Thèses sur le sujet « Cardiogenesi »

Finora, diversi studi hanno dimostrato l'importanza dei miRNAs, in fase di sviluppo embrionale e nell’insorgenza di molte patologie. Nel sistema cardiaco, il ruolo del miR-133a è stato ampiamente caratterizzato dall’embriogenesi allo sviluppo di difetti cardiaci. Tuttavia, resta ancora molto da caratterizzare circa le funzioni del miR-133. Lo scopo principale della mia tesi di dottorato è stato indagare queste funzioni aggiuntive del miR-133 nello sviluppo cardiaco, in particolare sulla sua capacità di controllare vie di trasduzione a livello trascrizionale. La seconda fase della mia ricerca è stata quella approfondire il suo ruolo in patologie cardiache. In ultimo l'obiettivo finale della mia ricerca è stato quello di traslare l’importanza del miR-133 nel suo uso terapeutico con lo sviluppo di una nuova strategia che, basato sull'utilizzo dei nanomateriali al fine di sviluppare uno specifico e controllato rilascio di miR-133 nel sistema cardiovascolare.
So far, a plethora of studies demonstrated the importance of miRNAs, in embryo development and in the onset of basically all kinds of pathologies. In the cardiac system, the role of miR-133a was extensively characterized from embryogenesis to the development of cardiac defects. Nevertheless, much remains to be learned about the functions of miR-133. The main scope of my PhD thesis was to investigate these additional functions of miR-133 firstly in cardiac development, focusing on its potential ability to control signal pathways at the transcriptional level, and secondly in the already well characterized cardiac pathologies. Moreover, the ultimate goal of my research was to translate the additional roles of miR-133 into its therapeutic use by developing a new strategy that, based on the use of nanomaterials, allows for the specific and controlled delivery of miR-133 into the cardiac system.

Au cours de ma thèse, je me suis intéressée au développement du système cardio-vasculaire chez la drosophile afin de mieux comprendre la logique de régulation de ce processus. Au cours de l'embryogenèse, la cardiogenèse est réalisée grâce à un réseau de régulation génique (GRN) qui conduit à la formation d'un simple tube cardiaque linéaire. Ensuite, lors de la métamorphose, le tube cardiaque larvaire est remodelé pour former l'organe adulte.J'ai d'abord participé à l'évaluation et à l'amélioration d'une nouvelle méthode, cisTargetX, qui permet prédire des modules cis-régulateurs (CRM) présentant des caractéristiques communes à un groupe de gènes co-exprimés.En utilisant cette méthode, j'ai analysé le transcriptome du remodelage du cœur afin de prédire des motifs pouvant être liés par des TF impliqués dans le contrôle temporel de l'expression des gènes, ainsi que les CRM associés. Grâce aux validations in-vivo des CRM prédits, j'ai démontré qu'ils étaient capables de reproduire le patron d'expression temporel attendu. J'ai également démontré que la mutation du motif en question au sein de deux des CRM testés permet de supprimer son patron d'expression sauvage. Ce motif est reconnu par des facteurs de transcription (TF) de la famille des récepteurs nucléaires (NR). Dhr3, un NR fortement exprimé au début de l'induction des gènes analysés, est montré comme étant essentiel au patron d'expression temporel. Nos résultats suggèrent une architecture du GRN, dans lequel les régulations temporelle et spatiale sont distinctes.Par la suite, j'ai participé à la caractérisation du GRN impliqué dans la cardiogenèse. En combinant un transcriptome issu de la différenciation des cellules cardiaques avec des expériences ChIP-on-Chip sur le TF MEF2, j'ai prédit que certains TF appartenant aux familles bZIP et REL sont susceptibles de participer au GRN responsable de la différenciation cardiaque. La validation in-vivo de ces prédictions est en cours
During my thesis, I focused on the development of the cardiovascular system in Drosophila in order to investigate the regulatory logic of this process. During embryogenesis, cardiogenesis is mediated by a gene regulatory network which includes conserved signaling pathways and transcription factors, and leads to the formation of a linear cardiac tube. Then, during metamorphosis, the larval cardiac tube is remodeled to form the adult organ.I first participated in the evaluation and the improvement of a new method, cisTargetX, that uses a comprehensive library of motifs, combined with phylogenetic conservation, to identify potential cis-regulatory modules (CRM) presenting common features in a cluster of co-expressed genes.Using this method among other tools, I analysed cardiac remodeling during metamorphosis to predict motifs for transcription factors (TF) involved in the temporal control of gene expression, and also their associated CRM. I performed in-vivo validations of predicted CRM, and demonstrated that they reproduce the expected temporal expression pattern. In addition, I demonstrated that motifs mutation within selected CRM abrogate this expression pattern. This motif is predicted to be recognized by a TF that belong to the nuclear receptor (NR) family. Dhr3, a NR highly expressed at the onset of the induction of the analysed gene set, is demonstrated to be essential for CRM temporal pattern. Our results suggest a modular architecture of the regulatory machinery, in which the temporal and spatial regulations are distinct.Next, I participated in the characterization of the Gene Regulatory Network (GRN) involved in cardiac differentiation during embryogenesis. Combining transcriptome profiling of differentiating cardiac cells with Mef2 Chip-on-Chip experiments allowed me to predict that TF belonging to bZIP and REL family are likely to participate in the GRN driving cardiac differentiation. In-vivo validation of these predictions is in progress

An early event in animal development is the formation of the three primary germ layers that define the body plan. During gastrulation, cells migrate through the primitive streak of the embryo and undergo changes in morphology and gene expression, thus creating the mesodermal and endodermal cell layers. Gastrulation requires expression of Fibroblast Growth Factor (FGF), Wnt, and Platelet-Derived Growth Factor (PDGF). Embryos treated with FGF inhibitors fail to gastrulate, as cell migration is completely halted. During gastrulation, 44 microRNAs are expressed in the primitive streak of G. gallus embryos, and six (microRNAs -let7b, -9, -19b, -107, -130b, and -218) are strongly upregulated when FGF signaling is blocked. The abundance of these six FGF-regulated microRNAs is controlled at various stages of processing: most are regulated transcriptionally, and three of them (let7b, 9, and 130b) are blocked by the presence of Lin28B, an RNA-binding protein upregulated by FGF signaling. These microRNAs target various serine/threonine and tyrosine kinase receptors. We propose a novel pathway by which FGF signaling downregulates several key microRNAs (partially through Lin28B), upregulating gene targets such as PDGFRA, which permits and directs cell migration during gastrulation. These findings add new layers of complexity to the role that FGF signaling plays during embryogenesis. FGF signaling is also required for the formation of the heartfields, and has an overlapping pattern of expression with BMP (Bone Morphogenetic Protein). A microarray experiment using inhibitors of FGF and BMP found that thousands of genes in pre-cardiac mesoderm are affected by FGF signaling, BMP signaling, or a cooperative effect of the two. The promoter regions of similarly regulated genes were queried for over-represented transcription factor binding sites or novel DNA motifs. Cluster analysis of over-represented sites determined candidate transcriptional modules that were tested in primary cardiac myocyte and fibroblast cultures. About 75% of predicted modules in FGF-upregulated genes proved to be functional enhancers or repressors. Functional enhancers among FGF-upregulated genes contained clusters of CdxA and NFY sites, and increased transcription in the presence of a constitutively active FGF receptor.

The Wnt/ -catenin and the Wnt/planar cell polarity (Wnt/PCP) signalling pathways have been shown to play important roles in cardiogenesis and their disruption has been shown to cause severe disturbances in heart development. Spatially and temporally complex interplays between the two pathways have been described. One component of the PCP pathway is Jnk, a member of the highly conserved mitogenactivated protein kinase (MAPK) family. This stress responsive mitogen is known to control a variety of cellular behaviours such as proliferation, apoptosis and cell migratory behaviour and as such, is likely to be of pivotal importance in cardiac development. The aim of this study was to investigate the role played by Jnk in vertebrate heart formation and the relationships between Jnk signalling and canonical Wnt signalling, using in silico and in vivo approaches in zebrafish and an in vitro approach on a mouse embryonic stem (ES) cell model of cardiogenesis. Firstly, using a range of bioinformatic methods, an analysis of jnk genes, splice variants and proteins, and an investigation of their phylogenetic relation with other species was undertaken. This suggested conservation of Jnk family members, but suggested that there were additional orthologues of jnk1 present in the zebrafish transcriptome. The spatial and temporal expression profiles of these genes were then examined by semi-quantitative PCR and in situ hybridisation. The functional role of Jnk proteins during zebrafish development was subsequently investigated using a specific chemical inhibitor, SP600125. Inhibition of Jnk signalling during gastrulation and somitogenesis caused a convergence extension-like phenotype and severe cardiac defects, including looping anomalies and alterations in atrial versus ventricular cell numbers. ES cells have the capacity to differentiate in vitro and give rise to cells of many different lineages, including cardiomyocytes. Canonical Wnt and Jnk components were manipulated during specific windows of differentiation as ES cells formed beating embryoid bodies. Examination of the spontaneous contractile behaviour of differentiating ES cells as they entered the cardiogenic lineage, and analysis of their developmental gene expression profiles, showed the beating behaviour of ES cellderived cardiac cells was enhanced in a temporally specific manner after inhibition of the non-canonical Wnt/Jnk pathway, while there was marked alteration of canonical Wnt signalling. To investigate whether there were reciprocal interactions between the two pathways, analysis of the system after activation of the canonical pathway was also undertaken. These studies indicated that the beating behaviour of ES cell-derived cardiac cells was enhanced in a temporally specific manner after inhibition of Jnk, while after activation of canonical Wnt/ -catenin signalling, the cardiogenic potential of differentiating ES cells was severely suppressed. The findings of this study extend our understanding of the role played by canonical and non-canonical Wnt signalling pathways in heart morphogenesis and highlight the interacting effects of related signalling pathways activity in cardiogenesis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, February 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 114-127).
Cardiovascular diseases persist as the leading cause of mortality worldwide. Stem cell therapy, aimed to restore contractility and proper vasculature, has gained considerable attention as an attractive therapeutic option. However, proper cell differentiation, survival and integration in an infarcted zone remain elusive. This thesis aims to utilize in vitro techniques to obtain a systematic characterization of how individual stimulations can affect the cardiogenesis process of embryonic stem cells. First, a compliant microfluidic system was developed to study the individual and combined effects of culture dimensions and uniaxial cyclic stretch on the differentiation process. A smaller culture dimension, with a characteristic length scale of hundreds of micrometers, dramatically enhanced differentiation partly due to an accumulation of cell-secreted and cardiogenic BMP2. Uniaxial cyclic stretch, on the other hand, inhibited differentiation. With this microfluidic platform and a GFP-reporting differentiation cell line, effects of various external stimuli on differentiation were systematically studied. Next, the effects of collagen I and cell alignment, two biophysical signatures of the adult myocardium, on promoting phenotypic changes of isolated embryonic stem cell derived cardiomyocytes (ESCDMs) were investigated. Effects of collagen I depended on how it was presented to the cells and overlaying collagen gel impeded cell elongation. Binucleation. characteristic of maturing cardiomyocytes, was reduced with soluble collagen supplement and nanoscale topography and was associated with an increase in cytokinesis. Both nanoscale topography and microcontact printing resulted in aligned cardiomyocyte monolayers but produced different morphologies. Lastly, the lessons learned from studying the aforementioned processes were applied to test the utility of ESCDMs as biological actuators. Three proof-of-concept experiments were conducted: ESCDM monolayers were able to contract synchronously as a cell-assemble, force generated by the cell monolayer was estimated to be comparable to that by neonatal myocytes and lastly, the direction of contraction could be controlled with surface patterning. This work advances our understanding on the cardiogenic potential of murine embryonic stem cells and elucidated complex biological questions with well-characterized and controlled tissue engineering techniques.
by Chen-rei Wan.
Ph.D.

The heart is the first functioning organ to develop during embryogenesis to maintain the growing embryo with oxygen and nutrients. However, cardiogenesis is a complex and highly coordinated biological process, and any perturbation to this process can result in detrimental defects to the heart. Haemodynamics is known to play an important role in cardiac growth and vasculature remodelling. Congenital heart defects (CHDs) accounts for 0.4-1.3% of all live birth, whereas cardiomyopathy accounts for 8-11% of cardiovascular disease diagnoses detected in utero. Although the heart defects and cardiomyopathies are known to be attributed by genetic mutations, most cases have unknown etiology. Hence, OFT-banding model was employed to alter the haemodynamic loading via pressure overloading. Upon alteration of haemodynamics, enlargement of the heart with a spectrum of cardiac anomalies were found (e.g ventricular septal defects, thickened epicardium and dysmorphic atrioventricular valves) upon morphological and stereological analysis. A study of global differential expression of OFT-banded hearts by RNA sequencing revealed a number of differentially expressed genes and they were associated with cardioprotection, metabolism, shear stress and valve development; further, a reduction of apoptosis was seen in these banded hearts as well. One of the cardiac phenotypes seen upon OFT-banding, the abnormal primordial atrioventricular valve, was further characterized to provide an insight how the atrioventricular valve is affected upon alteration of haemodynamics. Aberrant expressions of extracellular matrix (ECM) genes such as TBX20, Aggrecan and Periostin alongside with the shear stress responsive genes (KLF2 and EDN1) were found, and a decrease in apoptosis was seen. Moreover, dysregulation of ECM proteins such as fibrillin-2, type III collagen and tenascin were further demonstrated in more mature primordial AV leaflets at HH35, with a concomitant decrease of ECM cross-linking enzyme, transglutaminase-2. In addition, for many years sarcomeric proteins have been associated with a range of cardiomyopathies, but only in recent years they have been linked to congenital heart defects (CHDs). To date, cardiac troponin T (TNNT2) has been associated with cardiomyopathies but not with isolated CHDs. TNNT2 encodes for cTnT regulatory proteins of the thin filament of the sarcomere and is vital for muscle contraction and force generation within cardiomyocytes. To investigate a role of TNNT2 in the early developing heart, targeted manipulation of TNNT2 was performed in embryonic chick to reduce the protein levels of cTNT (protein product of TNNT2) in ovo via translational block. Abnormal atrial septal growth, reduced ventricular trabeculation and ventricular diverticula were found upon TNNT2 morpholino treatment. The abnormal phenotype observed in the TNNT2 morpholino-treated groups was potentially suggested by differential expression of shear stress responsive gene, NOS3 gene.

L’objectif de cette thèse a été d’évaluer certains paramètres physiques et épigénétiques impliqués dans la différenciation cardiaque de cellules souches humaines pluripotentes induites. Un premier paramètre physique souvent sous-évalué a été étudié, celui de la rigidité. Classiquement, les cellules souches sont cultivées et adaptées à des rigidités in vitro allant de 1-10 GPa très éloignées des valeurs physiologiques, de l’ordre du kPa. L’impact de support de culture à 3, 12 et 25 kPa a été évalué sur les cellules souches initiales. Les résultats montrent que des rigidités inférieures à 25 kPa ne permettent pas le maintien de la pluripotence au bout de 6 passages. De plus, les colonies cellulaires se développent en 3D et créent leur propre microenvironnement. Un second paramètre étudié concerne les microRNAs appartenant à la famille let-7 dont la fonction exacte au niveau cardiaque reste à définir. Les résultats montrent qu’au cours de la différenciation son expression se caractérise par une augmentation transitoire précoce au moment de la formation du mésoderme, puis s’éteint pour ne ré-augmenter que plus tard lors de la maturation des cardiomyocytes. Des modulations via des mimics ou des inhibiteurs dans différents contextes cellulaires suggèrent qu’initialement let-7 contribue à une future spécification cardiaque, mais que plus tard cette famille devra être réprimée pour générer des progéniteurs cardiaques. À l’opposé, miR-1, spécifique au cœur, contribue toujours à la progression en cardiomyocytes. Ensemble, ces recherches contribuent à la recherche fondamentale sur le développement du cœur humain et à la recherche appliquée en ingénierie tissulaire cardiaque
The objective of this thesis was to evaluate some physical and epigenetic parameters involved during cardiac differentiation of human induced pluripotent stem cells. Environmentally, an often undervalued physical parameter remains, the stiffness defined by the Young’s modulus. Commonly stem cells are cultured and adapted to in vitro rigidities ranging between 1-10 GPa very far from physiological values, for instance 10-15 kPa for the heart. The impact of soft culture substrates with 3 kPa, 12 kPa and 25 kPa was studied on the initial stem cells. Globally, results indicated that rigidities lower than 25 kPa were not suited for total pluripotency maintenance after 6 passages. Also, cellular colonies started to grow in 3D suggesting that softness drove them to build their own microenvironment. Epigenetically, the exact role of one of the first discovered microRNAs, the let-7 family has not yet been fully elucidated. Throughout differentiation its expression was characterized by an early transient peak at the time of mesoderm formation, after which their expression extinguished to only gradually re-increase later in the course of cardiomyocytes maturation. Modulation experiments involving mimics or inhibitors of the let-7 family on different cellular contexts suggested that initially let-7 acted on future cardiac specification but later, this family had to be repressed in order for cardiac progenitors to emerge. Oppositely, the cardiac specific miR-1 always contributed to their progression into cardiomyocytes. Together these researches contribute to fundamental research on human heart development and to applied research on human engineered cardiac tissues

Aim: Recent interest in cardiogenesis has focused on the epicardium, the outer epithelial layer that envelops the heart. Epicardial-derived cells (EPDCs) contribute vascular smooth muscle to developing coronary vessels and provide critical signalling cues to facilitate myocardial functionality. However, the precise molecular mechanisms that underpin epicardial biology remain unclear. Ablation of Myh10 in the EHC mouse results in embryonic lethal cardiac malformations, including epicardial and coronary defects. We sought to establish the role of Myh10 in epicardial cell function to further dissect the coronary vessel developmental pathway, a deeper understanding of which may inform the design of therapeutics to regenerate and repair the injured heart. Methods: Utilising multiple cell and developmental biology techniques, we generated a pathological evaluation of the EHC phenotype. EPDC migration was investigated in vivo with Wt1 immunohistochemistry, and in vitro by performing scratch wound assays on epicardial cell cultures. Congruently, we examined the ability of epicardial cells to undergo EMT in vivo by employing Snail and Phosphohistone-H3 immunohistochemistry. Results: Our studies reveal that EHC epicardial cells have a reduced capacity to invade the ventricular myocardium. Furthermore, we discovered increased proliferation and reduced Snail expression specifically within the EHC epicardium, consistent with EMT dysregulation. Interestingly, epicardial cell function did not appear to be disrupted in vitro. Conclusion: These results demonstrate a novel role for Myh10 in both EPDC migration and the promotion of epicardial EMT. Our finding that migration is unaffected in vitro suggests that the unique properties of the in vivo epicardial microenvironment dictate a requirement for Myh10 in order to elicit correct epicardial function. Together, this research enhances our understanding of the dysfunctional processes that contribute to abnormal cardiogenesis; these insights may aid our ability to determine the molecular regulators of coronary vessel development, and create therapeutics to regenerate vessel growth and repair injured cardiac tissue in cardiovascular disease.

Dynamic histone modification has emerged as a robust and versatile regulator of gene expression in eukaryotic cells. One such modification, the trimethylation of lysine 27 on histone H3 (H3K27me3) is facilitated by the Polycomb repressive complex 2 (PRC2) and contributes to the localized repression of transcription. Subsequently, lysine specific demethylase Kdm6b (Jmjd3) can relieve the repressive H3K27me3 mark, allowing for transcriptional activation. In vitro studies have suggested a role for Kdm6b during mesodermal and cardiovascular differentiation in mammalian embryonic stem cells; however, this relationship has yet to be characterized in vivo. I utilized the advantages of the zebrafish model to investigate the in vivo roles of Kdm6b-family demethylases during development using a reverse genetic approach. I carried out two independent loss-of-function studies to analyze the role of Kdm6b-family demethylases during embryonic development in zebrafish. By comparing genetic loss-of-function and morpholino-mediated knockdown approaches, I found that morpholino–mediated knockdown of kdm6bb transcript produces off-target effects and does not portray an accurate representation of in vivo function. I then show that, while not required for early cardiogenesis, histone demethylases kdm6ba and kdm6bb function redundantly to promote late stage proliferation during heart ventricle trabeculation. These data reveal a previously unknown functional relationship and support the hypothesis that Kdm6b-family demethylases function primarily during later stages of development. Additionally, my description of morpholino-induced off-target effects supports the need to use extreme caution when interpreting morphant phenotypes. Due to the embryonic lethality exhibited by kdm6b-deficient embryos and the limited tools available for spatiotemporal transgene control in zebrafish, I was unable to investigate demethylase function within adult animals. I attempted to circumvent these limitations by creating an inducible gene expression system that uses tissue-specific transgenes that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. I showed that these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. I then demonstrated how this technology could be used to define developmental windows of gene function by spatiotemporally controlling expression of constitutively active Notch1 in embryos. This dissertation contains previously published co-authored material.

RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.

RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

Le muscle cardiaque est constitué de cellules contractiles, ou cardiomyocytes, majoritairement composées de myofibrilles. De nombreuses mutations affectant les protéines myofibrillaires sont responsables de cardiomyopathies congénitales. Jusqu'alors le processus de différenciation des cardiomyocytes en présence d'une telle mutation était mal connu. Dans la première partie de ce travail de thèse, nous avons établi un modèle in vitro de cellules souches embryonnaires de souris génétiquement modifiées reproduisant les premières étapes de la différenciation des cardiomyocytes en présence d'une mutation myofibrillaire. Dans les cellules mutées nous avons mis en évidence un défaut sévère dans la formation des myofibrilles et dans la gestion du calcium intracellulaire. La répartition du calcium intracellulaire en microdomaines finement régulés semble avoir un rôle primordial pour le bon déroulement de la myofibrillogenèse. Dans la deuxième partie de ce travail de thèse nous nous sommes intéressés à l'origine des battements cardiaques dans le cœur embryonnaire. En effet, comme le cœur adulte, le cœur embryonnaire se contracte en réponse à des oscillations calciques. Cependant, dans ces derniers, ces oscillations sont dues à l'activation d'une cascade intracellulaire impliquant l'IP3. Dans ce travail nous avons pu mettre en évidence l'implication de la voie FGF dans la génération des oscillations calciques induites par l'IP3. Ces résultats ont été obtenus par des approches pharmacologiques et génétiques sur des cœurs embryonnaires de souris en comparaison avec le modèle de différenciation in vitro. Notre modèle in vitro de cellules souches génétiquement modifiées est un excellent outil pour disséquer les processus physiopathologiques au cours des étapes précoces de la cardiogenèse.

Abstract Wnt genes encode secreted signalling molecules that control embryonic development including organogenesis, while dysregulated Wnt signalling is connected to many diseases such as cancer. Specifically, Wnts control a number of cellular processes such as proliferation, adhesion, differentiation and aging. Many Wnt proteins activate the canonical β-catenin signalling pathway that regulates transcription of a still poorly characterized set of target genes. Wnts also transduce their signaling in cells via β-catenin-independent “non-canonical” pathways, which are not well understood. In this study, Wnt-11 signalling mechanisms in a mammalian model cell line and roles of Wnt-11 in heart development were analyzed in detail. In addition the aim was to identify new Wnt target genes by direct chromatin immunoprecipitation and Affymetrix GeneChip assays in the model cells exposed to Wnt-3a. Our studies reveal that Wnt-11 signalling coordinates the activity of key cell signalling pathways, namely the canonical Wnt/β-catenin, the JNK/AP-1, the NF-κB and PI3K/Akt pathways in the CHO cells. Analysis of the Wnt-11-deficient embryos revealed a crucial role in heart organogenesis. Wnt-11 signalling coordinates cell interactions during assembly of the myocardial wall and Wnt-11 localizes the expression of N-cadherin and β-catenin to specific cellular domains in the embryonic ventricular cardiomyocytes. Collectively these studies reveal that the mammalian Wnt-11 behaves as a non-canonical Wnt and that it is a critical factor in the coordination of heart development. Specifically, it controls components of the cell adhesion machinery. Analysis of the Wnt target genes revealed a highly context-dependent profile in the Wnt-regulated genes. Several new putative target genes were discovered. Out of the candidate Wnt target genes, Disabled-2 was identified as a potential new direct target for Wnt signalling.

L'objectif général de ce travail de doctorat a été centré sur l'utilisation des cellules pluripotentes induites humaines dans la modélisation et l'évaluation thérapeutique des pathologies cardiaques. Depuis leur découverte en 2006, les iPSC offrent une opportunité pour le développement de modèles cellulaires humains et spécifiques de patients pour l'étude des mécanismes physiopathologiques, l'évaluation de réponses pharmacologiques et le génération de cellules redifférenciées (ici en cardiomyocytes) pour des applications thérapeutiques cellulaires. Dans ce travail nous avons démontré que la quantité mais aussi la qualité finale des cardiomyocytes dérivés d'iPSC dépend des conditions spatiales et pharmacologiques utilisées durant les différentes étapes de différenciation. L'utilisation d'un protocole de différentiation en monocouche avec blocage simultané et transitoire de l'ensemble des voies Wnt (canoniques et non canoniques) permet d'obtenir une maturation plus importante du sarcomère, étape essentielle pour la modélisation des sarcomèropathies La différenciation des iPSC en cardiomyocytes peut aussi être obtenue par une approche moléculaire ciblée visant à activer spécifiquement un programme cardiogénique. Celle-ci est obtenue via l'utilisation d'une protéine Cas9 mutée et couplée à un système transactivateur et permettant le ciblage simultané de 3 facteurs de transcription clés de la cardiogénèse (Gata4, Mef2c et Tbx5). Cette approche moléculaire est renforcée par la combinaison avec une stimulation pharmacologique ciblant la voie Wnt
The general objective of this work was centered on the use of human induced pluripotent cells in modeling and therapeutic evaluation of cardiac pathologies. Since their discovery in 2006, the iPSC provide an opportunity for the development of human cellular models and specific patients for the study of pathophysiological mechanisms, evaluation of pharmacological responses and the generation redifférenciées cells (cardiomyocytes here) for applications cellular therapeutic. In this work we demonstrated that the quantity but also the final quality of cardiomyocytes derived from iPSC depends on the spatial and pharmacological conditions used during the various stages of differentiation. The use of a monolayer differentiation protocol with simultaneous and transient blocking of all Wnt pathways (canonical and noncanonical) allows to obtain a higher maturation of the sarcomere, an essential step for modeling sarcomeropathies IPSC differentiation into cardiomyocytes can also be obtained by targeted molecular approach to specifically activate cardiogenic program. This is achieved through the use of a mutated Cas9 protein and coupled with transactivator system. This allows simultaneous targeting of 3 key cardiogenesis transcription factors (Gata4, MEF2C and Tbx5). This molecular approach is enhanced by the combination with a pharmacological stimulation targeting the Wnt pathway. Beyond modeling of monogenic cardiac disease, cardiomyocytes derived from iPSC can reproduce more complex and multigenic diseases

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

Durant ma thèse, j’ai étudié les bases génétiques de la cardiogenèse fonctionnelle en utilisant le cœur de la drosophile comme modèle. J’ai d’abord montré que la diversité cellulaire cardiaque dépend de l’activité des gènes homéotiques Ubx et abdA qui orientent notamment le choix du lignage cellulaire cardiaque. Les gènes Hox sont ensuite requis pour orchestrer la différenciation terminale du cœur de la mouche. J’ai identifié cinq cibles d’abdA, dont le canal ionique Ork1. J’ai notamment montré que Ork1 est un ‘pur' régulateur du rythme cardiaque. Enfin, j’ai montré que le système cardiaque larvaire est remodelé pour former le cœur de la mouche adulte. Ce processus dépend d’une modification de l’activité des gènes Hox induite par l’hormone stéroïde ecdysone. Ainsi, mes résultats présentent les gènes Hox comme des régulateurs majeurs des étapes majeures de la cardiogenèse chez la drosophile et établissent un nouveau système modèle pour l’analyser leur fonction
During my PhD, I have addressed the genetic basis of functional cardiogenesis, using the Drosophila heart as a model system. I have first shown that cardiac cell diversity is autonomously insured by the activity of the Hox genes Ubx and abdA, which notably direct the choice between specific cardiac cell lineages. Hox genes are then required to orchestrate terminal differentiation in the fly heart. I have identified five abdA targets, including the ion channel Ork1. Notably, I have shown that Ork1 is a pure regulator of heart rhythm. In a third work, I have shown that the larval heart is remodeled to form the adult one. This process is driven by an ecdysone-dependent modification of Hox activity. Together, my results identify Hox genes as master regulators controlling main steps of cardiogenesis in Drosophila, and establish a new model system to analyse their function

Un grand nombre de cardiomyopathies affectent l'organogenèse du muscle cardiaque soulignant l’importance d'approfondir nos connaissances de la cardiogenèse. Mon projet de thèse a consisté, par l’analyse de l’expression des gènes à grande échelle et l’identification in silico de séquences régulatrices putatives, en la caractérisation des mécanismes moléculaires contrôlant l’organogenèse du coeur chez la drosophile. J’ai, dans un premier temps, analyser la dynamique du transcriptome à 8 temps successifs au cours du remodelage du tube cardiaque larvaire en coeur adulte. L’analyse fonctionnelle des données de puces à ADN m’a permis de dresser une véritable cinétique transcriptionnelle et de mettre en évidence précisément les voies de signalisation potentiellement impliquées dans ce processus. Par une étude génétique de ces voies de signalisation identifiées, je montre d’une part que la voie de signalisation Wnt est nécessaire à la reprogrammation des myocytes cardiaques, et d’autre part que l’activation de la voie EGF-PDGF est requise pour la formation des valves cardiaques adultes. Dans un second temps, j’ai analysé l’expression différentielle des gènes entre l’aorte larvaire et le coeur larvaire, et identifié une quinzaine de gènes partageant spécifiquement le domaine d’expression du gène homéotique abd-A dans le coeur. Sous l’hypothèse que ces gènes soient régulés directement par abd-A et d’autres facteurs de transcription en commun, j’ai utilisé à la fois la technique de l’empreinte phylogénétique et une méthode de recherche de regroupement de motifs pour identifier les modules cis-régulateurs putatifs responsables de leur co-expression. Trois enhancers se sont révélés être positifs pour récapituler une expression spécifique dans le coeur, et une analyse plus approfondie de ces séquences devrait nous aider à déterminer si les gènes correspondants sont des cibles directes d’abd-A
A number of inherited cardiomyopathies affect cardiac muscle organogenesis emphasizing the need to improve our knowledge of heart formation. My thesis project concerned, by whole genome analysis and in silico identification of putative cis-regulatory sequences, the characterization of molecular mechanisms controlling heart organogenesis in drosophila. I have first drawn a molecular portrait of adult heart morphogenesis by whole-genome expression profiling at 8 successive time-points. This transcriptional map pointed out specific signalling pathways as potential players in the process. Phenotypic analysis confirmed they are involved in discrete steps of the remodelling. In particular, the Wnt signalling pathway is involved in cardiac myocyte trans-differentiation, while activation of the VEGF-PDGF pathway is required for adult cardiac valve formation. In a second work, I analyzed the differential expression between the aorta and the heart regions in larvae, and identified a dozen of genes that share the same expression pattern of the homeotic gene abd-A in the heart. As these genes are likely to be regulated by abd-A and other common transcription factors, I used a bioinformatic approach based both on the phylogenetic footprinting method and the research of motif clusters to identify the putative cis-regulatory modules responsible of their co-expression. Three enhancers have been found as positive to drive a specific expression in the heart part, and a more detailed analysis of these sequences should help us determine if the corresponding genes are direct targets of abd-A

Abstract Type XIII collagen is a type II oriented transmembrane protein with a short intracellular domain, a single transmembrane domain and a large, mostly collagenous extracellular domain. Tissue localization and cell culture studies have implicated that it is involved in cell adhesion. The spatio-temporal expression of type XIII collagen mRNA and protein during murine development is studied here. Type XIII collagen mRNAs were expressed at a constant rate during development, with an increase of expression towards birth. The strongest expression was detected in the central and peripheral nervous systems of the developing mouse fetus. Cultured primary neurons expressed this collagen, and recombinant type XIII collagen was found to enhance neurite outgrowth. Strong expression was also detected in the heart, with localization to cell-cell contacts and perinatal accentuation in the intercalated discs. Other sites of type XIII collagen expression included cartilage, bone, skeletal muscle, lung, intestine and skin. Clear developmental shifts in expression suggest a role in endochondral ossification of bone and the branching morphogenesis in the lung. To elucidate the function of type XIII collagen transgenic mice were generated by microinjection of a cDNA construct that directs the synthesis of truncated α1(XIII) chains with an in-frame deletion of the central collagenous COL2 domain. This construct was thought to disrupt the assembly of normal type XIII collagen trimers. Expression of shortened α1(XIII) chains by fibroblasts derived from mutant mice was demonstrated, and the lack of intracellular accumulation in immunohistochemical analysis of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to developmental arrest and fetal mortality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a failure in the fusion of the chorion and allantois membranes and subsequent disruption in placentation, while the late phenotype fetuses were aborted by day 13.5 of development due to cardiovascular and placental defects. Furthermore, it was shown that the heterozygous mice that were initially of normal appearance and bred normally had an increased susceptibility to develop T-cell lymphomas and angiosarcomas later in life. The results presented here increase the evidence that type XIII collagen is involved in cell adhesion, with several important tasks during development. A role of type XIII collagen in malignant transformation of certain mesenchymal cell populations is also implicated.

Abstract Type XIII collagen is a type II transmembrane protein which is expressed in many tissues throughout development and adult life. It is located in focal adhesions of cultured cells and in the adhesive structures of tissues such as the myotendinous junctions in muscle, intercalated discs in the heart and the cell-basement membrane interphases. To further characterize the function of this protein, we generated transgenic mice overexpressing it in normal and mutant forms. A large in-frame deletion in the COL2 domain of type XIII collagen led to synthesis of truncated α1(XIII) chains in transgenic mice, disrupting the assembly of normal type XIII collagen trimers. Fibroblasts derived from the mutant mice expressed shortened α1(XIII) chains, and no intracellular accumulation of the mutant protein was detected, suggesting that the mutant molecules were expressed on the cell surface. Transgene expression led to an embryonally lethal phenotype in offspring from heterozygous mating at two distinct stages of development. The early phenotype fetuses died due to the lack of chorioallantoic fusion and functioning placenta at 10.5 dpc, while the death of the late phenotype fetuses was caused by cardiac and placental defects around 13.5 dpc. The phenotype resembles closely several other cell adhesion molecule mutants, indicating that type XIII collagen has an essential role in certain adhesive interactions that are necessary for normal development. Mice overexpressing type XIII collagen with or without a point mutation developed postnatally an unexpected skeletal phenotype marked by a massive increase in bone mass. The cortical bone cross-sectional area and volumetric bone mineral density were highly increased, but trabecular bone volume was not significantly altered. The bone formation rate was several times higher in the mutant mice than in their normal littermates, while the osteoclast number and resorption activity were normal. Type XIII collagen was expressed highly in primary osteoblasts derived from the transgenic mice. Overexpression of type XIII collagen in osteoblasts enhanced both cell proliferation and differentiation while lack of it had opposite effects. Furthermore, mutant cells responded to mechanical strain differently than wild-type cells. The findings suggest that type XIII collagen has an important role in bone modeling, and it may in particular have a function in coupling the regulation of bone mass to mechanical usage.

Das Herz ist das erste Organ, das sich während der Embryonalentwicklung bildet und durch die Verteilung von Nährstoffen und Sauerstoff für die Lebenserhaltung von Geweben und Organen verantwortlich ist. Die Herzentwicklung benötigt die koordinierte Rekrutierung von zwei Herzvorläufer-Populationen, dem ersten und zweiten Herzfeld, welche sich aus einer gemeinsamen Vorläuferzellpopulation während der Gastrulation bilden. In der vorliegenden Arbeit wurde der Einfluss der Bmp- und Wnt-Signalwege auf die frühe Herzentwicklung in Mäusen untersucht. Dafür wurden mit Hilfe der Cre/LoxP-Technik inaktivierende und aktivierende Mutationen im Bmp-Rezeptor Ia (BmpRIa) und im zentralen Modulator des Wnt-Signalweges, beta-Catenin, in Zellen des Mesoderms eingeführt, aus dem beide Herzfelder hervorgehen. Inaktivierende Mutationen im BmpRIa führen zum Verlust von erster Herzfeldderivate und zum Expressionsverlust von Genen, welche für die Aufrechterhaltung und Spezialisierung des ersten Herzfeldes in den späteren linken Ventrikel wichtig sind. In Mäusen mit inaktivierenden Mutationen in beta-Catenin bildet sich das erste Herzfeld korrekt, während die Entwicklung des zweiten Herzfeldes, z.B. die rechtsgerichtete Windung des linearen Herzrohres sowie Bildung des Ausflusstrakts und rechten Ventrikels, gestört ist. Die Genexpression von Bmp4 und Islet1 in Vorläufern des zweiten Herzfeldes ist stark reduziert, während aktivierende Mutationen in beta-Catenin diese verstärken und die Bildung des linearen Herzrohres stören. Diese Ergebnisse zeigen, dass beta-Catenin für die Entwicklung des zweiten Herzfeldes entscheidend ist, und dass die Aktivierung des Wnt/beta-Catenin-Signalweges zeitlich und räumlich präzise reguliert werden muss, damit sich ein windendes lineares Herzrohr entwickeln kann. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die BmpRIa- und Wnt/beta-Catenin-Signalwege unterschiedliche Rollen während der Musterbildung in der frühen Herzentwicklung spielen.
The vertebrate heart is the first organ that forms during embryonic development. Heart formation requires the coordinated recruitment of multiple cardiac progenitor cell populations derived from both the first and second heart fields, which arise from a common progenitor at gastrulation. In this study we have ablated the Bmp receptor 1a (BmpRIa) and the Wnt effector beta-Catenin in the developing heart of mice using MesP1-cre, which acts in early mesoderm progenitors that contribute to both first and second heart fields. Remarkably, the entire cardiac crescent and later the primitive ventricle were absent in MesP1-cre; BmpR1a loss-of-function mutants. While myocardial progenitor and differentiation markers were detected in the small, remaining cardiac field in these mutants, first heart field markers, which are required for the maintenance and specification of first heart field derivatives, were not expressed. We conclude from these results that Bmp receptor signaling is crucial for the specification of the first heart field. In MesP1-cre; beta-Catenin loss-of-function mutants, cardiac crescent formation as well as first heart field markers were not affected, although cardiac looping and right ventricle formation were blocked. Expression of Isl1 and Bmp4 in second heart field progenitors was strongly reduced. In contrast, in gain-of-function mutation of beta-Catenin using MesP1-cre we revealed an expansion of Isl1 and Bmp4 expressing cells, although the heart tube was not formed. We conclude from these results that Wnt/beta-Catenin signaling regulates second heart field development, and that a precise amount and/or timing of Wnt/beta-Catenin signaling is required for proper heart tube formation and cardiac looping. In conclusion, we have shown that Bmp and canonical Wnt signaling have distinct roles during early cardiogenesis in mice.

L’environnement in utero, dont le statut en vitamine D, est un facteur crucial pour assurer le développement normal du fœtus, mais il participe également à la susceptibilité à développer des maladies métaboliques et cardiaques tout au long de la vie. Cette thèse a pour objectif d’étudier les interactions existantes entre la carence maternelle en vitamine D (VDD) et la programmation du devenir cardio-métabolique de la descendance. D’abord, chez la descendance juvénile, l’homéostasie énergétique et le poids de la descendance issue de mères VDD étaient altérés de manière sexe-dépendant. A l’âge adulte, une alimentation obésogène combinée à la VDD maternelle, altérait l’homéostasie glucidique et l’adiposité de la descendance mâle mais pas des femelles. De telles phénotypes été associés à des profils transcriptomiques différentiels dans le tissu adipeux, qui pourraient être associés à une modulation différentielle des taux circulants d’estradiol chez les femelles. La VDD maternelle module donc le devenir métabolique de la descendance, dans des proportions amplifiées, lorsque celle-ci est exposée à une alimentation obésogène au cours de sa vie. Ensuite, nous avons étudié l’impact de la VDD maternelle sur le devenir cardiaque de la descendance. Chez les embryons, la VDD maternelle induisait une hypertrophie ventriculaire gauche, modulait leur transcriptome cardiaque et de telles modifications semblait être liées à une modulation de la structure chromatinienne. La morphologie et le fonctionnement du cœur, étaient également altérés chez la descendance adulte. La VDD maternelle altère donc le développement cardiaque du fœtus et induit des altérations jusqu’à l’âge adulte
In utero environment, including vitamin D status, is crucial to ensure normal development of the foetus and to prevent any metabolic and cardiac diseases throughout the whole life. The aim of this thesis is to highlight the interactions existing between maternal vitamin D deficiency (VDD) and the potential programming of cardio-metabolic fate of the offspring. First, for the juvenile offspring, the energetic homeostasis and the weight of the offspring from deficient mother were sex-dependently altered. In adulthood, an obesogenic diet combined with maternal VDD, disrupted glucose homeostasis and adiposity in male offspring but not in females. Such phenotypes were associated to different transcriptomic profiles in adipose tissue, that could be related to differential modulation of circulating levels of estradiol in females. The maternal VDD modulates metabolic fate of the offspring, in exacerbated proportions, when the offspring was exposed to obesogenic diet during adulthood. Then, we studied the impact of maternal VDD on the cardiac fate of offspring. In embryos maternal VDD induced left ventricular hypertrophy, modulated their cardiac transcriptome and such modifications seemed to be related to the modulation of chromatin structure. Also, the morphology and cardiac function were altered in the adult offspring. Maternal VDD impairs the cardiac development of the foetus and programs cardiac outcomes in adulthood

Les malalties cardiovasculars són una de les majors causes de mortalitat a escala mundial. L’infart de miocardi és el principal responsable de les cardiopaties isquèmiques. La irrigació sanguínia al cor es veu bloquejada degut a una oclusió en un capil•lar sanguini provocant mort cel•lular massiva que genera una zona miocardíaca necròtica. En la última dècada, la medicina cardíaca regenerativa s’ha focalitzat en estratègies fonamentades en l’enginyeria de teixits i la teràpia cel•lular basada en cèl•lules mare. En aquest treball, hem caracteritzat el potencial cardíac de diferents tipus cel•lulars cultivats en bastides tri-dimensionals (3D) generades a partir de l’hidrogel peptídic RAD16-I. En primer lloc, hem estudiat l’adquisició de potencial mesenquimàtic de fibroblasts humans de dermis (hNDFs) en cultius 3D i la seva diferenciació subseqüent a llinatges adipogènic i cardiogènic. Únicament els hNDFs cultivats en hidrogels de RAD16-I adquireixen una potenciació mesenquimàtica. Les cèl•lules adopten espontàniament propietats semblants a les cèl•lules mare mesenquimàtiques mentre que la diferenciació a adipogènesis i cardiogènesis requereix medi d’inducció. En segon lloc, hem comparat el grau de diferenciació cardíaca de cèl•lules mare humanes pluripotents induïdes (hiPSCs) cultivades en ambients 2D versus 3D i hem avaluat l’efecte de l’àcid ascòrbic (AA) en el procés. En el nostre treball i com ja s’havia demostrat en publicacions prèvies, l’AA va resultar accelerar i millorar la diferenciació cardíaca de hiPSCs en cultius 2D. A més, els resultats presentats suggereixen que les hiPSCs cultivades en 3D augmenten el seu grau de diferenciació i adquireixen un potencial cardiogènic 105 vegades més elevat que en els cultius 2D. En tercer lloc, hem dissenyat un pegat cardíac basat en cultius 3D de cèl•lules adultes porcines progenitores del teixit adipós del mediastí (pMATPCs) injectats en matrius naturals (pericardi humà descel•lularitzat). Hem implantat la bio-pròtesis miocardíaca in vivo i hem determinat que la bio-bastida afavoreix la migració cel•lular i la regeneració de la zona infartada en el model porcí. En conclusió, hem analitzat el potencial cardiogènic de cèl•lules adultes somàtiques (hNDFs), cèl•lules mare adultes (pMATPCs) i cèl•lules mare pluripotents (hiPSCs) en cultius 3D basats en hidrogels de RAD16-I per a futures aplicacions en el tractament de malalties cardíaques.
Las enfermedades cardiovasculares son una de las mayores causas de mortalidad a escala mundial. El infarto de miocardio es el principal responsable de las cardiopatías isquémicas. La irrigación sanguínea al corazón se ve bloqueada debido a una oclusión en un capilar sanguíneo provocando muerte celular masiva que genera una zona miocárdica necrótica. En la última década, la medicina cardíaca regenerativa se ha focalizado en estrategias fundamentadas en la ingeniería de tejidos y la terapia celular basada en células madre. Es este trabajo, hemos caracterizado el potencial cardíaco de distintos tipos celulares cultivados en andamios tridimensionales (3D) generados a partir del hidrogel peptídico RAD16-I. En primer lugar, hemos estudiado la adquisición de potencial mesenquimático de fibroblastos humanos de dermis (hNDFs) en cultivos 3D y su diferenciación subsecuente a linajes adipogénico y cardiogénico. Únicamente los hNDFs cultivados en hidrogeles de RAD16-I adquieren una potenciación mesenquimática. Las células adoptan espontáneamente propiedades parecidas a las células madre mesenquimáticas mientras que la diferenciación a adipogénesis y cardiogénesis requiere medio de inducción. En segundo lugar, hemos comparado el grado de diferenciación cardíaca de células madre humanas pluripotentes inducidas (hiPSCs) cultivadas en ambientes 2D versus 3D y hemos evaluado el efecto del ácido ascórbico (AA) en el proceso. En nuestro trabajo y como ya se había demostrado en publicaciones previas, el AA resultó acelerar y mejorar la diferenciación cardíaca de hiPSCs en cultivos 2D. A demás, los resultados presentados sugieren que las hiPSCs cultivadas en 3D aumentan su grado de diferenciación y adquieren un potencial cardiogénico 105 veces más elevado que en los cultivos 2D. En tercer lugar, hemos diseñado un parche cardíaco basado en cultivos 3D de células adultas porcinas progenitoras del tejido adiposo del mediastino (pMATPCs) inyectados en matrices naturales (pericardio humano descelularizado). Hemos implantado la bio-prótesis miocárdica in vivo y hemos determinado que el bio-andamio favorece la migración celular y la regeneración de la zona infartada en el modelo porcino. En conclusión, hemos analizado el potencial cardiogénico de células adultas somáticas (hNDFs), células madre adultas (pMATPCs) y células madre pluripotentes (hiPSCs) en cultivos 3D basados en hidrogeles de RAD16-I para futuras aplicaciones en el tratamiento de enfermedades cardíacas.
Cardiac failure is the primary cause of mortality throughout the world. One of the leading causes of heart failure is myocardial infarction, which results from a reduced flow of blood to a part of the heart. This leads to cardiomyocyte death and myocardial necrosis. In the past decade, various strategies for cardiac reparative medicine have been investigated, from tissue engineering to stem cell-based therapy. Herein, we characterized the cardiac potential of different cell types cultured in three-dimensional (3D) scaffolds based on the peptide hydrogel RAD16-I. Firstly, we studied the mesenchymal potential acquisition of human Normal Dermal Fibroblasts (hNDFs) in 3D cultures and further commitment into adipogenic and cardiogenic lineages. We suggest that only hNDFs cultured in RAD16-I hydrogels undergo a mesenchymal potentiation. Cells spontaneously acquired mesenchymal stem cell-like properties whereas they required induction media to differentiate into adipogenic- and cardiogenic-like lineages. Secondly, we compared the degree of cardiac commitment of human induced Pluripotent Stem Cells (hiPSCs) when cultured in 2D versus 3D and the effect of ascorbic acid (AA), which has been proven to promote cardiac differentiation, on the process. In fact, AA seemed to accelerate and improve the cardiac commitment of hiPSCs in 2D cultures. Results suggested that hiPSCs in 3D cultures displayed an increased level of differentiation and acquired 105-fold more cardiogenic potential than cells cultured in 2D. Thirdly, we designed a cardiac patch based on 3D cultures of adult porcine Mediastinal Adipose Tissue Progenitor Cells (pMATPCs) injected into natural matrices (decellularized human pericardium). We implanted the myocardial bioprosthesis in vivo and determined that the bioscaffold supported cell migration and regeneration into the infarcted area in swine. In summary, we studied the cardiogenic potential of adult somatic cells (hNDFs), adult stem cells (pMATPCs) and pluripotent stem cells (hiPSCs) in 3D cultures based on RAD16-I hydrogels for potential future applications in the treatment of heart disease.

Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra
Heart formation involves the participation of various signaling pathways that crosstalk in a temporal and context–dependent manner. The molecular events taking place from pre-gastrulation up to formation of cardiomyocytes are recapitulated in vitro by differentiating mouse embryonic stem (mES) cells. Importantly, by closely following the kinetics of cell fate decisions occurring in the embryo, ES cells facilitate mechanistic studies aimed at the dissection of early lineage specification. Following previous findings demonstrating the role of the Notch pathway in specifying a cardiac fate from mesodermal progenitors and hemangioblasts1, our laboratory identified a novel function for Hes5, as a downstream effector of Notch1, at the onset of cardiogenesis (Freire, AG et al, unpublished). Loss and gain of function studies unveiled that Hes5 instructs ES cell-derived mesodermal progenitors to commit preferentially towards cardiac over a hematopoietic fate, in part by regulating the early cardiac transcription factor, Isl1. Interestingly, a short-pulsed Hes5 induction enhances cardiac specification, whereas a sustained activation impairs the emergence of contracting colonies. The herein Thesis aimed to further dissect the role of Hes5 in cardiogenesis. To this end, we proposed to understand the role of this bHLH regulator at different stages of the cardiomyocytic program. Given the robustness of the mES cell in vitro model system for cardiac differentiation, a mES cell line expressing exogenous Hes5 under the control of a Doxycycline (Dox)-inducible promoter was used. The data indicated that Hes5 expression maintains an undifferentiated cardiac progenitor state, in part by sustaining high Isl1 levels. These results demonstrated that after induction of cardiac fate, Hes5 withdrawal is required to allow cardiac differentiation, suggesting a confined transient temporal window for Hes5 participation in cardiogenesis. A second aim of this Thesis was the characterization of endogenous Hes5 expression during mES cell differentiation towards mesodermal derivatives, in a more close to physiological system. Interestingly, Hes5 levels upregulated from day 4 to day 6 of in vitro differentiation, correlating to the temporal window identified for enhanced cardiac differentiation induced by transient exogenous Hes5 overexpression. x Finally, aiming at the validation of Hes5 role in specifying cardiac fate in the developing mouse embryo, Hes5 expression was assessed in E6.5 and E7.5 mouse embryos. These results report for the first time Hes5 expression in the nascent mesoderm of gastrulating E6.5 embryos. Interestingly, Hes5 expression was not found or dramatically reduced in E7.5 embryos, further corroborating a transient role for Hes5 at the onset of cardiogenesis. Overall, the work performed in the frame of the herein Thesis contributed (i) a better understanding of Hes5 role at different stages of the cardiomyocytic differentiation program (i.e. at the specification of cardiac progenitors and during differentiation into cardiomyocytes), (ii) the indication that endogenous Hes5 is upregulated at the time cardiac progenitors are specified during in vitro mES cell differentiation, and (iii) the first report of Hes5 expression in the nascent mesoderm of E6.5 gastrulating embryos.
A formação do coração envolve a participação de várias vias de sinalização que interagem entre si de um modo tempo- e contexto-dependente. Os eventos moleculares que ocorrem desde a pré-gastrulação até à formação de cardiomiócitos são recapitulados in vitro através da diferenciação de células estaminais embrionárias (CEEs). Importante ainda é que, ao acompanharem de perto a cinética das decisões de destino celular que ocorrem no embrião, as EECs facilitam a execução de estudos mecanísticos com o objectivo de dissecar a especificação das linhagens celulares. Dado o papel, previamente demonstrado, da via de sinalização Notch na especificação de um destino cardíaco a partir de progenitores da mesoderme e hemangioblastos, o nosso laboratório identificou uma nova função para o Hes5, como efector do Notch1, na indução da cardiogénese (Freire, AG et al, não publicado). Estudos de perda e ganho de função revelaram que o Hes5, em parte por regular os níveis do factor de transcrição Isl1, determina uma decisão preferencial pela diferenciação em linhagens cardíacas em detrimento de hematopoiéticas nos progenitores da mesoderme derivados de CEEs. Interessantemente, a indução de um pulso curto de Hes5 aumenta a especificação cardíaca, enquanto que uma activação contínua diminui o aparecimento de colónias a contrair. Esta dissertação de Mestrado teve como objectivo dissecar o papel do Hes5 na cardiogénese. Com este propósito, propusemos compreender o papel deste factor de transcrição em diferentes etapas do programa de diferenciação cardiomiocítico. Dada a robustez do modelo in vitro de diferenciação de CEEs em células cardíacas, foi utilizada uma linha celular estaminal embrionária que expressa Hes5 exógeno sob o controlo de um promotor indutível de Doxiciclina (Dox). Os dados obtidos indicam que a expressão de Hes5 mantém as células num estado de progenitores cardíacos indiferenciados, em parte por manter os níveis de Isl1 também elevados. Estes resultados demonstraram que após a indução de um destino cardíaco é necessária uma diminuição de expressão de Hes5 para permitir diferenciação cardíaca, sugerindo que a participação do Hes5 na cardiogénese ocorre numa janela temporal transiente. xii Um segundo objectivo desta dissertação incluiu a caracterização da expressão de Hes5 endógeno durante a diferenciação in vitro de derivados da mesoderme, num sistema mais próximo do fisiológico. Interessantemente, os níveis de Hes5 aumentam desde o dia 4 até ao dia 6 de diferenciação in vitro, o que correlaciona com a janela temporal previamente identificada por indução de expressão de Hes5 exógeno na qual o Hes5 aumenta a diferenciaçao cardíaca. Finalmente, a expressão de Hes5 foi avaliada em embriões de murganho com 6,5 e 7,5 dias com o objectivo de validar o papel do Hes5 na especificação de um destino cardíaco no desenvolvimento embrionário de murganho. Estes resultados descrevem pela primeira vez expressão de Hes5 na mesoderme nascente de embriões com 6,5 dias. Importante ainda, é o facto de a expressão de Hes5 não ter sido detectada, ou estar dramaticamente reduzida em embriões com 7,5 dias, corroborando o papel transiente do Hes5 na indução da cardiogénese. Em resumo, o trabalho realizado no âmbito da presente dissertação contribuiu (i) um melhor conhecimento do papel do Hes5 em diferentes etapas do programa de diferenciação cardiomiocítio (i.e. na especificação de progenitores cardíacos e durante a diferenciação em cardiomiócitos), (ii) a indicação que a expressão de Hes5 endógeno está aumentada durante o tempo de especificação dos progenitores cardíacos durante a diferenciação in vitro de CEEs, e (iii) uma primeira descrição de expressão de Hes5 na mesoderme nascente em embriões com 6,5 dias.
PTDC/SAL-ORG/118297/2010
Pest-C/SAL/LA0002/2011
Pest-C/SAL/LA0002/2013

博士
國立清華大學
生物科技研究所
104
Abstract 　　Heart disease is the leading cause of human death in the 21st century. Heart transplantation is a promising way to treat this. Because donor resources are limited, cell-based therapy has been developed as an alternative. Therefore, genes that trigger cardiogenesis could have potential in the treatment of heart disease. FGF1 is reported to stimulate cardiomyocyte proliferation under conditions of myocardial infarction; but little is known about its function during cardiac differentiation. In this study, we established an in vitro cardiogenesis model through a reliable chemical induction protocol to determine whether FGF1 and its gene expression are involved in cardiogenesis. Oxytocin, a well-known hormone but also a cardiac differentiation inducer, was used in a mouse embryonic stem cell line E14Tg2a to achieve cardiac differentiation. After differentiation, beating cell clusters appeared and the expression of FGF1B mRNA was upregulated in the late differentiation stage (differentiation days 8–14). Interestingly, FGF1B expression patterns during cardiac differentiation were similar to those of a mature cardiomyocyte marker, troponin T2, cardiac. The blockage of FGF1-FGFR signaling reduced not only the appearance of beating cluster formation but also the expression levels of cardiomyocyte-associated genes. Moreover, by investigating FGF1 downstream signaling cascades, we observed that the efficiency of beating cluster formation was mainly regulated via the FGF1-FGFR-PKC signaling axis. Taken together, we provide evidence to support that FGF1 could regulate cardiogenesis primarily through the PKC signaling, but not through the MAPK signaling pathway.

碩士
國立臺灣大學
分子與細胞生物學研究所
93
Glycogen synthase kinase 3β (GSK3β) encodes a multifunctional serine/threonine protein kinase, which is ubiquitously expressed in organisms. Although GSK3β is known to play roles in many biological processes, including cell survival, tumorigenesis and developmental patterning, it remains unclear that the function of GSK3β in cardiogenesis in vivo. We used a GFP-tagged heart transgenic zebrafish to address this question. In order to specifically inhibit GSK3β, gsk3β antisense morpholino oligonucleotide (gsk3β-MO) was injected into one-celled embryos to block the translation of the gsk3β mRNA. In gsk3β-MO -injected embryos, we found that heart precursor cells lined up at the midline at 24 hpf, failed to complete the heart positioning, and then stretched slowly to a thin, ‘string-like’ shape about 96 hpf. In addition, in this morphants, the heart rate was slower, the contraction was weaker, and pericardial edema was commonly observed. Knowdown of GSK3β resulted in a severe disruption of early (jogging) and late (looping) aspects of cardiac left-right asymmetry. The degree of cardiac defects due to GSK3β attenuation was dose-dependent. But these defectives could be rescued by injecting synthetic gsk3β mRNA. Consistent with the morphological change of heart, the expression of bmp4, a heart-asymmetry marker and a target of Wnt/β-catenin signaling pathway, was upregulated in GSK3β morphants: the asymmetry of heart was completely disrupted. Interestingly, we found that cardiac defects happened in the gsk3β-MO-injected embryos were similar to those observed in axin1 morphants. Therefore, our findings strongly suggest that GSK3β plays a role in L/R-biased heart positioning through Wnt/β-catenin pathway during zebrafish cardiogenesis.

Dissertação mest., Ciências Biomédicas, Universidade do Algarve, 2009
The heart is the first organ that becomes functional in the vertebrate embryo. Heart morphogenesis is a complex process, with precise control developmental mechanisms, that can nevertheless fail. There are morphological aspects as polarity of the heart intrinsically related with the three body axes, anterior?posterior (A-P), dorsal?ventral (D-V), and left?right (L-R). The L-R axis has became subject of many studies in recent years and was found that the heart undergoes multiple morphogenetic processes, which are governed by this axis. Development of internal organs proceeds across the L-R axis and gain shape during organogenesis as a result of the early asymmetric activation of the conserved Nodal signalling cascade, in the left lateral plate mesoderm (Hamada et al., 2002). A Cerberus/Dan family member, mouse cerberus-like2 (cerl-2) is asymmetrically expressed on the right side of the mouse node and encodes for a secreted protein that binds directly to nodal restricting the Nodal signalling pathway towards the left side by preventing its activity in the right side (Marques et al, 2004). Preliminary studies showed that cerl-2 knockout (KO) mice display multiple laterality defects including heart?s rotation failure and randomization of organs? position due to L/R axis disruption. In addition, was observed several cardiac defects as severe hyperplasia of the myocardium and incomplete atria formation and ventricular septation that may not be explained by laterality abnormalities. In this study, were conducted morphological analyses of cerl-2 KO newborns, histological sections of newborn hearts and WISH with Gata-4, mefc2, hand and fgf8 probes on embryos throughout heart development (7,5dpc to 10.5dpc). Furthermore, a new compound mouse line cerl- 2KO::mlc1vlacZ was generated which will help to identify the contribution of the Secondary Heart Field (SHF) to the cerl-2KO heart defects. This body of work leads to the suggestion that, in addition to the previously described laterality-related defects, another distinct mechanism may contribute to the spectrum of complex cardiac defects in cerl-2 KO mice that cannot be explained only by the disruption of the nodal cascade in LPM. Problems in heart morphogenesis lead to congenital heart disease, which is the most common form of birth defect in humans (Harvey, 2002; Olson and Schenider, 2003). Abstract

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.
Sendo o coração o primeiro órgão a ser formado, é também o primeiro a funcionar com o objectivo de suprir as necessidades do embrião vertebrado. No entanto, a morfogénese cardíaca é bastante complexa, e perturbações podem originar doenças cardíacas congénitas, sendo os defeitos com maior incidência à nascença. Com o objectivo de melhor compreender a intrincada base molecular que controla a formação e o desenvolvimento deste órgão e assim promover a união entre prevenção e tratamento, é de importância fundamental aprofundar os conhecimentos nesta área de investigação. Apesar das óbvias diferenças entre o humano e o ratinho, o coração nos dois modelos animais é constituído por quatro câmaras (dois átrios e dois ventrículos) e os respectivos funcionamentos são semelhante tendo contudo algumas ressalvas ao serem equiparados. Além disto, 99% dos genes no ratinho têm equivalentes em humanos, logo o ratinho é considerado um ótimo modelo para o estudo de doenças humanas. Durante o desenvolvimento, o coração recebe informação para se posicionar ao longo dos três eixos do corpo. É o terceiro eixo, o esquerdo-direito (E/D), que determina a direção de rotação do coração e que portanto influencia a sua morfologia final, como exemplo, ventrículo e átrio esquerdo posicionados no lado esquerdo do embrião. Embora já se saiba através de recentes estudos em peixe-zebra como o sinal assimétrico do eixo E/D direciona a rotação cardíaca, ainda é desconhecido a influência deste eixo no posicionamento dos outros órgãos viscerais. Nodal, membro da família de factores de crescimento TGF-ß, tem sido reconhecido como um elemento chave durante o estabelecimento do eixo E/D e do desenvolvimento do embrião sendo especialmente requerido para a formação da mesoderme. O membro da família Cerberus/Dan, mouse cerberus-like2 (Cerl2), é expresso assimetricamente no lado direito do nó em ratinhos e codifica a proteína que uma vez secretada é capaz de se ligar diretamente a Nodal. Dessa maneira, o antagonismo entre Cerl2 e Nodal é essencial para restringir assimetricamente a atividade do Nodal para o lado esquerdo do nó levando a indução da sua cascata de sinalização na placa lateral da mesoderme esquerda. Esta restrição assimétrica para o lado esquerdo do embrião vai determinar a orientação assimétrica dos órgãos. Em ratinhos knock-out para o gene Cerl2 (Cerl2-/-) foi demonstrado que o gene Nodal também pode ser expresso no lado direito do nó levando a indução da cascata de sinalização Nodal na placa lateral da mesoderme direita. Como consequência os ratinhos Cerl2-/- manifestam uma variedade de defeitos de lateralidade (DL) conhecidos como situs inversus, isomerismo e heterotaxia. Adicionalmente estão associadas aos DL malformações cardiovasculares, sendo as mais comuns rotação alterada do eixo cardíaco, defeitos no septo ventricular e atrial, bem como alterações na rotação e diferenciação das artérias, estas últimas conhecidas como transposição das grandes artérias, tronco arterioso comum, dupla saída do ventrículo direito e dupla via de saída do ventrículo esquerdo. Assim, estes fenótipos podem ser a causa de morte nos primeiros dias de vida dos animais Cerl2 mutantes. Adicionalmente, resultados preliminares revelaram um novo fenótipo nos Cerl2-/- recém-nascidos caracterizado pelo aumento da espessura das paredes do ventrículo esquerdo ao qual nestes animais não foram associados defeitos de lateralidade. De forma a caracterizar detalhadamente este fenótipo e identificar o papel do Cerl2 durante a formação do coração, procedeu-se à análise do fenótipo cardíaco em embriões e em ratinhos no começo da vida pós-natal 0 (P0). Com os dados apresentados neste estudo, demonstrou-se que o aumento da espessura do miocárdio no ventrículo esquerdo (VE) e no septo interventricular (SIV) em Cerl2 mutantes sem DL é causado pela hiperplasia dos cardiomiócitos no ventrículo esquerdo. Além disso, nos mutantes de Cerl2 o aumento da expressão relativa de ciclina D1 no VE foi detectada no estádio embrionário 13 (E13). Este resultado pode estar relacionado com a expressão específica de Cerl2 no VE, tal como detectada em animais controlo, indicando assim um possível papel regulatório do Cerl2 durante a formação do coração e mais especificamente, do ventrículo esquerdo. Além disso, os ratinhos Cerl2-/- apresentaram expressão alterada de genes cardíacos durante o estádio embrionário e nas primeiras horas após o nascimento, o que é incompatível com a função cardíaca normal também confirmada pela redução da função sistólica em ratinhos neonatais de Cerl2-/-. Para além de investigarmos o mecanismo celular responsável pelo aumento da massa ventricular esquerda, também sugerimos dois mecanismos moleculares pelos quais o Cerl2 pode estar envolvido. Sendo Cerl2 antagonista da via de sinalização TGFßs/Nodal/Activin/Smad2, em corações embrionários (E13) e neonatais de Cerl2-/- observou-se um aumento da fosforilação de Smad2 (pSMAD2). Estes resultados sugerem que a via de sinalização TGFßs/Nodal/Activin/Smad2 pode estar ativamente aumentada na ausência de Cerl2. Interessantemente, tem sido relatado que esta via é essencial para a regulação da cardiogénese uma vez que também desempenha um papel relevante como mediador na patogénese cardíaca em corações de ratinho adulto após lesão. A segunda hipótese tem como base dados recentes os quais reportam que a via de sinalização Wnt e Cerl2 estão interligadas no nó através de um feedback negativo, onde o mRNA do Cerl2 é regulado após a transcrição por Wnt3 levando à degradação de Cerl2 no lado esquerdo do nó e portanto estabelecendo a sua expressão de forma assimétrica. Em contrapartida, Cerl2 é capaz de inibir a auto-regulação da proteína Wnt3. Adicionalmente, também foi revelado que a via de sinalização Wnt/ß-catenin é essencial para estimular a proliferação de cardiomiócitos na camada compacta de ambos os ventrículos. Apesar de em nenhum estudo até agora ter sido demonstrada a relação entre Wnt/ß-catenin e Cerl2 no coração e de serem necessárias confirmações adicionais, sugerimos que a hiperplasia do VE encontrado em Cerl2-/- pode também ser devida ao aumento da via de sinalização Wnt/ßcatenin. Uma vez que as células estaminais embrionárias derivadas da estirpe 129 de ratinho colonizam eficientemente as linhas germinativas, esta linhagem celular tem sido utilizada com frequência para a produção de linhas de animais geneticamente modificados. Como diferentes fundos genéticos podem originar diferentes fenótipos, o estabelecimento de valores de referência para cada estirpe de ratinhos é uma ferramenta útil nos dias atuais. A ecocardiografia não-invasiva permite avaliar a função cardíaca e a morfometria ventricular esquerda e o seu uso tem crescido na última década. Dessa maneira, foram criados valores de referência para a estirpe 129/Sv de ratinhos juvenis (3 semanas) e adultos (8 semanas). Com o objectivo de analisar se os ratinhos Cerl2-/- continuam a manifestar um aumento da massa ventricular esquerda com a redução da função cardíaca, procedeu-se à monitorização, através de ecocardiografia, de ratinhos recém-nascidos até à fase de jovens adultos (P60). Uma parte significante dos mutantes Cerl2 morrem no primeiro dia de vida, na presença e na ausência de DL. Tem sido relatado que a presença de DL é incompatível com um longo tempo de vida e portanto é esperado que a grande maioria destes mutantes com DL não sobrevivam. Já nos mutantes de Cerl2 que morrem nas primeiras horas de vida e não apresentaram defeitos de lateralidade, foi detectada uma tendência para o aumento da massa ventricular esquerda a qual é indicativa de hipertrofia. Além disto este grupo revelou uma dramática redução da função cardíaca como foi demonstrado pela diminuição dos batimentos cardíacos por minuto e pela diminuição do pico da velocidade da artéria pulmonar. Curiosamente, os mutantes de Cerl2 que sobrevivem conseguem recuperar a sua função cardíaca como também demonstrado pela fração de ejeção, a fração de encurtamento e a fração de alteração das áreas (sistólica e diastólica) quando comparados com os animais controlo. Este resultado foi confirmado pela manutenção dos níveis de expressão dos indicadores de hipertrofia e stress (como por exemplo o Anp, Bnp e Ankrd1). Dessa maneira, concluiu-se que o primeiro dia de vida pós-natal é determinante para os mutantes que não apresentam DL. Contudo é desconhecido o(s) factor(es) que determina(m) a morte ou sobrevivência destes animais. Apesar da melhora na função cardíaca nos adultos de Cerl2-/-, o débito cardíaco obtido no ventrículo esquerdo quando normalizado pelo peso do corpo, revelou uma redução quando comparado com animais wild-type, indicando portanto que a recuperação da função sistólica não é completa. Curiosamente os ratinhos adultos Cerl2-/- quando comparados com os ratinhos controlo apresentaram diferentes padrões de dimensão nas paredes do VE, como exemplo afinamento da parede anterior e posterior. De acordo com a literatura tem sido sugerido que a alteração aeróbica regional do metabolismo cardíaco pode levar á uma diferença assimétrica na espessura das paredes dos ventrículos, sendo no entanto necessária uma investigação detalhada. Embora ainda seja desconhecido o mecanismo pelo qual os ratinhos Cerl2-/- conseguem adaptar-se a vida pós-natal, sugerimos que o estes ratinhos mutantes podem ser um modelo interessante para estudar os mecanismos moleculares, celulares e fisiológicos que estão por trás da restauração da função cardíaca. Portanto análises detalhadas neste modelo poderia ajudar a desenvolver abordagens terapêuticas para o tratamento de insuficiência cardíaca. Em conclusão, os resultados apresentados nesta tese proporcionam um conjunto importantes de novos dados relacionados com a cardigénese, durante e após a vida intrauterina, os quais revelam a importância de Cerl2 na regulação da formação do coração independentemente da sua bem conhecida função no estabelecimento do eixo E/D.
Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina

碩士
國立臺灣師範大學
生命科學研究所
93
One of the major functions of the Renin-angiotensin system (RAS) is to maintain the homeostasis of blood pressure in vertebrates. Members of the Angiotensin-converting enzyme (ACE) gene family are also involved in heart development and morphogenesis. Previous studies have demonstrated that mammalian ACE2 and its Drosophila counterpart, Acer, are expressed in heart and associated tissues. Since cardiogenesis and regulatory genes that involved in heart development of fly and mammals are evolutionary conserved, and Drosophila as an excellent model system to dissect the genetic factor participated in heart development, we would like to unravel the function of Acer in heart development and dissect up-stream factors that regulate the expression of Acer. The dynamic expression pattern of Acer has been revealed by in situ hybridization. It has been shown that Acer is a maternal gene as it has been detected in fertilized egg. Its maternal expression late is disappeared, and it is expressed in dorsal mesoderm, which late contributes to cardial mesoderm, at germ extension stages. The mesodermally expressed Acer is persisted in heart precursors and persisted till the cardial cells fused to form a cardiac tube underneath the dorsal mid line. As Acer is mainly expressed in heart-specific manner, it suggests that it may function in heart development. Pervious studies have demonstrated that cardial as pericardial cells are missing in Acer mutant embryos. To further understanding the upstream factors that regulate the cardial expression of Acer, we have identified the cardial enhancer of Acer is located between -188 and 1211. The expression pattern of the reporter gene is similar to that of Acer. Further studies had demonstrated that Acer is under the direct control of tinman and pannier. Since ACE is also control the homeostasis of blood pressure, which affects the cardiovascular function of mammals, we would like to learn whether Acer is also function in adult fly. Previously studies suggested that heart beat as well as life span are not affect when Acer is overexpressed. Less than 5% of embryos are dead at late embryogenesis with ectopically cardial cells as Acer activity is elevated. Nevertheless, cardiac performance is great affected when Acer is mis-regulated, suggesting Acer is also function at adult stages.

The teleost zebrafish (danio rerio) has a highly elevated regenerative capacity compared to mammals, with the ability to quickly and correctly regenerate complex organs such as the fin and the heart following amputation. Studies in other highly regenerative systems suggest that regenerative capacity is directly related to the homeostatic demands of a given tissue, such as high basal levels of cell turnover or the ability to modify tissue size in response to homeostatic changes. However, it is not known if this relationship is present in vertebrate tissues with blastema-based regeneration. To test this idea, we investigated whether markers associated with regeneration are expressed in uninjured zebrafish tissues, and if treatments that block regeneration also lead to homeostatic defects over long periods.

We found that regenerative capacity is generally required for homeostasis in the fin, as multiple genetic treatments that block regeneration also led to a degenerative loss of distal fin tissue in uninjured animals. In addition, we found that there is extensive cell turnover in the distal fin tissues, accompanied by expression of critical effectors of blastemal regeneration. Both cell proliferation and gene expression were sensitive to changes in Fgf signaling, a factor that is critical for fin regeneration.

In the heart, we found that although there is little cell turnover in uninjured adult animals, the zebrafish heart can undergo rapid, dramatic cardiogenesis in response to animal growth. These growth conditions induce cardiomyocyte hyperplasia similar to regeneration, and induce gene expression changes in the epicardium, a tissue that is critical for cardiac regeneration. We find that the epicardium continually contributes cells to the uninjured heart, even in the absence of cardiac growth. If this contribution is prevented via a long-term block of Fgf signals, scarring can result, indicating that continual activity of epicardium derived cells (EPDCs) is critical for cardiac homeostasis. We have generated reagents that allow us to visualize EPDCs, and find that they contribute cardiac fibroblasts and perivascular cells during rapid cardiac growth. Uncovering the fate of EPDCs during cardiac homeostasis and regeneration will allow us to better understand their function, and may lead to the development of regenerative therapies for human cardiovascular diseases.

Dissertation

Indiana University-Purdue University Indianapolis (IUPUI)
The heart is the first organ to form during development in vertebrates and Nkx2-5 is the first marker of cardiac specification. In Xenopus laevis, Nkx2-5 is essential for heart formation, but early targets of this homeodomain transcription factor have not been fully characterized. In order to discover potential early targets of Nkx2-5, synthetic Nkx2-5 mRNA was injected into eight-cell Xenopus laevis embryos and changes in gene expression measured using microarray analysis. While Xenopus laevis is a commonly used model organism for developmental studies, its genome remains poorly annotated. To compensate for this, a cross-species annotation database called CrossGene was constructed. CrossGene was created by exhaustively comparing UniGene transcripts from Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans using the BLAST family of algorithms. Networks were then assembled by recursively combining reciprocal best matches into groups of orthologous genes. Gene ontology annotation from all organisms could then be applied to all members of the reciprocal group. In this way, the CrossGene database was used to augment the existing genomic annotation of Xenopus laevis. Combining cross-species annotation with differential gene expression analysis of Nkx2-5 overexpression led to the discovery of 99 potential targets of Nkx2-5.

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016
Das suas quatro cavidades à sua síncrona rede elétrica, o coração foi perfeitamente projetado para servir de interface entre cada órgão presente no corpo humano. Devido à sua complexidade, as doenças cardiovasculares englobam também um grande conjunto de manifestações clínicas incluindo miocardites, hipertensão arterial, defeitos congénitos cardíacos e doenças isquémicas. Muitas destas patologias traduzem-se geralmente na perda de tecido cardíaco funcional e por outro lado pela formação de tecido fibrótico não funcional. Similarmente ao que ocorre nos países desenvolvidos, em Portugal também as doenças cardiovasculares continuam a ser uma das maiores causas de morbidade e mortalidade. Devido à limitada capacidade regenerativa do coração e ao facto das terapias existentes para tratar doenças cardiovasculares serem ineficientes ou implicarem enormes riscos para o paciente, é urgente desenvolver novas terapias mais eficazes. Nesse sentido, o uso de células multi e pluripotentes tem contribuído na última década para um franco avanço nesta área. Muitos ensaios clínicos têm sido feitos, ou decorrem ainda, onde se avalia a capacidade regenerativa de células estaminais de diferentes origens na reposição dos tecidos cardíacos danificados. Além disto pensa-se que certos nichos de células progenitoras de cardiomiócitos residentes no coração adulto possam representar um mecanismo endógeno de regeneração. De modo a explorar este mecanismo tem-se recorrido a técnicas de isolamento destas células para transplante em doentes cardíacos. No entanto, até agora as melhorias evidenciadas por essas terapias celulares parecem estar associadas a efeitos parácrinos que as células transplantadas exercem sobre os tecidos envolventes, em detrimento da sua implantação no tecido danificado e consequente diferenciação em novo tecido cardíaco. Em paralelo às terapias celulares tem-se feito um esforço para desenvolver patches e scaffolds que possam complementar estas terapias por facilitar o homing de células transplantadas ao constituírem uma matriz onde estas células possam ser envolvidas e desempenhar a sua função. Outra alternativa ao uso de células estaminais para uso em terapias de regeneração cardíaca é o uso de células já diferenciadas com identidade semelhante à do tecido a ser substituído. No caso do miocárdio, será potencialmente interessante o uso de cardiomiócitos como fonte em transplantes para a regeneração do tecido danificado. Tal abordagem é especialmente interessante visto terem sido identificadas no coração populações de novos cardiomiócitos derivados de cardiomiócitos já existentes, que contribuem para o turnover normal do miocárdio. No entanto, para explorar este mecanismo é necessário criar e otimizar protocolos eticamente aceitáveis para experimentação humana de derivação em grande escala de cardiomiócitos a partir de células pluripotentes. Tal objetivo pode ser alcançado através do uso de fatores segregados que possam ser utilizados para estimular o potencial cardiogénico das células pluripotentes. A procura de genes envolvidos na cardiogénese têm-se tornado cada vez mais importante com o objetivo de identificar potenciais fatores que possam modular este processo biológico quer in vitro como in vivo. De facto, é possível modelar in vitro com grande rigor os estadios iniciais da cardiogénese através da diferenciação de células estaminais. Tal como ocorre in vivo, a especificação das linhagens cardiovasculares in vitro implica uma transição para populações de células progenitoras cardíacas com potencial de diferenciação cada vez mais restrito e específico. Começando num estado de pluripotência, durante a sua diferenciação estas especificam-se em mesoderme cardíaca e posteriormente em células de todas as outras linhagens cardíacas. Para monitorizar o seguimento deste processo biológico e para assegurar o correto comprometimento nas várias linhagens cardíacas recorre-se à expressão génica de marcadores genéticos específicos para cada linhagem esperada em cada ponto específico de tempo. Através desta monitorização é possível identificar células de mesoderme cardíaca pela expressão dos genes Mesp-1 e Isl-1 a dia 4 de diferenciação das células estaminais, e também diferentes populações de células progenitoras cardíacas pela expressão concomitante de genes como Isl-1 e Nkx2.5 em dias posteriores. Assim é possível estabelecer em laboratório um modelo fidedigno e manipulável para se estudar a cardiogénese. Num rastreio génico efetuado pelo nosso laboratório em células progenitoras cardíacas de galinha com expressão do marcador Nkx2.5, foram identificados genes não caracterizados, mas com um potencial envolvimento na cardiogénese. Um destes novos genes identificados foi o collagen and calcium binding EGF domains 1 ou Ccbe1. Na literatura, é possível hoje ver que em modelos animais knockout para este gene, um outro processo biológico é afetado i.e. a linfangiogénese. Estes animais apresentam uma total ausência de vasos linfáticos. Este fenótipo deve-se em parte ao papel já identificado que o CCBE1 tem na maturação do fator pro-linfangiogénico VEGF-C. Em humanos a síndrome de Hennekam (associado também a mutações em CCBE1), é caracterizada pela existência de uma rede linfática disfuncional fazendo com que estes apresentem um edema generalizado. Não obstante estes estudos, recentemente verificou-se em ratinho e galinha a expressão deste gene nas regiões embrionárias que dão origem ao coração, sugerindo assim também um potencial papel neste processo. De facto, trabalho efectuado no nosso laboratório veio a demonstrar que o silenciamento deste gene em galinha leva ao desenvolvimento de defeitos cardíacos incompatíveis com a vida, associados a uma redução da proliferação das células cardiacas. Também, em ratinhos knockout para este gene é possível identificar um miocárdio subdesenvolvido pelo estreitamento da camada compacta do miocárdio também associado a problemas na proliferação. Assim, no presente trabalho propusemo-nos a estudar mais detalhadamente o envolvimento deste gene nos estadios iniciais da cardiogénese. Como este gene codifica para uma proteína secretada, a verificar-se um importante papel na cardiogénese, a sua manipulação como um fator de crescimento torna-se de grande interesse visando a otimização de protocolos para derivação de cardiomiócitos. Para estudar os estadios iniciais da cardiogénese recorremos ao uso de uma linha de células estaminais duplamente transgénica que nos permite acompanhar o processo de diferenciação para linhagens cardíacas pois expressam a proteína fluorescente GFP sob o controlo do promotor de Nkx2.5 e a proteína fluorescente dsRed sob um promotor específico de cardiogénese de Mef2c. Assim pode-se confirmar que é possível obter células progenitoras cardíacas in vitro correspondentes aos estadios iniciais do desenvolvimento do coração de ratinho. De seguida analisámos o padrão de expressão de Ccbe1 e verificou-se que coincide com o aparecimento da expressão dos marcadores genéticos cardíacos, mostrando que in vitro a sua expressão ocorre aquando da especificação das células para as linhagens cardíacas. Posteriormente gerámos duas linhas estáveis de células estaminais com silenciamento de Ccbe1 para avaliar o seu impacto na cardiogénese. Os resultados demonstram que ao diferenciar estas células em agregados 3D conhecidos como corpos embrióides (nome dado devido à sua semelhança física e funcional com um embrião nos estadios iniciais do desenvolvimento), estas células são incapazes de se especificar em mesoderme cardíaca pois apresentam a expressão de Mesp-1 e Isl-1 reduzida. Em paralelo com estes resultados, foi possível verificar que os corpos embrióides gerados a partir de células estaminais com silenciamento de Ccbe1 apresentam um tamanho muito reduzido. Este defeito é devido não a um aumento da morte celular mas sim a um défice na proliferação das células estaminais silenciadas. Estes defeitos na proliferação estão de acordo com outros estudos efetuados pela nossa equipa, em que fibroblastos embrionários derivados de ratinhos knockout apresentam grandes problemas na proliferação. Adicionalmente, em embriões de galinha foi verificado necessidade de Ccbe1 para a correta proliferação de células precursoras cardíacas para formar o tubo cardíaco. Em conjunto, estes resultados demonstram que CCBE1 tem um papel importante em proliferação. Tais resultados são corroborados por experiências onde foi feita a adição de CCBE1 recombinante ao meio de cultura e se observou a recuperação parcial dos corpos embrióides silenciados. Apesar das dificuldades em produzir quantidades elevadas desta proteína recombinante, os resultados indicam que CCBE1 foi capaz de aumentar a proliferação dos corpos embrióides silenciados. No entanto, as células demonstram-se incapazes de se especificar em mesoderme cardíaca, sugerindo que para além deste papel que Ccbe1 tem em proliferação, o seu papel na cardiogénese é independente deste mecanismo. Conclui-se assim que Ccbe1 é indispensável para a especificação das células em diferenciação em mesoderme cardíaca. Para vir a ser utilizado no futuro como fator de crescimento em células estaminais em diferenciação, para derivar grandes quantidades de células cardíacas, é necessário desenvolver ainda mais estudos que permitam ultrapassar as limitações associadas à sua produção e à sua bioatividade. Paralelamente a estes estudos, uma outra parte do meu trabalho incidiu numa colaboração com uma equipa de bioinformática, na qual nos propusemos a analisar o transcriptoma de diferentes tipos de células progenitoras cardíacas. O objetivo desta análise seria primariamente identificar através de sequenciação RNA novas isoformas de genes envolvidos na cardiogénese, e adicionalmente identificar novos genes não caracterizados com potencial impacto na cardiogénese. Para tal utilizámos a linha de células estaminais duplamente transgénica já referida, da qual isolámos diferentes populações de células progenitoras cardíacas em dias de diferenciação diferentes. Conseguimos analisar o dataset resultante utilizando algumas ferramentas bioinformáticas, que nos permitiu construir uma lista de genes potencialmente envolvidos em cardiogénese ainda não caracterizados. Deste trabalho resultam alguns genes que merecerão um estudo funcional mais detalhado visto estarem claramente expressos nas regiões embrionárias cardiogénicas.
The identification and use of new growth factors to stimulate the cardiogenic potential of pluripotent cells is a safe and alternative approach to develop cell therapies to address the limited regenerative capacity of the heart. Collagen and calcium binding EGF domains 1 (Ccbe1) was firstly identified in our laboratory, which encodes for a secreted protein with potential involvement in cardiogenesis. Knockout animal models for this gene and humans with mutations in CCBE1, have lymphangiogenic defects, resulting in the absence of lymphatic vessels. This is in part due to the known described role that CCBE1 has in the processing of the pro lymphangiogenic factor VEGF-C. However, Ccbe1 is also expressed in the embryonic cardiogenic regions of both mouse and chick and in fact, silencing this gene in chick embryos leads to the development of heart defects incompatible with life. Noteworthy, knockout mice show an underdeveloped myocardium. The objective of the present work is to perform a detailed study of the involvement of this gene in the early stages of cardiogenesis. The results demonstrate that silencing the expression of Ccbe1 or blocking CCBE1 in differentiating stem cells, impairs their specification towards cardiac mesodermal lineages. Additionally, we found that differentiating Ccbe1 KD ESCs have a reduced proliferation rate that leads to smaller EBs. In agreement with this result, when supplementing the differentiating Ccbe1 KD ESCs lines with recombinant CCBE1, we were able to partially rescue the size of the EBs, but the expression of the cardiac mesoderm markers remained downregulated. These data suggest that those defects are independent from each other, but are intimately related to the disruption of Ccbe1, placing CCBE1 as a direct regulator of cell proliferation and cardiac mesoderm specification during ESC differentiation.

Le cœur des vertébrés est un organe modulaire qui requiert le " patterning " complexe des champs morphogénétiques cardiogènes et la convergence coordonnée des diverses sous-populations de progéniteurs cardiogéniques. Au moins 7 facteurs de transcription de la famille T-box coopèrent au sein de ces nombreuses sous-populations de progéniteurs cardiogéniques afin de réguler la morphogenèse et l’agencement de multiples structures le long de l’ébauche cardiaque, ce qui explique que les mutations humaines de ces gènes engendrent diverses malformations congénitales cardiaques (MCCs). L’un de ces gènes T-box, Tbx5, dont l’haploinsuffisance génère le syndrome de Holt-Oram (SHO), intervient dans une grande variété de réseaux de régulation géniques (RRGs) qui orchestrent la morphogenèse des oreillettes, du ventricule gauche, de la valve mitrale, des septums inter-auriculaire et inter-ventriculaire, ainsi que du système de conduction cardiaque. La diversité des RRGs impliqués dans la formation de ces structures cardiaques suggère que Tbx5 détient une profusion de fonctions qui ne seront identifiables qu’en répertoriant ses activités moléculaires dans chaque lignée cardiaque examinée isolément. Afin d’aborder cette problématique, une ablation génétique de Tbx5 dans l’endocarde a été réalisée. Cette expérience a démontré le rôle crucial de Tbx5 dans la survie des cellules endocardiques bordant le septum primum et des cardiomyocytes au sein de cette structure embryonnaire qui contribuera à la morphogenèse du septum inter-auriculaire. En outre, cette étude a révélé l’existence d’une communication croisée entre la sous-population de cellules endocardiques Tbx5+ et le myocarde au niveau du septum primum, afin d’assurer la survie des cardiomyocytes, et ultimement de garantir la maturation du septum inter-auriculaire. Nos résultats confirment aussi l’importance de l’interdépendance génétique (Tbx5 et Gata4 ainsi que Tbx5 et Nos3) entre différents loci dans la morphogenèse de la cloison inter-auriculaire, et particulièrement de l’influence que peut avoir l’environnement sur la pénétrance et l’expressivité des communications inter-auriculaires (CIAs) dans le SHO. En outre, puisque les fonctions d’un gène dépendent ordinairement des différents isoformes qu’il peut générer, une deuxième étude a focalisé davantage sur l’aspect transcriptionnel de Tbx5. Cette approche a mené à la découverte de 6 transcrits alternatifs exhibant des fonctions à la fois communes et divergentes. La caractérisation de 2 de ces isoformes a révélé le rôle de l’isoforme long (Tbx5_v1) dans la régulation de la croissance des cardiomyocytes durant la cardiogénèse, tandis que l’isoforme court (Tbx5_v2), préférentiellement exprimé dans le cœur mature, réprime la croissance cellulaire. Il est donc entièrement concevable que les mutations de TBX5 entraînant une troncation de la région C-terminale accroissent la concentration d’une protéine mutée qui, à l’instar de Tbx5_v2, interfère avec la croissance de certaines structures cardiaques. En revanche, la divergence de fonctions de ces isoformes, caractérisée par les disparités de localisation subcellulaire et de d’interaction avec d’autres cofacteurs cardiaques, suggère que les mutations affectant davantage un isoforme favoriseraient l’émergence d’un type particulier de MCC. Finalement, un dernier objectif était d’identifier le ou les mécanisme(s) moléculaire(s) par le(s)quel(s) Tbx5 régule son principal gène cible, Nppa, et d’en extraire les indices qui éclairciraient sa fonction transcriptionnelle. Cet objectif nécessitait dans un premier lieu d’identifier les différents modules cis-régulateurs (MCRs) coordonnant la régulation transcriptionnelle de Nppa et Nppb, deux gènes natriurétiques dont l’organisation en tandem et le profil d’expression durant la cardiogénèse sont conservés dans la majorité des vertébrés. L’approche d’empreinte phylogénétique employée pour scanner le locus Nppb/Nppa a permis d’identifier trois MCRs conservés entre diverses espèces de mammifères, dont un (US3) est spécifique aux euthériens. Cette étude a corroboré que la régulation de l’expression du tandem génique Nppb/Nppa requérait l’activité transcriptionnelle d’enhancers en complément aux promoteurs de Nppa et Nppb. La concordance quasiment parfaite entre les profils d’expression de Tbx5 et de ces deux gènes natriurétiques chez les mammifères, suggère que le gradient d’expression ventriculaire de Tbx5 est interprété par le recrutement de ce facteur au niveau des différents enhancers identifiés. En somme, les études présentées dans cette thèse ont permis de clarifier la profusion de fonctions cardiaques que possède Tbx5. Certaines de ces fonctions émanent de l’épissage alternatif de Tbx5, qui favorise la synthèse d’isoformes dotés de propriétés spécifiques. Les diverses interactions combinatoires entre ces isoformes et d’autres facteurs cardiaques au sein des diverses sous-populations de progéniteurs cardiogènes contribuent à l’émergence de RRGs cardiaques divergents.
The vertebrate heart is a modular organ, which requires the complex patterning of the morphogenetic heart fields and the coordinated convergence of the diverse subpopulations of cardiogenic progenitors. At least 7 transcription factors of the T-box family cooperate within these numerous subpopulations of cardiogenic progenitors to regulate the morphogenesis and the layout of multiple structures along the primordial heart tube, which explains that the human mutations of these genes induce various congenital heart defects (CHDs). One of these T-box genes, Tbx5, whose haploinsufficiency generates the Holt-Oram syndrome (HOS), intervenes in a wide variety of gene regulatory networks (GRNs) that orchestrate the morphogenesis of the atria, the left ventricle, the mitral valve, the inter-atrial and inter-ventricular septa, as well as the cardiac conduction system. The diversity of GRNs involved in the formation of these cardiac structures suggests that Tbx5 holds a profusion of functions which will be identifiable only by indexing its molecular activities in each separately examined cardiac lineage. To address this problem, a conditional knockout of Tbx5 in the endocardium was generated. This experiment demonstrated a crucial role of Tbx5 in the survival of the endocardial cells lining the septum primum and the cardiomyocytes within this embryonic structure, which will contribute to the morphogenesis of the inter-atrial septum. Moreover, this study revealed a crosstalk between the Tbx5-positive endocardial cells subpopulation and the myocardium at the level of the septum primum to ensure the survival of cardiomyocytes, and ultimately to guarantee the maturation of the inter-atrial septum. Our results also confirmed the importance of genetic interdependence (Tbx5 and Gata4 as well as Tbx5 and Nos3) between different loci in the morphogenesis of the inter-atrial septum, and particularly the influence that the environment can have on the penetrance and the expressivity of atrial septal defects (ASDs) in the HOS. Besides, since the functions of a gene usually depend on the different isoforms it can generate, a second study focused more on the transcriptional aspect of Tbx5. This approach led to the discovery of 6 alternative transcripts exhibiting both common and specific functions. The characterization of 2 of these isoforms revealed the role of the long isoform (Tbx5_v1) in the regulation of cardiomyocytes growth during cardiogenesis, whereas the short isoform (Tbx5_v2), preferentially expressed in the mature heart, represses cell growth. It is thus entirely conceivable that TBX5 mutations leading to a C-terminal truncation increase the concentration of a mutated protein, which, like Tbx5_v2, interferes with the growth of certain cardiac structures. On the other hand, the divergence of functions of these isoforms, characterized by the disparities of subcellular localization and interaction with other cardiac cofactors, suggests that mutations affecting more one isoform would favor the emergence of a particular type of CHD. Finally, a last objective was to identify one or several molecular mechanism(s) by which Tbx5 regulates its main target gene, Nppa, and to extract clues that might clarify its transcriptional function. This objective required in a first place to identify the various cis-regulatory modules (CRMs) coordinating the transcriptional regulation of Nppa and Nppb, two natriuretic genes whose tandem organization and expression pattern during cardiogenesis are preserved in most vertebrates. The phylogenetic footprint approach employed to scan the Nppb/Nppa locus allowed the identification of three CRMs evolutionary conserved between different mammals species, one of which (US3) is specific to eutherians. This study confirmed that the regulation of the tandem genes Nppb/Nppa required the transcriptional activity of enhancers in complement to Nppa and Nppb promoters. The almost perfect concordance between the expression profiles of Tbx5 and these two natriuretic genes in mammals, suggests that the ventricular expression gradient of Tbx5 is interpreted by the recruitment of this factor to the identified enhancers. Altogether, the studies presented in this thesis allowed clarifying the profusion of Tbx5 cardiac functions. Some of these functions emanate from the alternative splicing of Tbx5, which favors the synthesis of isoforms endowed with specific properties. The diverse combinatorial interactions between these isoforms and other cardiac factors within the various cardiogenic progenitor subpopulations contribute to the emergence of distinct cardiac RRGs.