Medfödda hjärtfel är globalt en av de vanligaste defekterna vid födsel, trots det är den underliggande mekanismen och dess sjukdomsförlopp fortfarande inte välkända. Flera hundra sjukdomsgener har identifierats vilket gör sjukdomen inte bara komplex, men också heterogen. För att kunna avancera i förebyggande syfte, i patient-diagnostik och behandling behövs en djupare förståelse av dess etiologi. Noterbart är att många patienter med medfödda hjärtfel också upplever samsjuklighet som utvecklingsneurologiska störningar, neurodegenerativa sjukdomar samt psykiska hälsoproblem. I den här studien undersöktes tre potentiella sjukdomsgener: ANK3, COPZ1 och DCTN1 för deras roll i både hjärtat och hjärnans utveckling i zebrafisk som experimentell modell. Detta gjordes genom att göra gen-knockouts på generna individuellt med hjälp av F0 Crispant CRISPR/Cas9 tekniken i följd av in-situ hybridisering, för att undersöka potentiella laterala defekter i hjärtat samt bildanalys för att mäta storleken på hjärnan. Alla tre gener verkade orsaka en signifikant ökning av hjärtfel genom att laterala defekter observerades, där två av generna också visade en signifikant minskad hjärtfrekvens. Angående hjärnan så verkade två av generna orsaka en mindre hjärna medan en av generna orsakade en förstorad hjärna. Dessa resultat ger en preliminär inblick i deras medverkan i hjärtat och hjärnans utveckling, dock behövs vidare studier inte bara för att stärka resultaten men också för att kunna grundligt säkerställa korrelationen till samsjukligheten med utvecklingsneurologiska störningar, neurodegenerativa sjukdomar samt psykiska hälsoproblem. / Congenital heart defects (CHD) are amongst the most common birth defects globally, yet the underlying mechanism and the disease trajectory of it is still poorly understood. Several hundred disease genes have been discovered, making it not only complex but also heterogeneous. In order to advance in prevention, patient diagnosis and treatment, a deeper understanding of its etiology is needed. Notably, many patients with CHD experience comorbidities such as neurodevelopmental- and neurodegenerative disorders as well as mental health issues. In this project, three candidate disease genes: ANK3, COPZ1 and DCTN1 were chosen to further investigate their role in both heart- and brain development in zebrafish as the experimental model. This was done by knocking out the genes individually using the F0 Crispant CRISPR/Cas9 method followed by whole mount in situ hybridization (WISH), to assess potential defective lateral phenotypes of the heart and image analysis to measure the size of the brain. All three genes seemed to cause a significant increase in overall heart defects by showing laterality defects whereas two of them also showed significantly slower heart rate. As for the brain, two of the genes seemed to cause a smaller brain, whereas one gene seemed to cause an enlarged brain. These findings give a preliminary insight into their involvement in the heart- and brain development, however, further research is necessary to not only strengthen the findings but to also more thoroughly establish the correlation to the comorbidity of the neurodevelopmental, neurodegenerative disorders and mental health issues.

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.

As coronárias derivam do proepicárdio, uma estrutura formada por precursores dos constituintes de vasos coronários, células endoteliais e musculares lisas (CoSMC). In vivo observa-se um marcante atraso entre a diferenciação endotelial e a integração de CoSMC à parede do vaso. O objetivo deste trabalho foi identificar os mecanismos que inibem a diferenciação a CoSMC in vivo. Baseados na perda progressiva da expressão de raldh2, a principal enzima de síntese de ácido retinóico (AR), nós exploramos a sinalização por AR como um possível inibidor da diferenciação a CoSMC. Através de um vetor adenoviral de expressão de raldh2 e da inibição in vivo da síntese de AR nós demonstramos que a sinalização por AR bloqueia a diferenciação a CoSMC dos precursores coronários. Nós também identificamos o VEGF como um fator chave no controle da diferenciação a CoSMC. Em conjunto, nossos dados suportam o modelo que a síntese de AR e VEGF durante o desenvolvimento cardíaco foi co-optada para o bloqueio da diferenciação a CoSMC até o estabelecimento de uma vasta malha vascular. / Coronary vessels derive from the proepicardium (PE), a structure formed by precursor of coronary vessels cells, endothelial and smooth muscle cells (CoSMC). In vivo there is a clear gap between the endothelial differentiation and the integration of CoSMC into the vascular tubes. The aim of this work was to understand the mechanisms controlling the delayed in vivo CoSMC differentiation. Based on the progressive loss of expression of raldh2, the main retinoic acid (RA) synthesizing enzyme, we explored the RA signaling as a possible candidate inhibitor of CoSMC differentiation. Using a adenoviral raldh2 expression system and in vivo inhibition of RA synthesis we showed that RA signaling act as a brake to slow CoSMC differentiation in PE-derived cells. We also identified VEGF as key factor acting on the control of CoSMC differentiation. Together our results support a model that AR and VEGF synthesis during cardiac development was co-opted to block the CoSMC differentiation of coronary precursors before an extensive endothelial network of tubes is established.

As coronárias derivam do proepicárdio, uma estrutura formada por precursores dos constituintes de vasos coronários, células endoteliais e musculares lisas (CoSMC). In vivo observa-se um marcante atraso entre a diferenciação endotelial e a integração de CoSMC à parede do vaso. O objetivo deste trabalho foi identificar os mecanismos que inibem a diferenciação a CoSMC in vivo. Baseados na perda progressiva da expressão de raldh2, a principal enzima de síntese de ácido retinóico (AR), nós exploramos a sinalização por AR como um possível inibidor da diferenciação a CoSMC. Através de um vetor adenoviral de expressão de raldh2 e da inibição in vivo da síntese de AR nós demonstramos que a sinalização por AR bloqueia a diferenciação a CoSMC dos precursores coronários. Nós também identificamos o VEGF como um fator chave no controle da diferenciação a CoSMC. Em conjunto, nossos dados suportam o modelo que a síntese de AR e VEGF durante o desenvolvimento cardíaco foi co-optada para o bloqueio da diferenciação a CoSMC até o estabelecimento de uma vasta malha vascular. / Coronary vessels derive from the proepicardium (PE), a structure formed by precursor of coronary vessels cells, endothelial and smooth muscle cells (CoSMC). In vivo there is a clear gap between the endothelial differentiation and the integration of CoSMC into the vascular tubes. The aim of this work was to understand the mechanisms controlling the delayed in vivo CoSMC differentiation. Based on the progressive loss of expression of raldh2, the main retinoic acid (RA) synthesizing enzyme, we explored the RA signaling as a possible candidate inhibitor of CoSMC differentiation. Using a adenoviral raldh2 expression system and in vivo inhibition of RA synthesis we showed that RA signaling act as a brake to slow CoSMC differentiation in PE-derived cells. We also identified VEGF as key factor acting on the control of CoSMC differentiation. Together our results support a model that AR and VEGF synthesis during cardiac development was co-opted to block the CoSMC differentiation of coronary precursors before an extensive endothelial network of tubes is established.

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...

No description available.

Phenylethanolamine-N-methyltransferase (Pnmt) catalyzes the conversion of noradrenaline to adrenaline and is the last enzyme in the catecholamine biosynthetic pathway. Pnmt serves as a marker for adrenergic cells, and lineage-tracing experiments have identified the embryonic heart and hindbrain region as the first sites of Pnmt expression in the mouse. Pnmt expression in the heart occurs before the adrenal glands have formed and prior to sympathetic innervation, suggesting that the heart is the first site of catecholamine production in the mouse. The function of these Pnmt+ cells in heart development remains unclear. In the present study, we test the hypothesis that (i) a genetic ablation technique utilizing a suicide reporter gene selectively destroys Pnmt cells in the mouse, and (ii) Pnmt cells are required for normal cardiovascular and neurological function. To genetically ablate adrenergic cells, we mated Pnmt-Cre mice, in which Cre-recombinase is under the transcriptional regulation of the Pnmt promoter, and a Cre -activated diphtheria toxin A (DTA) mouse strain (ROSA26-eGFP-DTA), thereby causing activation of the toxic allele (DTA) in Pnmt-expressing (adrenergic) cells resulting in selective "suicide" of these cells in approximately half of the offspring. The other half serve as controls because they do not have the ROSA26-eGFP-DTA construct. In the Pnmt+/Cre; R26+/DTA offspring, we achieve a dramatic reduction in Pnmt transcript and Pnmt immunoreactive area in the adrenal glands. Furthermore, we show that loss of Pnmt cells results in severe left ventricular dysfunction that progressively worsens with age. These mice exhibit severely reduced cardiac output and ejection fraction due to decreased LV contractility and bradycardia at rest. Surprisingly, these mice appear to have a normal stress response, as heart rate and ejection fraction increased to a similar extent compared to controls. In addition to baseline cardiac dysfunction, these mice fail to gain body weight in a normal manner and display gross neurological dysfunction, including muscular weakness, abnormal gaiting, and altered tail suspension reflex, an indicator of neurological function. This work demonstrates that selective Pnmt cell destruction leads to severe left ventricular dysfunction, lack of weight gain, and neurological dysfunction. This novel mouse is expected to shed insight into the role of Pnmt cells in the heart, and suggests a role for Pnmt cells in neurological regulation of feeding behavior, metabolism, and motor control.

Collective cell migration is a defining feature of many morphogenetic processes. Diseases such as congenital heart diseases and cancer arise due to mis-regulation of collective migratory behaviour and animal models have played a pivotal role in dissecting the molecular mechanisms which underlie this process. During embryonic heart development, cardiac precursors undergo a stage of collective migration in both vertebrates and invertebrates. We developed a paradigm to quantitatively assess collective cell migration of cardiac precursors in live embryos of Drosophila, which is the simplest genetic model organism with a heart. Therefore, we studied processes which are commonly observed in most collective cell migration models such as guidance signalling and extracellular matrix remodelling. Our results demonstrate that leading edge of migrating cardioblasts is highly active and that this behaviour is regulated by guidance cues, Slit and Netrin and their respective receptors Robo/Robo2 and Frazzled/Uncoordinated5. These molecules cooperatively promote leading edge motility and epithelial characteristics of the cardioblasts. Next, we determined that matrix restructuring around the cardioblasts requires proteases Mmp1 and Mmp2, which are members of the highly conserved Matrix Metalloproteinase family. We demonstrate that Mmp1 and Mmp2 have distinct roles during lumen formation, however, both Mmp1 and Mmp2 are required for collective motility of the cardioblast leading edge. Hence, we propose that embryonic heart development in Drosophila is an effective and amenable model of collective cell migration which can be applied to discover unique mechanisms which coordinate cell movement in groups. / Thesis / Doctor of Philosophy (PhD)

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L’acide rétinoïque (AR), dérivé actif de la vitamine A, agit comme un morphogène dans de nombreux processus de développement. Des études antérieures chez l’embryon de poulet (Hochgreb et al., 2003) ont montré que l’ AR est impliqué dans la régionalisation antéro-postérieure du tube cardiaque. Au cours de ma thèse, j’ai cherché à définir le rôle de l’AR dans le développement du coeur et plus particulièrement dans la régionalisation antéropostérieuredu territoire cardiaque. Pour cela, j’ai utilisé les mutants souris déficients pourl’enzyme RALDH2 permettant la synthèse d’AR. L’utilisation de marqueurs spécifiques des progéniteurs cardiaques nous a permis de montrer que l’AR est requis pour établir la bordure postérieure du second champ cardiaque (mésoderme splanchnique).Dans le but de mieux comprendre comment la voie de l’AR agit sur la spécification cardiaque, nous avons voulu identifier ses cibles dans le mésoderme splanchnique. Pour la première fois, nous montrons l’implication des gènes Hox dans la cardiogenèse précoce.L’analyse du lignage des cellules exprimant Hoxa1, Hoxa3 et Hoxb1 nous a permis demontrer que les pôles artériels et veineux ont la même origine au niveau du territoire cardiaque.Nous avons aussi étudié le rôle de l’AR dans la morphogenèse des arcs aortiques et de sesdérivés, en particulier son influence sur le développement de la quatrième artère des arcspharyngés. Cette étude a mis en évidence l’interaction génétique de Raldh2 et du facteur àboîte T, Tbx1, lors de la morphogenèse du quatrième arc aortique. En effet, la diminution de l’AR accélère la récupération des défauts de la quatrième artère des arcs pharyngés chez le modèle murin pour le syndrome de Di George (Tbx1+/-). Nos résultats suggèrent que l’AR estun modificateur de la micro-délétion 22q11 (syndrome de DiGeorge) chez l’homme, ceci pouvant expliquer en partie la grande variabilité des malformations cardiaques des patients DiGeorge.J’ai aussi participé à l’étude du rôle de l’AR dans la différenciation des progéniteurs du myocarde ventriculaire. Ces résultats montrent que l’AR est nécessaire à la différenciation de la population de cellules progénitrices du myocarde. La portée de ces résultats est importante et pourra conduire à plus long terme à la thérapie et la réparation du muscle cardiaque. Enfin,la dernière partie de l’étude se concentre sur le rôle de l’AR dans le développement de la vasculature coronaire. Ce morphogène semble influencer le positionnement des ostia coronaires à l’aorte. / Retinoic acid (RA), the active derivative of vitamin A (retinol), acts as a morphogen inseveral developmental processes. Previous studies in the chick embryo (Hochgreb et al.,2003) have indicated that RA signaling is required to antero-posterior patterning of the cardiac tube. The aim of my thesis was to define the role of RA signaling in heart development and in particular in the establishment of antero-posterior identity of the cardiac field. Thus, we used Raldh2 (Retinaldehyde dehydrogenase 2) mutants that are deficient for RA synthesis. To understand the role of RA, we examined the contribution of the second heart field to pharyngeal mesoderm, atria and outflow tract in Raldh2-/- embryos. Our findingsshown that embryo lacking RA synthesis enzyme RALDH2 have expansion of the secondheart field (splanchnic mesoderm).To better understand the mechanism by which RA signaling regulates the cardiac progenitors,we have identified its targets in the splanchnic mesoderm. We have shown for the first timethat Hox genes contribute to cardiogenesis. Moreover, genetically labeled cells analysis reveals a common origin of the arterial and venous poles in the cardiac field.Then, we have analyzed the role of RA in aortic arch remodeling, in particular its influence onfourth aortic arch arteries. This work demonstrates a genetic interaction between Raldh2 and the T-box factor, Tbx1, during fourth aortic arch formation. Our results shows that decreasedon RA level accelerates recovery of fourth aortic arch artery defects seen in Tbx1-/-, which is amodel of DiGeorge syndrome. Moreover, this study suggests that RA is a modifier of 22q11microdeletion (DiGeorge syndrome) in patient.In a collaborative work, we have analyzed the role of RA in differentiation of ventricular myocardium progenitors. Our results showed that the differentiation of the myocardial progenitor cells required RA. The impact of these results is crucial and would lead to therapyand cardiac muscle repair.The last part of my thesis focuses on the role of RA on coronary vascular development. This morphogen seems to influence the position of coronary ostia to the aorta.