5 Abstract: Aims: The main objective of this work was to analyze in detail the effects of acute temperature changes on the function of isolated chick embryonic heart in vitro in comparison with natural conditions in ovo. Methods: The effects of temperature change (34 řC, 37 řC and 40 řC - hypo-, normo- and hyperthermia, respectively) on calcium dynamics in four days old isolated chick hearts in vitro were investigated by high-speed calcium optical imaging. For comparison and validation of in vitro measurements, experiments were also performed in ovo using videomicroscopy. Artificial electrical stimulation experiments were performed in vitro and in ovo to uncover conduction limits of different heart segments. Results: We observed almost linear dependence of sinus frequency on temperature in our temperature range. Sinus frequency during hypothermia and hyperthermia in vitro and in ovo changed about 20% in comparison with normothermia. We observed no significant changes in amplitude of calcium transients during temperature change to hypothermia but hyperthermia caused a significant decrease in amplitude of calcium transients (atria 35%, ventricles 38%). We observed a wide spectrum of arrhythmias, which occurred spontaneously even during normothermia in vitro. Occurrence of arrhythmias in vitro significantly...

Numerous initiatives to improve our understanding of cancer biology have been lunched in different laboratories that aim to describe the interactome and gene-expression profile in different tumour cell line. It is now clear that the different strategies of cell migration observed in cancer are reminiscent of the different migratory strategies observed during embryo development. These similarities suggest that developmental program that has to be kept off after embryogenesis may be induced by spontaneous genetics modifications that produce tumour cells. In this study we went inside the genetic network/profile that controls how neural crest cells eventually switch on the migration program and how they are able to arise into different lines with the propose of getting new ideas on how to prevent dissemination of tumour cells or how to treat advanced tumour that have already spread. Neural progenitors of the dorsal neural tube that acquire the expression of specific neural crest determinants, delaminate from the neural tube and follow precise migratory pathways, to terminally differentiate into the various neural crest derivatives. Here we developed a novel resource for lineage trace and isolation of neural crest cells that allowed for a genome-wide expression screen in pre-migratory and migratory neural crest progenitors. We efficiently identified previously known neural crest specific genes. Expression profiling revealed new neural crest genes belonging to a wide range of cellular functions, with high representation of genes associated to cell motion. Additionally, we identified chick genes for which the human orthologues and/or paralogues are associated to Neuroblastoma formation. In my thesis we identified new genes specifically expressed in the developing neural crest, and proposed a revised genomic signature for the normal neural crest cells. Furthermore, mutations on some of these genes are markers for Neuroblastoma tumour formation. Thus we propose this as a valid screen to identify candidates genes that contribute to the characterization of the Neuroblastoma cancer stem cells, and thus to the identification of specific targets to design new therapeutic strategies. Getting in more detail into this genetic network, Wnt canonical signalling response has to been shown to be a key event in both cancer and neural crest cell development. Traditionally Wnt canonical pathway has been involved in neural crest induction process, but here we demonstrated that it is also critical for the onset migration of the neural crest cell. In fact, high levels of Wnt canonical activity prevents neural crest cell to delaminate and only through the inhibition of this activity mediate by dapper protein, neural crest cells can undergo into their normal migration pathways. If this process has an implication in cancer is still unknown, but Dapper expression proteins have been already associated to different types of cancer.

Une cascade de facteurs de transcription composée de SIM1, ARNT2, OTP, BRN2 et SIM2 est requise pour la différenciation des cinq types cellulaires qui peuplent le noyau paraventriculaire (PVN) de l’hypothalamus, un régulateur critique de plusieurs processus physiologiques essentiels à la survie. De plus, l’haploinsuffisance de Sim1 est aussi une cause d’hyperphagie isolée chez la souris et chez l’homme. Nous désirons disséquer le programme développemental du PVN, via une approche intégrative, afin d’identifier de nouveaux gènes qui ont le potentiel de réguler l’homéostasie chez l’individu adulte. Premièrement, nous avons utilisé une approche incluant l’analyse du transcriptome du PVN à différents stades du développement de la souris pour identifier de tels gènes. Nous avons comparé les transcriptomes de l’hypothalamus antérieur chez des embryons de souris Sim1+/+ et Sim1-/- à E12.5 issus de la même portée. De cette manière, nous avons identifié 56 gènes agissant en aval de Sim1 dont 5 facteurs de transcription - Irx3, Sax1, Rxrg, Ror et Neurod6. Nous avons également proposé un modèle de développement à deux couches de l’hypothalamus antérieur. Selon ce modèle, les gènes qui occupent un domaine médial dans la zone du manteau caractérisent des cellules qui peupleront le PVN alors que les gènes qui ont une expression latérale identifient des cellules qui donneront plus tard naissance aux structures ventrolatérales de l’hypothalamus. Nous avons aussi démontré que Sim1 est impliqué à la fois dans la différenciation, la migration et la prolifération des neurones qui peuplent le PVN tout comme Otp. Nous avons également isolé par microdissection au laser le PVN et l’hypothalamus médiobasal chez des souris de type sauvage à E14.5 pour en comparer les transcriptomes. Ceci nous a permis d’identifier 34 facteurs de transcription spécifiques au PVN et 76 facteurs spécifiques à l’hypothalamus médiobasal. Ces gènes représentent des régulateurs potentiels du développement hypothalamique. Deuxièmement, nous avons identifié 3 blocs de séquences au sein de la région 5’ d’Otp qui sont conservés chez l’homme, la souris et le poisson. Nous avons construit un transgène qui est composé d’un fragment de 7 kb contenant ces blocs de séquences et d’un gène rapporteur. L’analyse de 4 lignées de souris a montré que ce transgène est uniquement exprimé dans le PVN en développement. Nous avons généré un deuxième transgène dans lequel le fragment de 7 kb est inséré en amont de l’ADNc de Brn2 ou Sim1 et de Gfp. Nous avons obtenu quatre lignées de souris dans lesquels le profil d’expression de Brn2 et de Gfp reproduit celui d’Otp. Nous étudierons le développement du PVN et la prise alimentaire chez ces souris. En parallèle, nous croisons ces lignées avec les souris déficientes en Sim1 pour déterminer si l’expression de Brn2 permet le développement des cellules du PVN en absence de Sim1. En résumé, nous avons généré le premier transgène qui est exprimé spécifiquement dans le PVN. Ce transgène constitue un outil critique pour la dissection du programme développemental de l’hypothalamus. Troisièmement, nous avons caractérisé le développement de l’hypothalamus antérieur chez l’embryon de poulet qui représente un modèle intéressant pour réaliser des études de perte et de gain de fonction au cours du développement de cette structure. Il faut souligner que le modèle de développement à deux couches de l’hypothalamus antérieur semble être conservé chez l’embryon de poulet où il est aussi possible de classer les gènes selon leur profil d’expression médio-latéral et le devenir des régions qu’ils définissent. Finalement, nous croyons que cette approche intégrative nous permettra d’identifier et de caractériser des régulateurs du développement du PVN qui pourront potentiellement être associés à des pathologies chez l’adulte telles que l’obésité ou l’hypertension. / A cascade of transcription factors composed of SIM1, ARNT2, OTP, BRN2 and SIM2 is required for the differentiation of the five major cell types populating the paraventricular nucleus (PVN) of the hypothalamus, a critical integrator of several homeostatic processes that are required for the survival of vertebrates. Haploinsufficency of Sim1 also causes isolated hyperphagia in mice and humans. The goal of our study is to dissect the developmental program of the PVN using an integrative approach in order to identify new genes that could potentially be implicated in the regulation of homeostasis in adults. First, we used a comparative approach to analyse the PVN transcriptome at different developmental stages in mice embryos in order to identity new genes implicated in PVN development. We compared gene expression in the anterior hypothalamus of E12.5 Sim1-/- and Sim1+/+ littermate embryos using a microarray approach. We identified 56 genes acting downstream of Sim1 including 5 transcription factors - Irx3, Sax1, Rxrg, Ror and Neurod6. We proposed a model for the development of the anterior hypothalamus. In this model, the genes expressed in the medial domain of the mantle layer characterise cells that will form the PVN and genes expressed in the lateral domain identify cells that will give rise to ventrolateral areas of the hypothalamus. We also showed that Sim1, like Otp, is implicated in the differentiation, migration and proliferation of the neurons populating the PVN. Furthermore, we have isolated by laser captured microdissection the PVN and ARC nucleus in wild type mice at E14.5 and compared their transcriptomes. This technique allowed us to identity 34 transcription factors specific to the PVN and 76 factors specific to the ARC. These genes represent potential regulators of hypothalamic development. Second, we identified 3 blocks of sequence in the 5’ region of Otp that are conserved between human, mouse and fish. We constructed a transgene which included a 7 Kb fragment encompassing these sequences followed by a reporter gene. The analysis of 4 mice strains showed that this transgene is specifically expressed in the prospective PVN. We have generated a second transgene in which the 7 Kb fragment is located upstream of the cDNA encoding Brn2 or Sim1 and Gfp. We obtained 4 mice strains in which the Brn2 and Gfp expression pattern is similar to the Otp expression pattern. These mice will be used to study PVN development and food intake. Also, to determine if Brn2 expression only – without Sim1 gene expression - allows the development of PVN cells, we are presently crossing these mice with Sim1 deficient mice. In conclusion, we have generated the first transgene that is specifically expressed in the PVN. This transgene constitutes a critical tool for dissecting the developmental program of the hypothalamus. Third, we have characterised the development of the anterior hypothalamus of chick embryos which represent an interesting model for loss and gain of function experiments during the development of this brain region. Interestingly, our proposed model for the development of the anterior hypothalamus seems to be conserved in chick embryos. As a matter of fact, it is possible to classify genes according to their medio-lateral expression patterns and the outcome of the regions that they are defining. Finally, we believe that this integrative approach will allow us to identify and characterize factors implicated in PVN development. From a clinical point of view, these factors could potentially be associated with pathologies such as obesity or arterial hypertension.

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