<p>Vertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration. </p><p>Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.</p><p>During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.</p> / Dissertation

Contrairement aux mammifères adultes, l’amputation d’un membre de vertébrés capables de régénérer, est suivie de la formation d’une structure hautement proliférative et hétérogène : le blastème. Les conditions de formation de ce blastème sont encore mal connues. La sécrétion de facteurs par les cellules dérivées de la crête neurale CCN, seraient à l’origine de la prolifération du blastème. De plus, les macrophages sont recrutés sur le site d’amputation et participeraient à la régénération mais leurs mécanismes d’actions et interactions avec les CCN n’ont jamais été étudiés dans ce contexte. Mon projet de thèse avait pour but d’élucider ces mécanismes en s’appuyant sur deux modèles de régénération : la régénération de la nageoire chez la larve de zebrafish et le membre supérieur de l’embryon de souris au stade E10.5. Ces travaux ont permis :• Chez la larve de zebrafish : d’identifier deux sous-types de macrophages recrutés de manière séquentielle au cours de la régénération, de montrer que l’activation de la voie TNFa/TNFR1 par les macrophages était nécessaire à la prolifération du blastème, d’identifier une population de CCN foxd3+ dans la nageoire dont la présence est indispensable au recrutement et à la polarisation des macrophages ainsi qu’à la prolifération des cellules du blastème.• Chez l’embryon de souris : d’identifier un stade régénératif (E10.5) et non régénératif (E12.5), de montrer l’accumulation de CCN au niveau du site d’amputation au stade E10.5 et de démontrer le rôle de ces cellules dans le processus de régénération. / Unlike in adult mammals, in regenerative species, appendage amputation is followed by the formation of a highly proliferative and heterogeneous structure called the blastema. The required conditions for its formation are still not completely understood. Paracrine factors produced by neural crest derived cells (NCC) have been proposed to be responsible for blastemal cell proliferation. Moreover, macrophages are recruited to the wound site and could participate to the regeneration process. However, their exact functions and interactions with NCC during regeneration have never been investigated. My thesis project consisted in deciphering those mechanisms using two different models: zebrafish larva caudal fin regeneration and forelimb bud regeneration of the E10.5 mouse embryo. This work allowed us:• In zebrafish larva: to identify two subpopulations of macrophages, to highlight their roles during regeneration, to demonstrate the role of the TNFa/TNFR1 axis in the blastemal cell proliferation and to identify a new foxd3+ NCC population in the caudal fin, which is required for macrophage recruitment, polarization and for blastemal cell proliferation. •In mouse embryo: to identify a regenerative (E10.5) and non-regenerative (E12.5) stage of development, to demonstrate the accumulation of NCC at the wound site in E10.5 embryos and demonstrate the crucial role of NCC during epimorphic regeneration in mammals.

We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.

Urodele Amphibien besitzen außergewöhnliche Regenerationsfähigkeiten, die es ihnen ermöglichen, verlorene Körperteile vollständig nachzubilden. Besonders der Axolotl ist aufgrund seiner einfachen Haltung im Labor ein wichtiger Modellorganismus für die Regenerationsforschung. Allerdings schränkt seine pädomorphe Lebensweise – das Ausbleiben einer natürlichen Metamorphose – die Übertragbarkeit der Ergebnisse auf andere Salamanderarten ein. Um Unterschiede und Gemeinsamkeiten in zugrundeliegenden Mechanismen von Regenerationsprozessen zu entschlüsseln sowie evolutionäre und ökologische Einflüsse besser zu verstehen, sind vergleichende Studien mit weiteren Salamanderarten entscheidend. Kapitel I vergleicht die Regenerationsfähigkeit des Axolotls mit der des metamorphosierenden Tigersalamanders. Dabei zeigen larvale Tigersalamander ähnlich beeindruckende Regenerationsfähigkeiten wie Axolotl, während postmetamorphe Individuen eine unvollständige Frakturheilung und verzögerte Regeneration mit Skelettanomalien aufweisen. Kapitel II untersucht die Schwanzregeneration des Tigersalamanders während der Metamorphose und zeigt, dass sie in und auch nach diesem Entwicklungsstadium fortgesetzt wird, obwohl die Geschwindigkeit und die strukturelle Qualität der Regeneration in den verschiedenen Entwicklungsstadien variieren, was den Einfluss der Metamorphose auf den Regenerationsprozess unterstreicht. Kapitel III analysiert die Gliedmaßenregeneration bei sechs plethodontiden Salamanderarten mit verschiedenen Lebensweisen und Habitaten. Alle Arten zeigen erhebliche Regenerationsfähigkeiten, wenngleich häufig anatomische Anomalien auftreten. Zudem werden Korrelationen zwischen der Regenerationsgeschwindigkeit und dem jeweiligen Habitat festgestellt. Kapitel IV untersucht die Flossenregeneration bei Lungenfischen. Dabei auftretende Anomalien ähneln denen von Salamandern und stützen damit die Hypothese, dass epimorphe Regenerationsfähigkeiten einen tiefen evolutiven Ursprung besitzen. / Urodele amphibians possess remarkable regenerative abilities, allowing them to fully restore lost body parts. The axolotl, in particular, has become a key model organism for regeneration research due to its easy maintenance in laboratory settings. However, its paedomorphic life history pattern—the absence of natural metamorphosis—limits the generalizability of findings to other salamander species. To decipher differences and similarities in the underlying mechanisms of regeneration processes and to better understand evolutionary and ecological influences, comparative studies with other salamander species are essential. Chapter I compares the regenerative abilities of the axolotl with those of the metamorphosing tiger salamander. Larval tiger salamanders exhibit similarly impressive regenerative capacities as axolotls, while post-metamorphic individuals show incomplete fracture healing and delayed regeneration with skeletal anomalies. Chapter II investigates tail regeneration in the tiger salamander during metamorphosis, demonstrating that regeneration continues both during and after this developmental stage, although its speed and structural quality vary. This highlights the impact of metamorphosis on the regeneration process. Chapter III analyzes limb regeneration in six plethodontid salamander species with different lifestyles and habitats. All species exhibit significant regenerative abilities, though anatomical anomalies are common after regeneration. Additionally, correlations between regeneration speed and habitat are identified. Chapter IV examines fin regeneration in lungfish. The observed anomalies resemble those found in salamanders, supporting the hypothesis that epimorphic regenerative abilities have a deep evolutionary origin.

No description available.

<p>Regeneration is a remarkable feat of developmental regrowth and patterning. The blastema is a mass of progenitor cells that enables complete regeneration of amputated salamander limbs or fish fins. Despite years of study, methodologies to identify and track blastemal cell progenies have been deficient, restricting our understanding of appendage regeneration at a cellular and molecular level. To bridge this knowledge gap, gene expression analysis, the generation of transgenic and mutant zebrafish, qualitative and quantitative analyses, morphological measurements, and chemical treatments were used to assess molecular and cellular processes involved in fin regeneration. Two main findings arose from these methods. The first provides evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures, and that amputation stimulates resident cells to reset proximodistal positional information. The second identifies a fibroblast subpopulation near uninjured fin joints that contributes to the blastemal progenitor population. These findings reveal insights on cellular and molecular mechanisms of appendage regeneration and provide a basis for work exploring how cells in an adult vertebrate bone appendage coordinately rebuild a new structure.</p> / Dissertation

La tumeur de Wilms (WT) est la tumeur du rein la plus fréquente chez l'enfant âgé de moins de 5 ans. Bien que la majorité des enfants avec WT soit aujourd'hui soignée, 10 à 15% rechutent. L'identification de nouveaux marqueurs pronostic au diagnostic est donc nécessaire. Nous avons centralisé le matériel biologique de toutes les WTs françaises inclus dans le protocole SIOP-2001. Le blastème, la composante histologique la plus agressive de ces tumeurs, a été caractérisé par une analyse qualitative (architecture et aspect cytologique, index mitotique et prolifératif) et quantitative (volume et pourcentage). En collaboration avec les équipes européennes du Renal Tumor Study Group nous avons caractérisé les anomalies structurales de la WT par Multiple Ligation Probe Amplification (MLPA). De plus, nous nous sommes focalisés sur la région 11p15, où le gène IGF2 est localisé, en analysant les anomalies structurales et de méthylation au niveau de différents loci (IGF2-DMR0, ICR1, ICR2, H19), par MLPA et par ASMM RTQ- PCR (TaqMan allele-specific methylated multiplex real-time quantitative PCR). Nous avons de plus étudié l'expression d'IGF2 par RT-QPCR. Une dernière partie de notre étude a porté sur la caractérisation de la réponse immune intratumorale par immunohistochimie (CD3, CD4, CD8, PD1, PDL1). En conclusion, nous avons identifié des potentiels marqueurs pronostiques concernant la prise en charge des WTs: volume et pourcentage de blastème, index mitotique et de prolifération, perte de méthylation d'IGF2-DMR0 et rapport CD4/CD8. Ce travail pourrait contribuer à la détermination d'une nouvelle classification bio-pathologique de la WT. / Wilms tumor (WT) is the most common tumor of the kidney in children aged under 5 years. Although the majority of children with WT survives, 10-15% relapse. The identification of new prognosis markers at diagnosis is necessary. We centralized the biological material of all French WTs included in the SIOP-2001 protocol. The blastema, the most aggressive histological component of these tumors, was characterized by qualitative (architectural and cytological aspect, proliferative and mitotic index) and quantitative analysis (amount and percentage). In collaboration with European teams Renal Tumor Study Group we characterized the structural abnormalities of the WT by Multiple Ligation Probe Amplification (MLPA). In addition, we focused on the 11p15 region, where the IGF2 gene is located, analyzing the structural abnormalities and methylation at different loci (IGF2-DMR0, ICR1, ICR2, H19) by MLPA and ASMM RTQ - PCR (TaqMan allele-specific methylated multiplex quantitative real-time PCR). We also studied the expression of IGF2 by RT-QPCR. A final part of our study focused on the characterization of intratumoral immune response by immunohistochemistry (CD3, CD4, CD8, PD1, PDL1). In conclusion, we identified potential prognostic markers for the management of WTs: volume and percentage of blastema, mitotic and proliferation index, loss of IGF2 methylation-DMR0 and CD4 / CD8 ratio. This could contribute to the determination of a new bio-pathological classification of WT.