Le système de conduction ventriculaire (SCV) est responsable de la propagation rapide de l’activité électrique dans le cœur. Il est composé du faisceau de His, des branches droite et gauche, et des fibres de Purkinje. 1) Le développement du SCV a été étudié par l’utilisation de souris transgéniques exprimant la recombinase Cre inductible, insérée par recombinaison homologue au locus du gène de la connexine-40 (Cx40). Ce gène code pour une protéine exprimée dans les trabécules ventriculaires et le SCV. La recombinaison est observée uniquement dans les cellules exprimant la Cx40 et leur descendance. Mes résultats révèlent une restriction progressive du destin des trabécules Cx40-positives en cardiomyocytes conducteurs. Les progéniteurs Cx40-positifs participent à la formation du myocarde contractile et du SCV de E10.5 à E14.5 ; alors qu’ils ne donnent que des cardiomyocytes conducteurs après E16.5. 2) L’analyse du SCV a été étudiée dans un modèle de malformation cardiaque congénitale. Le mutant Tbx1-/-, modèle du syndrome de DiGeorge humain, présente une communication inter-ventriculaire. Des défauts morphologiques du SCV sont détectés chez ces mutants : absence de branche droite et un faisceau de His moins compacté. Ceux-ci sont corrélés avec des défauts de conduction. Le phénotype observé ne résulte pas d’un défaut d’activation du programme génique à l’origine de l’établissement du SCV ; mais semble dû à la présence de la communication inter-ventriculaire qui empêcherait les cellules progénitrices du SCV de rejoindre le sommet du septum inter-ventriculaire. / The ventricular conduction system (VCS) is responsible for the rapid propagation of electrical activity in the heart. The VCS is composed of the His bundle, the left and right bundle branches, and the peripheral Purkinje fibers. 1) The development of the VCS has been studied by using transgenic mice expressing the inducible Cre recombinase, introduced by homologous recombination at the locus of gene of connexin-40 (Cx40). Cx40 encodes for a protein expressing in the ventricular trabeculae and the VCS. The recombination is observed in the cells expressing Cx40 and in their descendants. My results suggest a progressive restriction of the fate of Cx40-positive trabeculae in conductive cardiomyocytes. Cx40-positive progenitors give rise to the formation of the compact myocardium and of the VCS when they are induced between E10.5-E14.5; while they participate only in the cardiomyocytes of VCS after E16.5. 2) The analysis of the VCS has been studied in a model of congenital heart malformation. The mutant Tbx1-/-, model of the DiGeorge syndrome, present a ventricular septal defect. Morphological defects of the VCS are found in Tbx1-/- hearts: an absence of right bundle branch and a non-compacted His bundle; which are correlated with functional defect. The phenotype observed in these mutants does not result from a defect of the activation in the genetic program being at the origin of the establishment in the VCS, but seems to be explain by the presence of a large ventricular septal defect, because it could block the progression of the progenitors of the VCS along the crest of the inter-ventricular septum.

Le cœur des vertébrés se développe à partir du tube cardiaque et de la participation des cellules progénitrices mésodermiques du second champ cardiaque (SHF). Une perturbation de l’addition des cellules du SHF conduit à des malformations cardiaques congénitales (MCC). Chez l’embryon, l’outflow tract (OFT) dérivé du seul SHF est formé par deux domaines complémentaires qui formeront le myocarde sous-aortique et sous-pulmonaire. Ce travail analyse les cellules progénitrices du SHF qui contribuent aux deux domaines de l’OFT pour former la base de l’aorte et du tronc pulmonaire, l’identité transcriptionnelle des domaines et leur régulation. Nous avons mis en évidence une sous-population de cellules progénitrices Notch-dépendantes, situées en région antérieure du mésoderme pharyngé, qui contribue au myocarde sous-aortique. Nous avons démontré que des cascades de régulation croisées impliquant Notch/Hes1 et Tbx1/Pparg sont importantes pour former les deux domaines fonctionnels régionalisés de l’OFT. Des expériences de culture d’explants et d’embryons ont démontré que Pparg est nécessaire au déploiement des cellules du SHF et pour la régulation transcriptionnelle du futur myocarde sous-pulmonaire. Dans le domaine complémentaire, futur myocarde sous-aortique, nous avons observé l’expression de Dlk1, un régulateur négatif de Pparg. Dlk1 est en amont de la voie de régulation Notch et participe probablement à l’identité régionale de l’OFT. Dans son ensemble, ce travail identifie de nouvelles voies de signalisation et gènes qui régulent l'identité régionale du mésoderme cardio-pharyngé et de nouvelles cibles pour l’étude clinique des MCC. / The vertebrate heart develops from the heart tube and the contribution of mesodermal progenitors termed second heart field (SHF). Perturbation in SHF addition leads to congenital heart defects (CHD). The outflow tract (OFT) myocardium is entirely derived from the SHF. Distinct regions of the embryonic OFT have been shown to give rise to subaortic and subpulmonary myocardium of the heart. The work described here focuses on SHF progenitor subpopulations in mouse giving rise to distinct OFT domains and characterizes the regional transcriptional identity and regulation of future subaortic and subpulmonary myocardium. We identified Notch-dependent subaortic myocardial SHF progenitors in anterior pharyngeal mesoderm. We demonstrated that Notch/Hes1 and Tbx1/Pparg cross regulatory cascades are important to establish functionally important OFT regional domains. Explant and embryo culture experiments revealed that Pparg is required for both the deployment of SHF cells and transcriptional regulation of the future subpulmonary myocardial domain. We also found that Dlk1, a negative regulator of Pparg, is expressed in the complementary subaortic domain upstream of Notch receptor activation and potentially participates in the establishment of OFT regional identity. We also report an overlapping transcriptional profile between future subaortic myocardium and subpopulation of epicardial cells at fetal stages. Finally, we provide evidence for the existence of conserved bipotential myogenic progenitors in cardiopharyngeal mesoderm coexpressing Nkx2-5 and Tbx1. Overall this work identifies novel pathways and genes in cardiopharyngeal mesoderm that may contribute to clinically relevant CHD.

Mit dem Ziel, die denaturierende Hochdruck-Flüssigkeitschromatographie (DHPLC) als standardisierte Methode in der molekulargenetischen Diagnostik des TBX1-Gens einzuführen, wurde an 35 Patienten mit begründetem klinischen Verdacht oder Nachweis eines congenitalen Herzfehlers die Mutationsanalyse etabliert und durchgeführt. Die erzielten Ergebnisse bestätigten die kosteneffiziente und zugleich einfache Durchführung des Verfahrens. In der Evaluation wurde für die DHPLC zudem eine hohe Sensitivität und Spezifität nachgewiesen, womit der zukünftige Einsatz dieser Methode im routinemäßigen Mutationsscreening gerechtfertig ist. Mittels DHPLC konnten im Rahmen der vorliegenden Dissertation insgesamt 14 verschiedene Veränderungen im TBX1-Gen identifiziert und in der anschließenden Sequenzanalyse als Polymorphismen (Normvarianten) charakterisiert werden.

Mit dem Ziel, die denaturierende Hochdruck-Flüssigkeitschromatographie (DHPLC) als standardisierte Methode in der molekulargenetischen Diagnostik des TBX1-Gens einzuführen, wurde an 35 Patienten mit begründetem klinischen Verdacht oder Nachweis eines congenitalen Herzfehlers die Mutationsanalyse etabliert und durchgeführt. Die erzielten Ergebnisse bestätigten die kosteneffiziente und zugleich einfache Durchführung des Verfahrens. In der Evaluation wurde für die DHPLC zudem eine hohe Sensitivität und Spezifität nachgewiesen, womit der zukünftige Einsatz dieser Methode im routinemäßigen Mutationsscreening gerechtfertig ist. Mittels DHPLC konnten im Rahmen der vorliegenden Dissertation insgesamt 14 verschiedene Veränderungen im TBX1-Gen identifiziert und in der anschließenden Sequenzanalyse als Polymorphismen (Normvarianten) charakterisiert werden.

Sensory neurons are key mediators of the transduction of external stimuli from the ear to the brain, essential for the sense of balance and hearing. Understanding when, where and how the sensory nervous system is assembled during development can provide insights on deafness and balance disorders. Here, I show in zebrafish that Her9 transcription factor is a key element in the regulation of the otic neurogenesis. Loss of Her9 function leads to the ectopic expression of neurogenic genes neurod and neurod4. Moreover, I show that Her9 acts downstream of Tbx1, and both genes are activated by retinoic acid signaling emanating from the paraxial mesoderm and negatively regulated by Hedgehog signaling. Altogether, the data demonstrates a role of retinoic acid in axial patterning and the establishment of a neurogenic domain through Tbx1 and Her9. At later stages, retinoic acid has an additional role by regulating neuronal differentiation in the statoacoustic ganglion. / Les neurones sensorials de l’oïda interna són mediadores claus en la transducció dels estímuls externs des de l’oïda interna al cervell. Entendre a on, quan i com el sistema nerviós sensorial s’organitza durant el desenvolupament embrionari pot ajudar en l’estudi de les malalties neurosensorials. En el present treball, mostro en peix zebra que el factor de transcripció Her9 és un element clau en el control de la neurogènesi òtica i que Her9 es troba sota el control directe del factor Tbx1. A més, ambdos factors estan regulats de manera positva per la via de senyalització de l’àcid retinoic i negativament per la vía de hedgehog. En resum, la tesis demostra un paper de l’àcid retinoic en la regionalització axial del primordi òtic en l’eix anteroposterior i l’establiment d’un domini neurogènic a través de Tbx1 i Her9. En estadis tardans, l’àcid retinoic regula la diferenciació neuronal en el gangli estato-acústic.

Background: The 22q11 deletion syndrome (22q11DS), also known as DiGeorge Syndrome, affects ~1/5000 live born children. Congenital heart defects (typically outflow tract and interrupted aortic arch) are present in 75% of individuals with 22q11DS and are the major cause of mortality. Other defects are cleft palate, thymus hypoplasia, inner ear defects and neuropsychiatric abnormalities. Df(16)1 mice carry a ~1 Mb hemizygous deletion on mouse chromosome 16 in a region syntenic with 22q11 and phenocopies 22q11DS. TBX1 is a DNA-binding transcription factor located in this interval and is required for neural crest cell proliferation and migration and for cardiac development. TBX1 point mutations have been identified in patients with DiGeorge syndrome. Thus TBX1 is thought to be a major gene responsible for the cardiac phenotype in 22q11DS. A key unresolved issue is the mechanism of reduced penetrance of cardiac malformations. One possibility is environmental variation during cardiogenesis. A second possibility is that variation in the TBX1 protein interaction network results in variable penetrance of the phenotype. Mutations in TBX1 or interacting partners could affect the structure of this protein interaction network. Aim: The aim of this thesis is to characterize the molecular mechanism of TBX1 function using biochemical and genetic approaches and to define the role of environmental variation on the DiGeorge phenotype. Results First part. Interaction of Df(16)1 with high-fat maternal diet. To determine if a maternal high-fat diet affects the penetrance of cardiac and thymus malformations in the Df1 deletion mouse model, wild-type and Df1 heterozygous embryos from control and high-fat diet groups were analyzed. No significant difference in the penetrance or the severity of cardiac malformations between these groups was found. These results do not support the idea that change in the fat content of maternal diet affects phenotype in this model. Thus, it is possible that high-fat diet interacts specifically with left-right patterning rather than with the genetic control of pharyngeal arch development and neural crest cell migration and survival. Second part. George, a novel ENU induced mutation in Tbx1. The George mutation, identified and mapped to Chr16 between rs4161352 and D16Mit112, results in fully penetrant cleft palate, cardiac malformations (VSD, IAA, CAT), absent cochlea and abnormal semicircular canals, and absent thymus resembling the human DiGeorge phenotype. Tbx1 lies in this interval and sequencing identified a G > A point mutation in exon 3 which is predicted to cause a Arginine to Glutamine change at amino acid position 160. George fails to genetically complement a Tbx1 null allele, confirming that it is causative and that George is functionally a null allele. RT-PCR showed that the George mutation affects splicing, resulting in a transcript lacking exon 3. This causes the loss of 34 amino acids within the TBX1 T-box domain, thus predicting that it affects DNA binding. Transactivation assays show that while the R160Q amino acid substitution significantly reduces the transactivation capacity of TBX1, surprisingly the loss of exon 3 does not affect this function. Analysis of endogenous TBX1 in developing embryos by Western blot showed that the protein expression is absent or significantly reduced. This finding suggests that the observed George phenotype is caused primarily by a loss of TBX1 protein expression. Third part. Investigation of the protein interaction network surrounding TBX1. In order to get a better insight into the protein network surrounding TBX1, a TBX1 split renilla-luciferase protein complementation assay was set up which allowed to test the physical interaction between TBX1 and several putative interactors. It was found that GATA4, SMARCAD1, RBBP5 and PTDSR interact with wild-type TBX1 in HEK293T cells. The R160Q point mutation and the loss of exon 3 affect some of these interactions supporting the idea that variation in the protein interaction network may, at least in part, be responsible for the DGS phenotype.

Une partie du cœur est formée à partir des cellules progénitrices du second champ cardiaque, qui permettent une élongation rapide du tube cardiaque. Des défauts dans le développement de ces cellules entrainent des malformations cardiaques congénitales. Ces cellules sont localisées dans le péricarde dorsal au sein du mésoderme pharyngé. Mon travail de thèse a permis de démontrer pour la première fois que ces cellules sont épithéliales et polarisées, et qu’elles forment des filopodes dynamiques du côté basal. La délétion du facteur de transcription Tbx1 perturbe la polarité des cellules et la formation des filopodes, et augmente le niveau de la protéine apicale aPKCζ. Le traitement avec un activateur de aPKCζ montre le lien entre l’intégrité épithéliale, la polarité et la formation des filopodes, et l’état progéniteur des cellules. J’ai également analysé la polarité planaire dans l’épithélium, et montrais que les cellules sont anisotropiques, étirées et allongées en direction du pole artériel. Cet étirement crée une tension orientée, révélée par une accumulation polarisée d’actomyosine, jouant le rôle de rétrocontrôle négatif. En absence d‘élongation du tube cardiaque cette tension orientée est absente. Nous avons identifié une région postérieure de l’épithélium où se trouvent une tension et une prolifération élevées, ainsi qu’une forte activité YAP/TAZ qui jouerait le rôle de relai entre tension et prolifération. La tension orientée oriente les divisions cellulaires et oriente ainsi la croissance du tissu, promouvant l’addition des cellules au pole artériel. La biomécanique des cellules du second champ cardiaque semble ainsi un moteur important pour l’élongation du cœur. / A major part of the heart is formed by progenitor cells called the second heart field, that contribute to rapid elongation of the heart tube. Defects in second heart field development leads to congenital heart malformations. Second heart field cells are localised in pharyngeal mesoderm in the dorsal pericardial wall. This study focuses on the epithelial properties of second heart field cells and first shows that these progenitors in the dorsal pericardial wall are epithelial and polarised, and form dynamic basal filopodia. Deletion of the transcription factor Tbx1 perturbs epithelial polarity and filopodia formation and upregulates the apical determinant aPKCζ. Treatment with an activator of aPKCζ reveals that epithelial integrity, polarity and basal filopodia are coupled to the progenitor status of second heart field cells. Next we evaluated planar polarity of second heart field cells in the dorsal pericardial wall. Cells are anisotropic, being stretched and elongated on an axis directed towards the arterial pole. This stretch results in oriented epithelial tension revealed by polarised actomyosin accumulation through a negative feedback loop. In the absence of cell addition to the cardiac poles oriented tension is absent. We identified a posterior region in the epithelium with high tension, elevated proliferation and a high level of active YAP/TAZ that may act as relay between tension and proliferation. Oriented tension orients the axis of cell division and the growth of the tissue on an axis toward the arterial pole, further promoting addition of the tissue to the pole. Biomechanical feedback may thus be an important driver of heart tube elongation.

<p>Despite its lack of septation, the tissue patterning of the arterial pole of the zebrafish is remarkably similar to the patterning of pulmonary and aortic arterial poles observed in mouse and chick. The secondary heart field (SHF) is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the cardiac arterial pole. This field is part of the overall heart field, and its myocardial component has been fate mapped from the mesoderm to the heart in both mammals and birds. In this study I demonstrate that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field. This zebrafish SHF contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. I show that this field expresses Isl1, a transcription factor associated with the SHF in other species. I further show that differentiation, induced by Bmp signaling, occurs in this progenitor population as cells are added to the heart tube. Some molecular pathways required for SHF development in birds and mammals are conserved in teleosts, as Nkx2.5 and Nkx2.7 as well as Fgf8 regulate Bmp signaling in the zebrafish heart fields. Additionally, the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.</p> / Dissertation

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.