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.

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Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

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.

Newts have the incredible ability to regenerate many different organs and tissues as adults, including the limbs. Limb regeneration occurs via the dedifferentiation of stump tissue and the formation of a blastema, which provides the majority of cells for the regenerate. Despite all that we have learned about dedifferentiation and blastema formation, the cellular and molecular mechanisms underlying these processes are still poorly understood. We used representational difference analysis (RDA) to identify genes involved in the early dedifferentiation process in newt forelimb regeneration. Our analysis identified approximately 410 unique genes that were differentially regulated during this process. Microarray analysis was used to determine the expression profile of these genes throughout limb and tail regeneration. We used quantitative PCR (qPCR) to validate the expression of a subset of these genes [β-catenin, wntless, dapper, thymosin-β 4 (Tβ4), and thymosin-β 10/15 (Tβ10/15)] in regenerating limb and tail tissue, as well as in differentiating newt myoblasts. We also verified the expression of these genes in the regenerating newt limb using immunohistochemistry (IHC) and in situ hybridization (ISH). Finally, we performed a functional analysis on β-catenin, wntless, dapper, and Tβ4 by overexpressing these genes in mouse myoblasts to examine their effects on differentiation and potential roles in dedifferentiation. Quantitative PCR verified the expression of β-catenin, wntless, dapper, and Tβ4 during limb regeneration and IHC/ISH localized the β-catenin and Tβ4 proteins to the blastema during regeneration. Tβ10/15 was shown by qPCR to be expressed in the tail during regeneration. Overexpression of newt β-catenin, wntless, dapper, and Tβ4 in mouse myoblasts showed that each of these genes has an inhibitory effect on the differentiation of myoblasts into myotubes and, therefore, may play a role in promoting or maintaining the dedifferentiated state. Our work has identified a large number of genes with potential roles in regulating the dedifferentiation process during newt forelimb regeneration. We have also laid a framework from which much more work can be done by drawing on the genes we have identified and the microarray data, which indicate ideal follow-up candidates. Our analysis of specific genes has also increased our understanding of the molecular events occurring during the dedifferentiation process in the regenerating newt limb.

Indiana University-Purdue University Indianapolis (IUPUI) / Biological systems behave differently under different conditions. Advances in sequencing technology over the last decade have led to the generation of enormous amounts of condition-specific data. However, these measurements often fail to identify low abundance genes/proteins that can be biologically crucial. In this work, a novel text-mining system was first developed to extract condition-specific proteins from the biomedical literature. The literature-derived data was then combined with proteomics data to construct condition-specific protein interaction networks. Further, an innovative condition-specific differential analysis approach was designed to identify key differences, in the form of subnetworks, between any two given biological systems. The framework developed here was implemented to understand the differences between limb regeneration-competent Ambystoma mexicanum and –deficient Xenopus laevis. This study provides an exhaustive systems level analysis to compare regeneration competent and deficient subnetworks to show how different molecular entities inter-connect with each other and are rewired during the formation of an accumulation blastema in regenerating axolotl limbs. This study also demonstrates the importance of literature-derived knowledge, specific to limb regeneration, to augment the systems biology analysis. Our findings show that although the proteins might be common between the two given biological conditions, they can have a high dissimilarity based on their biological and topological properties in the subnetwork. The knowledge gained from the distinguishing features of limb regeneration in amphibians can be used in future to chemically induce regeneration in mammalian systems. The approach developed in this dissertation is scalable and adaptable to understand differential subnetworks between any two biological systems. This methodology will not only facilitate the understanding of biological processes and molecular functions which govern a given system but also provide novel intuitions about the pathophysiology of diseases/conditions.

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.