<p> PEA3 is a member of the expanding Ets family of transcription factors. In the adult mouse, pea3 mRNA is expressed at highest levels in the brain, epididymis and at lower levels in the mammary gland, testes, ovary and uterus. PEA3 is overexpressed in 93% of all HER2/Neu positive human breast tumors and in 77% of mouse multiple intestinal (Min) tumors. Many of these tumors have disruptions in the Ras/MAPK and Wnt signaling pathways. Analysis of the influence of these pathways on pea3 promoter activity revealed that effectors of both pathways increased transcription from this promoter. Deletion mutations of the pea3 promoter linked to a luciferase reporter gene were used to localize the DNA sequences that are responsible for the effect of the Ras/MAPK pathway on its expression. A Ras-responsive element (RRE), composed of an ETS and an AP-1 binding site, was identified between sequences -247 and -227 and its importance was confirmed through mutational analysis. </p> <p> CYCLIN Dl is a potent oncogene involved in different types of tumors. The CYCLIN D1 gene is amplified in 20% of human mammary carcinomas, and its mRNA is overexpressed in 50% of human breast cancers. The CYCLIN Dl (CDJ) promoter was shown to be responsive to PEA3 transactivation and to dominant-negative PEA3 inhibition in co-transfection experiments in Cos-1 cells. Of the 4 Ets-binding sites (EBS) in the CDJ promoter, one site was shown to be important for the activity of the promoter and for its capacity to respond to PEA3 transactivation. It was also determined that PEA3, ~-catenin, Lef-1 and c-Jun cooperated synergistically to activate the CDJ promoter. PEA3 was absolutely required for the manifestation of this synergy among these transcription factors. These findings collectively illustrate the key role of PEA3 as an effector of multiple oncogenic signaling pathways. </p> / Thesis / Master of Science (MSc)

PEA3, ERM, and ER81 comprise a subfamily of ETS transcription factors that upregulate genes correlated with an increased metastastic potential of tumors. In mouse embryo fibroblast (MEF) cells, PEA3 is required for transformation by activated Ras or Neu, but the means by which PEA3 mediates Ras-transformation is not clear. Osteopontin (OPN) expression is induced upon B-ras-transformation and purified PEA3 can bind the OPN promoter by gel-shift analysis. In this study, OPN expressed higher transcript levels in the wildtype MEF 4 cell line than the PEA3 null MEF 1 cell line and was further characterized as a potential PEA3 target gene by Northern blot analyses and transient transfection studies. Northern blot analyses of 4 wildtype MEF (4, 100, 101, 104), 5 FEA3 null MEF (1, 115, B5, B10, B12), and 5 MEF 1 retransformant cell lines that stably reexpress PEA3 showed a good correlation between OPN and ERM transcript levels in 9/11 cell lines although at least 2 PEA3 subfamily members were coexpressed in 8/11 cell lines that expressed high OPN transcript levels. This suggested that the PEA3 subfamily additively regulated OPN and that ERM protein was more abundant than PEA3 and ER81 protein levels in the MEF cell lines. The relative PEA3 subfamily protein levels remain to be clarified. Transient transfection assays in the HEK 293-1 C cell line indicated that the OPN promoter was responsive to PEA3 and that the promoter region between -258 to -88 was required for maximal OPN promoter activity. There are 16 candidate core ETS binding sites in the -777 /+79 OPN promoter which could be responsible for PEA3 subfamily transactivation. The OPN promoter was more active in the MEF 4 cell line than the MEF 1 cell line, corresponding to their relative number of expressed PEA3 subfamily members. Ectopic expression of dominant negative PEA3 suppress ed OPN promoter activity in the MEF 4 cell line. Furthermore, ectopic expression of PEA3, ERM, or ER81 increased OPN promoter activity in the MEF 1 or COS-1 cell line. Thus OPN is transcriptionally regulated by the PEA3 subfamily and represents a target gene that can mediate the progression of tumor cells. / Thesis / Master of Science (MSc)

Les Facteurs de transcription du groupe PEA3 (Pea3, Erm et Er81) font partie de la famille d’oncogènes ETS. Leur expression est souvent observée lors de la mise en place des organes par morphogenèse de branchement tels que les poumons ou encore la glande mammaire. De plus une expression aberrante de ces facteurs de transcription est corrélée au caractère cancéreux de nombreux tissus tels que le côlon, les poumons ou encore le sein. Ainsi, l’expression d’Erm dans les tumeurs du sein est associée à un mauvais pronostic pour les patientes et celle de Pea3 constitue un marqueur de l’agressivité tumorale. Enfin, en qualité de facteur de transcription Pea3 module l’expression de gènes spécifiques alors appelés gènes cibles. Même si certains de ces gènes sont déjà bien caractérisés beaucoup de choses restent à faire pour comprendre les mécanismes moléculaires régulés par Pea3. Dans ce contexte lors de ma thèse je me suis intéressé à l’étude du rôle du facteur de transcription Pea3 dans les processus de morphogenèse et de tumorigenèse mammaires selon deux approches complémentaires : 1- l’étude des propriétés morphogénétiques modulées par Pea3 lors des étapes de morphogenèse et de tumorigenèse mammaires, 2- la recherche et la caractérisation de gènes régulés par Pea3 dans ce même contexte, par une analyse transcriptomique à grande échelle en utilisant des puces à ADN. Ces deux points sont développés grâce à l’utilisation de modèles cellulaires dans lesquelles nous modulons l’expression de Pea3. Les cellules épithéliales mammaires TAC 2.1 modèle de morphogenèse mammaire dans lesquelles nous surexprimons Pea3 et les cellules mammaires transformées MMT, modèle de tumorigenèse mammaire dans lesquelles nous inhibons l’expression du facteur de transcription Pea3. Au cours de ma thèse nous avons ainsi pu montrer l’importance du facteur de transcription Pea3 dans le contrôle des propriétés de migration, d’invasion et de prolifération des cellules cancéreuses TAC et MMT. En accord avec ces données, la recherche des gènes dont l’expression est régulée par Pea3 dans nos deux modèles cellulaires suite à la modulation de Pea3, a permis d’identifier de nombreux gènes capables de réguler la prolifération, la migration et l’invasion des cellules. Parmi ces gènes nous nous sommes intéressés au gène cycline d2 bien connu pour son implication dans le contrôle de la progression du cycle cellulaire. Nous avons pu montrer que le gène cycline d2 est un gène cible direct du facteur de transcription Pea3 qui module l’expression dans le modèle cellulaire TAC des deux transcrits (cycline d2 et cycline d2 trc) issus de ce gène et décrits à ce jour. L’étude de la fonction des protéines Cycline D2 et Cycline D2 Trc dans les cellules TAC a été entreprise. Tout d’abord la surexpression de l’une ou l’autre de ces isoformes dans les cellules TAC 2.1 modifie de façon opposée leur capacité à s’organiser dans un gel de collagène mimant l’environnement d’une glande mammaire, la Cycline D2 réprimant cette capacité et la Cycline D2 Trc l’augmentant. L’utilisation de petits ARN interférents permettant de réprimer l’expression de ces deux protéines a permis de montrer une relation fonctionnelle, toujours opposée, des deux isoformes avec le facteur de transcription Pea3 pour le contrôle de la progression du cycle cellulaire mais aussi pour l’induction d’une transition épithélio-mésenchymateuse étroitement reliée au pouvoir de migration des cellules épithéliales lors du développement des organes comme la glande mammaire mais aussi lors de la progression tumorale. Notre étude a ainsi permis de mieux définir l’implication du facteur de transcription Pea3 lors des événements de morphogenèse et de tumorigenèse de la glande mammaire. De plus elle ouvre la réflexion sur le rôle du gène cycline d2 lors de ces événements. / "Proteins of the PEA3 group (Pea3, Erm and Er81) belong to the ETS family of transcription factors. They are expressed in organs that undergo an epithelial branching morphogenesis process such as the lungs and the mammary gland. Moreover, in these organs, they are aberrantly expressed during cancer progression. Indeed, during breast cancer high Pea3 or Erm expression is respectively associated with cancer metastatic potential and a lower patient survival rate. Finally, as transcription factors they control the expression of specific genes called target genes. Even though some of these genes are known, more work is needed to understand the molecular mechanism governed by the PEA3 transcription factors. The main topic of my PhD is the study of the role of the Pea3 transcription factor during mammary morphogenesis and tumorigenesis using two complementary approaches: 1- characterize the morphogenetic properties that are controlled by Pea3 during the mammary morphogenesis and tumorigenesis events 2- find and characterize the genes that are regulated by Pea3 using a large scale transcriptomic analysis based on a microarray technology. The strategy is based on the utilization of two main cell lines in which we modulate Pea3 expression: an epithelial cell line, model of mammary morphogenesis (TAC 2.1) in which we overexpress Pea3 and a cancer cell line, model of mammary tumorigenesis (MMT) in which Pea3 expression is knocked down by means of small interfering RNA sequences. During my PhD we showed that Pea3 controls the proliferation, invasion and migration properties of TAC 2.1 and MMT cells. In agreement with these data, the transcriptomic analysis after Pea3 expression modulation in these cells (overexpression or knockdown) demonstrate that a large proportion of the Pea3 regulated genes are already known players in the regulation of the proliferation, invasion and migration processes. Amongst these genes, we focused on the cyclin d2 gene which is a well characterized actor in cell cycle progression and cell proliferation. We showed that cyclin d2 is direct Pea3 target gene. The cyclin d2 gene gives rise to two different isoforms generated by a splicing event (Cyclin D2 and Cyclin D2 Trc) that are both regulated by Pea3 at the mRNA and protein levels. We then evaluated the function of these two isoforms in the mammary epithelial cell line TAC 2.1. Overexpression of these proteins in the TAC2.1 cells leads to a modification, in an opposite fashion, of their ability to grow and organize in 3D structures within a collagen envirronement. The Cyclin D2 and the Cyclin D2 Trc respectively repressing and enhancing these abilities. The use of small interfering RNA sequences targeting specifically one or the other isoform allowed us to show a functional link between both isoforms and the Pea3 transcription factor , again in an opposite way, especially during cell cycle progression and during epithelial to mesenchymal conversion, a hallmark of development and cancer progression. Thus, this study gives new clues to understand the involvement of the Pea3 transcription factor during mammary morphogenesis and tumorigenesis, events in which the cyclin d2 gene seems to be a major player. The characterization of the molecular events governed by Pea3 should help in defining new therapeutic strategies against breast cancer progression. "

Mouse PEA3 is the founding member of the PEA3 subfamily of ETS transcription factors that includes ERM and ER81. Numerous studies implicate PEA3 subfamily members in a diversity of human cancers, especially breast cancer. Dominant-negative PEA3 (L1NPEA3En) effectively represses activated transcription by all three PEA3 subfamily members. When expressed under control of the MMTV promoter, L1NPEA3En significantly delays the appearance of mammary tumors and reduces their number and size in mouse models of HER2 mediated breast cancer. In addition, L1NPEA3En is not expressed in the mammary tumors that do develop in these mice. These findings strongly suggest a required role for PEA3 subfamily members or other ETS proteins with similar DNA binding specificity in HER2-mediated oncogenesis. The primary objective of this research was to identify the PEA3 subfamily target genes that could play a role in the initiation and progression of tumors, specifically in the breast. To achieve this, a recombinant adenovirus carrying L1NPEA3En was constructed to express L1NPEA3En in three human mammary tumor cell lines: MDA-MB-468, BT-549 and MDA-MB-361. Gene expression analysis using Affymetrix® GeneChip® technology identified a common set of 39 downregulated and 2 upregulated genes in cells expressing L1NPEA3En compared to control cells in all three tumor cell lines. Differentially expressed genes included some that have been shown to play key roles in tumorigenesis such as activating transcriptionfactor 3, heat shock 70kD protein lA and interleukin-8. In addition one colon carcinoma cell line, SW620, was used for gene expression analysis and 7 genes identified in the mammary tumor cell lines were also identified in the colon carcinoma cell line. The results suggest a role for PEA3 subfamily genes in a multiple human cancers mediated through a small subset of common target genes. The genes identified as being differentially expressed by ~NPEA3En hold potential value not only as targets for therapeautic drug discovery, but also as diagnostic or prognostic markers for human cancers, specifically breast cancer. / Thesis / Master of Biological Science (MBioSci)

PEA3 is the founding member of a subfamily of closely related Ets transcriptional regulatory proteins that includes ERM and ER81. The PEA3 subfamily members share greater than 95% identity in their ETS DNA binding domain and 500/o sequence similarity overall, suggesting these genes may serve redundant functions. The overexpression of each member is positively correlated with HER2 mediated breast tumorigenesis in humans and mice, suggesting a role for this subfamily in mammary development and oncogenesis. This study first addresses the role of PEA3 in cellular transformation mediated by oncogenic Ras and Neu. Wildtype and PEA3-null mouse embryo fibroblast cell lines were infected and tested for focus formation. PEA3-null fibroblasts are refractory to transformation as compared to their wildtype counterparts. Ras and Neu transformed foci show elevated PEA3 subfamily mRNA transcripts and PEA3 protein. ERM and ER81 are expressed in PEA3-null fibroblasts and do not appear to compensate for loss of function mutations in the PEA3 gene resulting in the transformation-defective phenotype. Expression of candidate PEA3 target genes (MMP-3 and MMP-9, which have known roles in transformation) is compromised in PEA3-null fibroblasts. Re-expression of PEA3 in these cells rescues the transformation-deficient phenotype and restores expression of MMP-3 and MMP-9. Hence, PEA3 appears to be a crucial effector in Ras and Neu mediated transformation, in addition to serving an important regulatory role of genes involved in cell motility and invasive tumor behaviour. This study also addresses the role of ER81 in normal mammary gland development. PEA3 is required for normal mammary gland development, as displayed by the reduced branching phenotype in PEA3-null female mice. Mice lacking functional ER81 were generated to determine if ER81 serves a similar role in mammary gland development. ER81 is expressed in the epithelial cells of mammary buds at E 1 0.5, when these structures first appear during mouse embryogenesis. ER81 is then differentially expressed during postnatal mammary gland development, with highest expression occurring at times of extensive epithelial branching. During puberty, expression is observed in undifferentiated cap and body cells of terminal end buds, in differentiated luminal and myoepithelial cells of ducts. During pregnancy, expression in luminal epithelial cells is lost, but persists in the myoepithelial cells within the ducts and alveoli. Targeted disruption of both ERSt alleles result in severely runted mice that die by 4 weeks of age, thereby precluding study of mammary gland development in these mice beyond this developmental stage. However, loss of a single ER81 allele results in healthy looking mice, comparable in size and lifespan to wildtype littermates. Studies employing ER81 heterozygous mice reveal a 50% allelic dose is sufficient for normal mammary gland development. Loss of a single ER81 allele did not result in any overt phenotypes in ductal branching, lobulo-alveolar development, or morphology of the surrounding fat pad. / Thesis / Master of Science (MSc)

PEA3 is a member of the expanding Ets family of transcription factors. In the adult mouse, PEA3 mRNA is expressed at highest levels in the brain, epididymis and at lower levels in the mammary gland, testes, ovary and uterus. PEA3 mRNA is expressed differentially during mouse embryogenesis and is down-regulated following retinoic acid induced differentiation in mouse embryonal carcinoma cell lines. PEA3 is overexpressed at the transcriptional level in 93% of all HER2/neu positive human breast tumors. The molecular basis for differential transcription of the PEA3 gene is not known. Sequence analysis revealed that the upstream region of the PEA3 gene has characteristics of a CpG island and does not possess a recognizable "TATA" element. Rapid amplification of 5' eDNA ends (5'RACE) reveals that transcription initiates from multiple sites, consistent with the absence of TATA elements. To localize cis-acting sequences required for PEA3 expression, deletions of the putative promoter were placed upstream of a luciferase reporter gene and tested for activity in the FM3A cell line. FM3A cells express substantial levels of PEA3 mRNA and protein, which suggests that all of the factors required for transcription are present in the cells. Transient transfections of 5' and 3' deletion mutants of the PEA3 promoter indicated that the efficiency of the PEA3 promoter depended on both negative and positive cis-elements, located upstream and downstream of the transcription start sites. A DNA fragment containing a region from -3 to +676, relative to the major start site of transcription, was sufficient for maximal promoter activity. Luciferase reporter plasmids containing more 5' flanking sequence had lower activity indicating the presence of silencer elements. To aid the identification of critical sequence elements within the minimal PEA3 promoter, we cloned and sequenced the putative human PEA3 promoter. Comparison of the mouse and human PEA3 DNAs revealed that sequences required for maximal promoter activity in the mouse were highly conserved in the human gene. Furthermore, these conserved sequences corresponded to a variety of consensus binding sites: 6 Sp1, 8 c-ets-1, 3 PEA3, 3 AP-2, 3 MZF-1, 2 MyoD, 2 Ik-1, 2 c/EBPB, 2 oEF-1/USF, 2 HSFI and one of each of the following: AP-4, Ik-2, SRY, CP2, HEN-I, CREB andE47. / Thesis / Master of Science (MS)

Les Facteurs de transcription du groupe PEA3 (Pea3, Erm et Er81) font partie de la famille d'oncogènes ETS. Leur expression est souvent observée lors de la mise en place des organes par morphogenèse de branchement tels que les poumons ou encore la glande mammaire. De plus une expression aberrante de ces facteurs de transcription est corrélée au caractère cancéreux de nombreux tissus tels que le côlon, les poumons ou encore le sein. Ainsi, l'expression d'Erm dans les tumeurs du sein est associée à un mauvais pronostic pour les patientes et celle de Pea3 constitue un marqueur de l'agressivité tumorale. Enfin, en qualité de facteur de transcription Pea3 module l'expression de gènes spécifiques alors appelés gènes cibles. Même si certains de ces gènes sont déjà bien caractérisés beaucoup de choses restent à faire pour comprendre les mécanismes moléculaires régulés par Pea3. Dans ce contexte lors de ma thèse je me suis intéressé à l'étude du rôle du facteur de transcription Pea3 dans les processus de morphogenèse et de tumorigenèse mammaires selon deux approches complémentaires : 1- l'étude des propriétés morphogénétiques modulées par Pea3 lors des étapes de morphogenèse et de tumorigenèse mammaires, 2- la recherche et la caractérisation de gènes régulés par Pea3 dans ce même contexte, par une analyse transcriptomique à grande échelle en utilisant des puces à ADN. Ces deux points sont développés grâce à l'utilisation de modèles cellulaires dans lesquelles nous modulons l'expression de Pea3. Les cellules épithéliales mammaires TAC 2.1 modèle de morphogenèse mammaire dans lesquelles nous surexprimons Pea3 et les cellules mammaires transformées MMT, modèle de tumorigenèse mammaire dans lesquelles nous inhibons l'expression du facteur de transcription Pea3. Au cours de ma thèse nous avons ainsi pu montrer l'importance du facteur de transcription Pea3 dans le contrôle des propriétés de migration, d'invasion et de prolifération des cellules cancéreuses TAC et MMT. En accord avec ces données, la recherche des gènes dont l'expression est régulée par Pea3 dans nos deux modèles cellulaires suite à la modulation de Pea3, a permis d'identifier de nombreux gènes capables de réguler la prolifération, la migration et l'invasion des cellules. Parmi ces gènes nous nous sommes intéressés au gène cycline d2 bien connu pour son implication dans le contrôle de la progression du cycle cellulaire. Nous avons pu montrer que le gène cycline d2 est un gène cible direct du facteur de transcription Pea3 qui module l'expression dans le modèle cellulaire TAC des deux transcrits (cycline d2 et cycline d2 trc) issus de ce gène et décrits à ce jour. L'étude de la fonction des protéines Cycline D2 et Cycline D2 Trc dans les cellules TAC a été entreprise. Tout d'abord la surexpression de l'une ou l'autre de ces isoformes dans les cellules TAC 2.1 modifie de façon opposée leur capacité à s'organiser dans un gel de collagène mimant l'environnement d'une glande mammaire, la Cycline D2 réprimant cette capacité et la Cycline D2 Trc l'augmentant. L'utilisation de petits ARN interférents permettant de réprimer l'expression de ces deux protéines a permis de montrer une relation fonctionnelle, toujours opposée, des deux isoformes avec le facteur de transcription Pea3 pour le contrôle de la progression du cycle cellulaire mais aussi pour l'induction d'une transition épithélio-mésenchymateuse étroitement reliée au pouvoir de migration des cellules épithéliales lors du développement des organes comme la glande mammaire mais aussi lors de la progression tumorale. Notre étude a ainsi permis de mieux définir l'implication du facteur de transcription Pea3 lors des événements de morphogenèse et de tumorigenèse de la glande mammaire. De plus elle ouvre la réflexion sur le rôle du gène cycline d2 lors de ces événements.

<p> PEA3 is a member of the ets family of transcription factors. It is expressed throughout embryonic development and in mouse mammary adenocarcinomas induced by expression of the receptor tyrosine kinase Neu. Mice lacking PEA3 due to a targeted disruption of the gene, develop normally, however, male mice fail to mate for yet undetermined reasons. To further understand the role of PEA3 in mammary gland development and tumorigenesis, the effects of loss of function of PEA3 were examined in tumor formation and in mammary gland development. </p> <p> Analysis of tumor formation in PEA3 +I+ and PEA3 -/-animals failed to show a statistically significant difference in tumor onset. Loss of PEA3 did not affect the tumor morphology, nor did it inhibit metastasis of these tumors to the lung. These data indicate that PEA3 is not required for tumor formation or metastasis. </p> <p> PEA3 deficient animals displayed defects in branching morphogenesis in the mammary gland. Decreased ductal branching was observed in virgin and pregnant females. Mice with decreased levels of PEA3 expression also exhibited defects in branching morphogenesis, indicating a dosage effect. PEA3 is expressed in the myoepithelial cells during puberty and pregnancy. It is also express in the highly proliferative cap cell layer of the terminal end bud. In the embryonic mammary gland, PEA3 is expressed as early as 10.5 days in the mammary epithelium and continues late in embryogenesis. Expression in the male mammary gland is lost at approximately embryonic day 16. </p> / Thesis / Candidate in Philosophy

Chicken and pufferfish genomic libraries were screened with the intent of isolating PEA3 orthologues from evolutionarily removed vertebrate species. The chicken PEA3 gene was found to reside within 15 kb of genomic sequence, and approximately 2 kb of promoter sequence has been identified. Although the pufferfish PEA3 genomic sequence has yet to be completed, exons 2, 3, 4, 5, 12 and 13 have been found, and approximately 1 kb of sequence upstream of the putative start codon has been determined. In addition to the genomic sequence that was isolated, 5' RACE using pufferfish heart RNA produced a 334 bp cDNA sequence encompassing exons 2 to 5 of pufferfish PEA3. A pufferfish homologue of the human RNA helicase 1 (HRH1) gene was also found 3' of the PEA3 gene. Given that HRH1 is also found 3' of human PEA3 (E1AF) on chromosome 17q21, this finding would seem to indicate that chromosomal synteny is maintained between the human and pufferfish PEA3 loci. A four-way alignment of the mouse, human, chicken and pufferfish PEA3 promoters revealed that a region spanning from +1 to -260, relative to the transcriptional start site of mouse PEA3, is well conserved across the four promoters. Conserved transcription factor binding sites for SRY, HNF3β, NFY, AP-1, TCF, AP-2, v-myb, δEF1, and c-Ets-1 were found in three, and in some cases four of the promoters. An additional outcome of the pufferfish genomic library screen was the isolation of a pufferfish orthologue of the Ets transcription factor ERM. The relevance of these findings to the issue of transcriptional regulation of PEA3 expression is discussed. / Thesis / Master of Science (MS)

Les signaux environnementaux ont une grande influence sur le devenir de certaines populations de motoneurones. J'étudie la population qui exprime le facteur de transcription PEA3, située au niveau brachial, caractérisée et spécifiée par ce facteur, et qui innerve les muscles dorsaux des membres (Livet et al., 2002 ; Vrieseling et al., 2006). Cette population représente un des exemples les mieux compris de l'acquisition d'une identité neuronale par des signaux provenant du muscle cible. Au cours du développement, l'expression de PEA3 se met en place de manière séquentielle. PEA3 est d'abord exprimé dans un premier sous-groupe de neurones localisé en position postérieure dans le domaine (neurones pionniers), puis dans un deuxième sous-groupe de neurones situé en position plus antérieure. Le développement de cette population implique des échanges de signaux entre les neurones pionniers, instruits par le muscle cible, et le deuxième groupe de neurones antérieurs, instruit par les neurones pionniers. Le GDNF, produit par les cellules du futur muscle cible, induit PEA3 dans les neurones pionniers (Haase et al., 2002). Puis le HGF, un autre facteur dérivé du membre, induit les neurones pionniers à sécréter un ‘signal de propagation’, qui agit à distance et induit l'expression de PEA3 dans le deuxième groupe de neurones (neurones recrutés) (Helmbacher et al., 2003). L'objectif initial de ma thèse a été basé sur l'identification de ce signal de propagation. J'ai d'abord utilisé une approche pharmacologique dans un système in vitro de cultures d'explants de moelles épinières d'embryons de souris. En y inhibant la voie EGF, j'ai démontré que le signal de propagation appartient à cette famille de molécules. Les récepteurs de la voie EGF (ErbB1 à ErbB4) sont exprimés chez l'embryon de poulet et de souris dans la moelle épinière brachiale, et spécifiquement dans les motoneurones, au moment où PEA3 est exprimé. Parmi les ligands de la voie EGF, je me suis intéressée aux neurégulines, une famille de glycoprotéines connue pour son implication dans la mise en place du système nerveux. J'ai montré que des isoformes du gène neuréguline1 (nrg1), possédant un domaine immunoglobuline (type I) sont capables d'induire l'expression de pea3 dans la moelle épinière brachiale, et spécifiquement dans les neurones recrutés. J'ai pu démontrer, en utilisant des souris mutantes pour le récepteur à l’HGF (metd/d), que le signal de propagation est vraisemblablement une isoforme NRG1, de type I. / Signals derived from the environment have an important influence on development of some motorneurons populations. I study the population that expresses the transcription factor PEA3, localized at the brachial level, characterized and specified by this factor. This population innervates the limb dorsal muscles (Livet et al., 2002 ; Vrieseling et al., 2006). This population represents one of the best understood examples of an acquisition of a neuronal identity induced by signals derived from the target muscle. During development, PEA3 expression is made in two times in motorneurons. Initially, PEA3 is expressed in a first population, localized in the posterior part of the domain (pionneers neurons), then in a second population, localized in a more anterior position. Development of this population implies exchanges of signals between pionneers neurons, instructed by the target muscle, and anterior neurons, instructed by pionneers neurons. GDNF, produced by cells of the future target muscle, induces PEA3 in the pioneers neurons (Haase et al., 2002). Then, HGF, another limb-derived factor, induces pioneers neurons to secrete a propagation signal’, which induces PEA3 expression in the second population of neurons (recruited neurons) (Helmbacher et al., 2003). The initial purpose of my phD was to identify this ‘propagation signal’. First, I used a pharmacological approach in an in vitro assay of mouse embryos spinal cord explants culture. I did inhibition of the EGF pathway in this assay, and I showed that the propagation signal belongs to this family. EGF receptors (ErbB1 à ErbB4) are expressed in chick and mouse embryos, and especially in motorneurons, when PEA3 is expressed. Among the EGF ligands, I studied neuregulins, a family of glycoproteins involved in the nervous system development. I showed that isoforms of neuregulin1 gene (nrg1), which have an immunoglobulin domain (type I) induce pea3 expression in the brachial spinal cord, and especially in the recruited neurons. I observed, by using mice mutants for HGF receptor (metd/d), that the propagation signal is plausibly a typeI NRG1 isoform. Keywords : motorneurons, PEA3, recruitment, ErbB.