit tip regeneration. The transcription factor MSX-1 has also been selected because of its ability to induce cellular dedifferentiation in vitro and its potential interaction with BMPs signaling. The results presented in this thesis show that BMP-2 and MSX-1 are expressed during phases of preparation and redevelopment of epimorphosis, and their spatio-temporal expression profiles are very similar at each stage of epimorphosis, suggesting an interaction of their signals during regeneration. In addition, in tetrapod amniotes, the expression of Shh is restricted to the posterior mesenchyme of developing limbs and overlaps with the expression of BMP-2. However, the expression of BMP-2 is not restricted to the posterior region but forms a posterior-anterior gradient. Shh is the main regulator of the anterior-posterior pattern formation of developing limbs. Given the overlapping expression domains of Shh and BMP-2, and the expression restriction of Shh in posterior, Shh is believed to iv regulate the pattern formation of developing limbs by the activation of BMP-2 expression. Interestingly, the axolotl also expresses Shh in the posterior region, but the limb develops from anterior to posterior rather than posterior to anterior as in other tetrapods, and this, during development and epimorphosis. We used this feature of the axolotl to demonstrate that Shh signaling does not regulate pattern formation through BMP-2. Indeed, the expression of BMP-2 is not regulated by the inhibition of hh signaling, and its expression is opposite to that of Shh during development and regeneration of the axolotl limb. It was observed, during limb development in mice that MSX-1 is regulated by Shh signaling. Our results suggest that in the axolotl, MSX-1 is not regulated by the inhibition of Shh signaling during limb regeneration. Furthermore, we demonstrated that unlike the expression of Shh, the expression of BMP-2 is correlated with the order of formation of the phalanges, is involved in cell condensation and apoptosis preceding chondrogenesis. Taken together, these results suggest a role for BMP-2 in the initiation of endochondral ossification. Finally, we demonstrated that BMP signaling is essential for the redevelopment phase of limb epimorphosis.

Les amphibiens urodèles (e.g. les axolotls) possèdent la remarquable capacité de régénérer plusieurs parties de leur corps. Ils peuvent, entre autres, régénérer parfaitement un membre amputé par épimorphose, un processus biphasique comprenant une phase de préparation, spécifique à la régénération, et une phase de redéveloppement, commune à l’épimorphose et au développement embryonnaire. Durant la phase de préparation, les cellules du moignon se dédifférencient en cellules pseudo-embryonnaires, prolifèrent et migrent distalement au plan d’amputation pour former un blastème de régénération. Parmi les vertébrés, la dédifférenciation est unique aux urodèles. Afin de mieux comprendre le contrôle moléculaire de la régénération chez les urodèles, nous avons choisi d’étudier BMP-2, un facteur de croissance, en raison de son implication dans la régénération des phalanges distales chez les mammifères. Le facteur de transcription MSX-1 a également été sélectionné en raison de sa capacité à induire la dédifférenciation cellulaire in vitro et de son interaction potentielle avec la signalisation des BMPs. Les résultats présentés dans cette thèse démontrent que BMP-2 et MSX-1 sont exprimés lors des phases de préparation et de redéveloppement de l’épimorphose, et que leur profil d'expression spatio-temporel est très semblable, ce qui suggère une interaction de leurs signaux. En outre, chez les tétrapodes amniotes, l’expression de Shh est restreinte au mésenchyme postérieur des membres en développement et chevauche l’expression de BMP-2. Toutefois, l’expression de BMP-2 n’est pas restreinte à la région postérieure mais forme un gradient postéro-antérieur. Shh est le principal régulateur de la formation du patron de développement antéro-postérieur du ii membre. Étant donné les domaines d’expression chevauchants de BMP-2 et Shh et la restriction postérieure d’expression de Shh, on croit que Shh régule la formation du patron de développement de postérieur à antérieur par l’activation de l’expression de BMP-2. Fait intéressant, l’axolotl exprime également Shh dans la région postérieure, mais le développement des pattes se fait de la région antérieure à la région postérieure au lieu de postérieur à antérieur comme chez les autres tétrapodes, et ceci durant le développement et la régénération. Nous avons utilisé cette caractéristique de l’axolotl pour démontrer que la signalisation Shh ne structure pas l’autopode via BMP-2. En effet, l’expression de BMP-2 n'est pas régulée par l'inhibition de la signalisation Shh, et son expression est du côté opposé à celle de Shh durant le développement et la régénération des pattes de l’axolotl. Il a été observé durant le développement du membre chez la souris que MSX-1 est régulé par la signalisation Shh. Nos résultats ont démontrés que chez l’axolotl, MSX-1 ne semble pas régulé par l'inhibition de la signalisation Shh au cours de la régénération du membre. De plus, nous avons démontré que contrairement à l’expression de Shh, l’expression de BMP-2 est corrélée avec l’ordre de formation des phalanges, est impliquée dans la condensation cellulaire et dans l'apoptose précédant la chondrogenèse. L’ensemble de ces résultats suggère un rôle de BMP-2 dans l’initiation de l’ossification endochondrale. Enfin, nous avons démontré que la signalisation BMP est indispensable pour l’épimorphose du membre durant la phase de redéveloppement. / Urodele amphibians (e.g. the axolotls) have a remarkable ability to regenerate parts of their body. They will, among other things, fully regenerate an amputated limb by epimorphosis, a biphasic process comprising a preparation phase, specific to the regeneration, and a redevelopment phase, common to epimorphosis and embryonic development. During the preparation phase, the cells of the stump dedifferentiate into embryonic-like cells, proliferate and migrate distally from the level of amputation to form a regeneration blastema. Among vertebrates, the process of dedifferentiation is unique to urodeles. To better understand the molecular control of regeneration in urodeles, we chose to study BMP-2, a growth factor, because of its involvement in mammalian digit tip regeneration. The transcription factor MSX-1 has also been selected because of its ability to induce cellular dedifferentiation in vitro and its potential interaction with BMPs signaling. The results presented in this thesis show that BMP-2 and MSX-1 are expressed during phases of preparation and redevelopment of epimorphosis, and their spatio-temporal expression profiles are very similar at each stage of epimorphosis, suggesting an interaction of their signals during regeneration. In addition, in tetrapod amniotes, the expression of Shh is restricted to the posterior mesenchyme of developing limbs and overlaps with the expression of BMP-2. However, the expression of BMP-2 is not restricted to the posterior region but forms a posterior-anterior gradient. Shh is the main regulator of the anterior-posterior pattern formation of developing limbs. Given the overlapping expression domains of Shh and BMP-2, and the expression restriction of Shh in posterior, Shh is believed to iv regulate the pattern formation of developing limbs by the activation of BMP-2 expression. Interestingly, the axolotl also expresses Shh in the posterior region, but the limb develops from anterior to posterior rather than posterior to anterior as in other tetrapods, and this, during development and epimorphosis. We used this feature of the axolotl to demonstrate that Shh signaling does not regulate pattern formation through BMP-2. Indeed, the expression of BMP-2 is not regulated by the inhibition of hh signaling, and its expression is opposite to that of Shh during development and regeneration of the axolotl limb. It was observed, during limb development in mice that MSX-1 is regulated by Shh signaling. Our results suggest that in the axolotl, MSX-1 is not regulated by the inhibition of Shh signaling during limb regeneration. Furthermore, we demonstrated that unlike the expression of Shh, the expression of BMP-2 is correlated with the order of formation of the phalanges, is involved in cell condensation and apoptosis preceding chondrogenesis. Taken together, these results suggest a role for BMP-2 in the initiation of endochondral ossification. Finally, we demonstrated that BMP signaling is essential for the redevelopment phase of limb epimorphosis.

Scar-free wound healing and regeneration are uncommon phenomena permitting the near complete restoration of damaged tissues, organs and structures. Although rare in mammals, many lizards are able to undergo scarless healing and regeneration following loss of the tail. This study investigated the spontaneous and intrinsic capacity of the leopard gecko (Eublepharis macularius) tail to undergo scar-free wound healing and regeneration following two different forms of tail loss: autotomy, a voluntary and evolved mechanism of tail shedding at fracture planes; and surgical amputation, involuntary loss of the tail outside the fracture planes. Furthermore, I investigated the ability of the regenerate tail to regenerate by amputating a regenerate tail (previously lost by autotomy). To investigate these phenomena I imaged wound healing and regenereating tails daily (following autotomy and amputation) to document gross morphological changes. I used histochemistry to document tissue structure and immunohistochemistry to determine the tissue/cellular location of my five proteins of interest (PCNA, MMP-9, WE6, α-sma, TGF-β3). Each of these proteins of interest has been previously documented during wound healing and/or regeneration in other wound healing/regeneration model organisms (e.g. mice, urodeles, lizards, zebrafish). Scar-free wound healing and regeneration occurred following autotomy, amputation of the original tail and amputation of the regenerate tail, indicating that the leopard gecko tail has an instrinsic scar-free wound healing and regenerative capacity that is independent of the mode of tail loss (autotomy or amputation). Furthermore immunohistochemistry revealed a conserved sequence and location of the expression of the five proteins of interest following both forms of tail loss. These results provide the basis for further studies investigating scar-free wound healing and regeneration in a novel amniote model, the leopard gecko. / NSERC

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.

Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate? Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration. The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb. As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo. In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.