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Craniofacial asymmetry is a dentofacial deformity with genetic influences. The genes PITX2, ENPP1 and ESR1 have multiple genetic associations with functional properties in muscle and bone. The objectives of this study are to investigate how PITX2, ENPP1 and ESR1 gene expression associates with four subclassifications of craniofacial asymmetry, temporomandibular disorders and fiber type differences compared between right and left masseter muscles. We developed an asymmetry classification that diagnosed four types of asymmetry with distinctive growth patterns: Group 1 – menton deviation without ramal difference (“mandibular body asymmetry”); Group 2 –menton deviation with shorter ramal height on the deviated side (“typical asymmetry”); Group 3 – shorter ramal height on the opposite side of menton deviation (“atypical asymmetry”); Group 4 – menton deviation with shorter ramal height and maxillary canting on the deviated side (“C-shaped asymmetry”). Some of these patients are at high risk for TMD; therefore, temporomandibular joint functioning is assessed as a routine part of the pre-surgical evaluation. TMD was diagnosed using the Diagnostic Criteria for TMD (DC/TMD). The clinical examination includes mandibular range of motion, palpation for pain, joint noise and bruxism. In addition, the Jaw Pain and Function (JPF) questionnaire was used to assess patient reported symptoms as an indication of perceived severity before and one year after orthognathic surgery. Masseter muscle samples were collected from 174 subjects undergoing surgical treatment for correction of malocclusion. Muscle serial cross-sections were mounted for immunostaining with five antibodies specific for myosin heavy chain (MyHC) isoform. We classified masseter fibers into 4 fiber type groups: type I, type I/II hybrid, type IIA and/or IIX, neonatal and atrial. With the remaining muscle samples, total RNA was isolated and PITX2, ENPP1, and ESR1 expression was quantified using TaqMan qRT-PCR. Average relative quantity gene expression values and percent differences between left and right masseter samples were calculated. In this population, there is a high prevalence of facial asymmetry (48%). Pre-surgical mean JPF scores are significantly different between symmetric (JPF=1.97) and asymmetric (JPF=6.9; p<0.001) patients; with scores ≥ 6 diagnostic for presence of TMD. ENPP1 and ESR1 expression is differentially expressed between right and left masseter muscle in patients with asymmetry. ENPP1 is differentially expressed in asymmetry group 4 (p=0.01) and ESR1 is differentially expressed in asymmetry group 1 (p=0.048), group 2 (p=0.004) and group 4 (p=0.02). Masseter fiber type properties of type I, type I/II hybrid and type II fibers associate with facial asymmetry and specific subclassifications, suggesting functional differences between type I, type I/II and type II fibers may be important factors in the development of symmetry between facial sides. There are significant differences in the left-right percent differences of fiber area of type I fibers in asymmetry group 3 (p=0.05), type I/II hybrid fibers in group 3 (p=0.02), and type II fibers in asymmetric patients (p=0.03), asymmetry group 2 (p=0.05) and group 4 (p=0.005). Additionally, there are significant differences in the left-right percent differences of percent occupancy of type I fibers in asymmetric patients (p=0.04), asymmetry group 2 (p=0.01) and group 3 (p=0.05) and type II fibers in asymmetry group 2 (p=0.04). By comparing gene expression with masseter muscle fiber type properties, we found significant results for PITX2 and ENPP1 suggesting their roles as genetic factors influencing jaw bone length and masticatory muscle strength in malocclusion. There are significant positive correlations between left-right percent differences of PITX2 and type I fiber area (r=0.86; p=0.03), type I/II hybrid fiber area (r=0.94; p=0.006), and type I/II hybrid fiber percent occupancy (r=0.90; p=0.01). Also, there are positive correlations approaching significance between left-right percent differences of ENPP1 and type I fiber area (r=0.80; p=0.06) and type I/II hybrid fiber area (r=0.75; p=0.09). Given the high prevalence of TMD in a population of patients with facial asymmetry, we compared differences in gene expression in masseter muscle of patients with specific TMD diagnostic conditions. Average PITX2 expression is significantly increased (p=0.0375) and average ENPP1 is increased, but not significantly, in all TMD patients diagnosed by the clinician. Average ESR1 is slightly increased compared to JPF scores and may be an essential factor for patient reported TMD symptoms. With these results, PITX2, ENPP1, and ESR1 should be considered biomarkers for asymmetry and TMD; however, further studies are needed to provide a more thorough understanding of the genetic influences on the craniofacial complex. / Oral Biology

La fibrillation atriale (FA) est un problème de santé publique majeur dans le monde entier. Le remodelage électrique, structurel et neuronal est sous-jacent à la myopathie atriale. La pharmacothérapie actuelle est souvent inefficace en raison du manque de connaissance de la pathophysiologie de la FA.Pour comprendre comment se réalise le remodelage atrial, une approche Omique qui explore le transcriptome, l'épigénome (méthylome et microOme) et le génome de patients atteints de FA a été réalisée. Parallèlement, le phénotype de vieux rats spontanément hypertendus (SHRs) a été caractérisé et une étude pharmacologique avec la décitabine (5-Aza-2'-deoxycitidine) a été menée. Les patients atteint de FA présentent un profil transcriptomique et d'expression de miRNA alteré dans l'oreillete gauche (OG), soulignant le rôle important d'un processus de "œmorphogénèse de la structure anatomique". L'expression réduite de Pitx2 était inversement corrélée à la taille de l'OG et ne pouvait pas être expliquée ni par le facteur de transcription ni par la surexpression de Smyd2, une cible de miR-519b. Les SHRs, similairement aux observations chez l'homme, ont développé des arythmies dépendantes de l'âge associées au remodelage atrial et ventriculaire gauche. La FA a été trouvée associée à l'hyperméthylation du promoteur de Pitx2 à la fois chez l'homme et chez les SHRs. L'agent hyperméthylant décitabine a amélioré le profil arhytmique de l'ECG et les activités SOD, et la réduction de la fibrose dans le ventricule gauche des SHRs. En utillisant une approche NGS basée sur un panel personnalisé de 55 gènes candidats à la myopathie atriale dans une cohorte de 94 patients atteints de FA, 11 nouvelles variantes faux-sens potentiellement pathogènes impliqués dans le remodelage structurel ont été identifiés. Des études fonctionnelles de ces variants ont débuté. Trois patients sont également des porteurs de variantes dans les gènes connus de FA. Les résultats actuels suggèrent que 1) la régulation épigénétique peut jouer un rôle dans la pathophysiologie de la FA 2) les agents hypométhylants doivent être considérés comme une nouvelle thérapie de la FA 3) une approche Omique peut aider à découvrir de nouveaux mécanismes sous-jacents à la myopathie atriale / Atrial fibrillation (AF) is a major public health care problem worldwide. Electrical, structural, and neural remodeling underlie atrial myopathy. Current pharmacotherapy is often ineffective due to the lack of knowledge of AF pathophysiology. To understand how atrial remodeling occurs, an Omic approach that explore the transcriptome, epigenome (methylome and microOme) and genome of AF patients was performed. In parallel, ageing spontaneously hypertensive rats (SHRs) were phenotypically characterised and a pharmacological study with decitabine (5-Aza-2’-deoxycitidine) was conducted. AF patients presented an altered transcriptomic and microRNA expression profile in the left atria (LA), emphasizing the important role of an "anatomical structure morphogenesis" process. The Pitx2 reduced expression was inversely correlated with LA size, and could not be explained by transcriptor factor. Smyd2 is a target of miR-519b-3p. SHRs, similar to what is observed in humans, developed age-dependent arrhythmias associated with left atrial and ventricular remodeling. AF was found to be associated with Pitx2 promoter hypermethylation both in humans and in SHRs. The hypomethylating agent decitabine improved ECG arrhythmic profiles and superoxide dismutase activities, and reduced fibrosis in the left ventricle of SHRs. Using a next-generation sequencing approach based on a custom panel of 55 atrial myopathy candidate genes in a cohort of 94 AF patients, 11 novel potentially pathogenic missense variants involved in structural remodeling were identified. Functional studies of these variants have started. Three patients were also carriers of variants in known AF-causing genes. The present results suggest that 1) epigenetic regulation may play a role in the pathophysiology of AF 2) hypomethylating agents have to be considered as a new AF therapy 3) an Omic approach may help to uncover new mechanisms underlying atrial myopathy

Les mutations du gène SCN5A, codant la sous-unité ? du canal Na+ cardiaque Nav1.5, sont responsables d'arythmies cardiaques héréditaires. La pénétrance incomplète observée dans ces maladies suggère l'existence d'autres facteurs modulant le phénotype associé à ces mutations. Dans ce travail de thèse, nous avons caractérisé deux mutations identifiées dans SCN5A. Le mutant R104W, identifié chez un patient atteint du syndrome de Brugada, est retenu dans le réticulum endoplasmique (RE), dégradé par le protéasome et abolit le courant Na+. Co-exprimé avec le canal sauvage, R104W conduit à la rétention de celui-ci dans le RE, résultant en un effet dominant négatif sur les canaux sauvages. Nous avons démontré que ce nouveau mécanisme mettait en jeu une interaction entre les sous-unités ? de Nav1.5. La mutation R1860Gfs*12 a été identifiée dans une famille présentant des arythmies auriculaires. Dans un système d'expression hétérologue, ce mutant induit à la fois une perte et un gain de fonction de Nav1.5. La modélisation informatique nous a permis de montrer que la perte de fonction était plus prononcée dans les cellules auriculaires que ventriculaires. De plus, nous avons montré que la présence de polymorphismes en amont du gène PITX2 dans cette famille pouvait expliquer la variabilité des phénotypes observés. En conclusion, l'interaction entre les sous-unités ? de Nav1.5, les propriétés électriques différentes entre oreillette et ventricule et la présence de polymorphismes chez les patients porteurs de mutations SCN5A sont des facteurs importants dans l'interprétation des effets fonctionnels de ces mutations, contribuant à la variabilité phénotypique des canalopathies Na+. / Mutations in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, are implicated in different inherited cardiac arrhythmias. The incomplete penetrance observed in these diseases suggests the existence of other factors modulating the phenotype of these mutations. In this thesis work, we characterized two mutations identified in SCN5A. The R104W mutant identified in a patient with Brugada syndrome is retained in the endoplasmic reticulum (ER), degraded by the proteasome and abolishes the sodium current. Co-expressed with wild type (WT) channels, R104W leads to WT channels ER retention, causing a dominant-negative effect. We demonstrated that interaction between Nav1.5 α-subunits is responsible for the retention and the dominant-negative effect. The R1860Gfs*12 mutation was identified in a family with atrial arrhythmias. In a heterologous system, this mutant induces both loss- and gain-of-function effects on Nav1.5. Computer-model simulation showed that the loss-of-function was more pronounced in atrial than in ventricular cells. In addition, we showed that the presence of polymorphisms upstream of the PITX2 gene could explain the observed phenotypic variability in this family. In conclusion, the interaction between the α-subunits of Nav1.5, the different electrical properties between atria and ventricles and the presence of polymorphisms in patients with SCN5A mutations, are important factors in the interpretation of the functional effects of these mutations, which could explain the phenotypic variability of sodium channelopathies.

Tooth development is precisely controlled by epithelium-mesenchyme interactions, coordinated signaling pathways and associated transcription factors. Although the processes involved in tooth development are well established, details of the cellular and molecular mechanisms that control tooth development are not fully understood. One of the primary unknown mechanisms is the regulation of dental epithelial stem cells (DESCs), including DESC specification, proliferation and differentiation. In this dissertation, I have addressed this gap in knowledge by studying the role of Pituitary homeobox 2 (Pitx2) and Iroquois 1 (Irx1) in teeth at the cellular and molecular level in mice. PITX2 contains mutations of which are associated for Axenfeld-Rieger syndrome (ARS) in humans and is also required for early tooth development. All the background knowledge is included in Chapter I. In Chapter II, I describe the conditional ablation of Pitx2 in the dental epithelium using a Krt14Cre driver line (Pitx2cKO mice). Knocking out Pitx2 in teeth led to delayed epithelial invagination at bud stage and disruption of tooth morphogenesis at cap stage. At the cellular level, Pitx2 mediates DESC differentiation, daughter cell proliferation in bud stage tooth and regulates enamel knot formation in cap stage tooth. At the molecular level, Pitx2 acts as an upstream regulator of the sonic hedgehog (Shh) signaling pathway by regulating the expression of Shh in the dental epithelial signaling center during early tooth development. In addition, I demonstrated that Pitx2 directly controls the transcription of Irx1. In Chapter III, I determined the cellular and molecular mechanisms of Irx1 in mice. Irx1 general knockout mice were generated by replacing the entire Irx1 gene body with a LacZ reporter gene. Irx1 null mice are neonatal lethal and this lethality is due to pulmonary immaturity with defective surfactant protein secretion. In teeth, Irx1 is expressed in the outer enamel epithelium (OEE) and stratum reticulum (SR) and mediates DESC to OEE and SR differentiation through regulation of Forkhead box protein J1 (Foxj1) and Sex determining region Y-box9 (Sox9). In summary, I identified a Pitx2-Irx1 regulatory network that controls DESC differentiation in teeth, which provided the field with a better understanding of tooth development and tooth regeneration.

Patients with Axenfeld-Rieger Syndrome (ARS) present various dental abnormalities. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in tooth development. However, it is unclear how its mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel) expression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is enhanced and controlled by co-factors and target genes of Pitx2. These co-factors include cooperative transcription factors such as Dlx2 and FoxJ1; chromatin-associated remodeler factor Hmgn2; and Wnt signaling components such as Lef-1, β-catenin and Dact2. We also unveil a novel Pitx2 target gene Irx1 that functions in dental epithelium differentiation. Consistent with a physiological significance to these modulations, we show that FoxJ1, Dact2, Irx1 knockout mice and K14-Hmgn2 transgenic mice display various types of amelogenesis defects including enamel hypoplasia - consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms and multi-level regulations involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carry PITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in novel mouse models of enamel hypoplasia. The impact of this study on current understanding of the dental epithelium development and the translational value lie in the gene network we identified. By manipulating components of the network, pluripotent dental cells can be reprogrammed and serve as new source for tooth regeneration. Our findings brought insights of novel gene therapy approach that can alleviate the dental problems of patients with ARS and other developmental anomalies.

Congenital heart disease (CHD) is the major cause of death in the first year of life, the estimated incidence being 0.5-5% of live births; therefore it is important to understand the genetic causes underlying the complex process of heart formation to help prophylaxis, diagnosis and treatment of affected patients. CHD is the commonest phenotype associated with left-right (LR) disorders. LR asymmetry is determined during embryonic development. The three major body axes ? antero-posterior, dorso-ventral and left-right ? are patterned at gastrulation. LR asymmetry is established shortly after the two other major axes are patterned. The process of LR determination can be sub-divided into four integrated steps: 1. breaking of molecular symmetry in the gastrulation organizer; 2. transfer or relay of this asymmetric information to the lateral plate mesoderm (LPM), from which most internal organs will be formed; 3. reinforcement and propagation of asymmetric cues throughout the LPM and 4. conversion of asymmetric molecular information into proper organ morphogenesis. The goal of this work is to investigate mechanisms involved at two specific points in the laterality pathway: the initial generation/maintenance of asymmetric gene expression in the LPM and the morphogenetic translation of these early events into correct heart formation in the mouse. My emphasis has been on the characterization of laterality targeted cells via careful analysis of Pitx2c expression using a Pitx2c-lacZ reporter transgene, the role of BMP signalling, via Smad1, in generation/maintenance of early asymmetric signalling in the LPM, and the later involvement of both Smad1 and Pitx2 in cardiac morphogenesis through analyses of knockout mice.

The spatial and temporal patterning of sequence specific transcription factors (SSTFs) contributes to cell type specification and organ formation during embryogenesis. Homeodomain transcription factors are evolutionally conserved among invertebrate and vertebrate animals. They are responsible for body segmentation and organogenesis. Lbx1 and Pitx2 both are homeodomain transcription factors contributing to SSTF pattern formation during multiple organ formations. We studied how homeodomain transcription factors regulate SSTF and non-SSTF genes in a population-specific manner using the Lbx1[superscript EGFP] and Pitx2[superscript LacZ] mouse models. We have studied the role of Lbx1 in dorsal horn interneuron specification and Pitx2 in forelimb muscle formation. The two top non-SSTF target genes, NPY and Chmp2b, of Lbx1 are studied for expression pattern and potential neuronal function in neural tube. The T box, Hox gene families and Pax genes were identified as Pitx2 target genes via microarray analysis and their expression pattern were analyzed in forelimb. The expression domains of signaling molecules were altered in absence of Pitx2, suggesting that Pitx2 played a general role in pattern formation in forelimb mesenchyme. / Graduation date: 2013

Congenital heart disease (CHD) is the major cause of death in the first year of life, the estimated incidence being 0.5-5% of live births; therefore it is important to understand the genetic causes underlying the complex process of heart formation to help prophylaxis, diagnosis and treatment of affected patients. CHD is the commonest phenotype associated with left-right (LR) disorders. LR asymmetry is determined during embryonic development. The three major body axes ? antero-posterior, dorso-ventral and left-right ? are patterned at gastrulation. LR asymmetry is established shortly after the two other major axes are patterned. The process of LR determination can be sub-divided into four integrated steps: 1. breaking of molecular symmetry in the gastrulation organizer; 2. transfer or relay of this asymmetric information to the lateral plate mesoderm (LPM), from which most internal organs will be formed; 3. reinforcement and propagation of asymmetric cues throughout the LPM and 4. conversion of asymmetric molecular information into proper organ morphogenesis. The goal of this work is to investigate mechanisms involved at two specific points in the laterality pathway: the initial generation/maintenance of asymmetric gene expression in the LPM and the morphogenetic translation of these early events into correct heart formation in the mouse. My emphasis has been on the characterization of laterality targeted cells via careful analysis of Pitx2c expression using a Pitx2c-lacZ reporter transgene, the role of BMP signalling, via Smad1, in generation/maintenance of early asymmetric signalling in the LPM, and the later involvement of both Smad1 and Pitx2 in cardiac morphogenesis through analyses of knockout mice.