No description available.

abstract: The development of the vertebrate musculoskeletal system is a highly dynamic process, requiring tight control of the specification and patterning of myogenic, chondrogenic and tenogenic cell types. Development of the diverse musculoskeletal lineages from a common embryonic origin in the paraxial mesoderm indicates the presence of a regulatory network of transcription factors that direct lineage decisions. The basic helix-loop-helix transcription factor, PARAXIS, is expressed in the paraxial mesoderm during vertebrate somitogenesis, where it has been shown to play a critical role in the mesenchymal-to-epithelial transition associated with somitogenesis, and the development of the hypaxial skeletal musculature and axial skeleton. In an effort to elucidate the underlying genetic mechanism by which PARAXIS regulates the musculoskeletal system, I performed a microarray-based, genome-wide analysis comparing transcription levels in the somites of Paraxis-/- and Paraxis+/+ embryos. This study revealed targets of PARAXIS involved in multiple aspects of mesenchymal-to-epithelial transition, including Fap and Dmrt2, which modulate cell-extracellular matrix adhesion. Additionally, in the epaxial dermomyotome, PARAXIS activates the expression of the integrin subunits a4 and a6, which bind fibronectin and laminin, respectively, and help organize the patterning of trunk skeletal muscle. Finally, PARAXIS activates the expression of genes required for the epithelial-to-mesenchymal transition and migration of hypaxial myoblasts into the limb, including Lbx1 and Met. Together, these data point to a role for PARAXIS in the morphogenetic control of musculoskeletal patterning. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2013

No description available.

No description available.

Oscillations are present at many different levels of biological organization. The cell cycle that directs the division of individual cells, the regular depolarization of neurons in the sinu-atrial node which underlies the regular beating of the heart, the circadian rhythms that govern the daily activity cycles of virtually all organisms, and the clocks that make entire populations of fireflies flash on and off in unison feature as prominent examples of biological clocks. During development, biological clocks regulate the patterning of growing tissues, as is the case in vertebrate somitogenesis, and potentially also in vertebrate limb outgrowth and axial segmentation of invertebrate embryos. During vertebrate segmentation, the embryonic axis is subdivided along its anterior-posterior axis into epithelial spheres of cells called somites. This rhythmic process is thought to be driven by a multicellular oscillatory gene network, the so-called segmentation clock. Oscillations of hairy and enhancer of split gene products have been proposed to constitute the core clockwork in individual cells, and these oscillators are coupled to each other by Delta-Notch intercellular signaling. The interaction of the segmentation clock with a posteriorly-moving arrest wavefront then translates the temporal information encoded by the clock into a spatial pattern of segments. In the framework of this Clock and Wavefront model, segment length is determined by both clock period and arrest wavefront velocity. How the period of the segmentation clock is regulated is presently unknown, and understanding the mechanism of period setting might yield insight into the nature and function of the segmentation clock. In this study, two different but complementary approaches were pursued to investigate how period is regulated in the zebrafish segmentation clock. First, it has been reported that zebrafish mind bomb (mib) mutant embryos form somites more slowly than their wt siblings, suggesting that Mib might be implicated in period setting. Mib is an E3 ubiquitin ligase required for ubiquitination and endocytosis of the Notch ligand Delta, and Notch signaling is impaired in mutants with defective Mib. It has been suggested that the mechanistic basis for the requirement of Delta endocytosis in Notch signaling is a need for Delta to enter a particular endocytic compartment, potentially a recycling endosome, in a ubiquitin-dependent manner, where its signaling ability might be established or amplified by an as yet unknown posttranslational modification. In the present study, Delta trafficking through the endocytic pathway was analyzed in the PSM of wt and mib embryos through colocalization studies with endocytic markers. The rationale of this approach was that if Delta gained access to a particular endocytic compartment through Mib-dependent endocytosis, the presence of Delta in this compartment would be expected to be reduced in mutants with defective Mib, thereby revealing the compartment’s identity. However, no qualitative changes in colocalization with different endocytic markers could be detected in mib mutants, and the methods available did not allow for quantification of colocalization in wt or mutant backgrounds. However, Delta colocalized with 13 markers of recycling endosomes, consistent with the hypothesis that these are functionally important in Notch signaling. More refined techniques will be necessary for a quantitative analysis of normal as compared to impaired Delta trafficking. A genetic approach to period regulation proved to be successful for the Drosophila circadian clock, where the identification of period mutants advanced the understanding of the clock’s genetic circuitry. This motivated a screen for period mutants of the segmentation clock, which was carried out by measuring somitogenesis period, segment length and arrest wavefront velocity in a pool of candidate mutants. A subset of Delta-Notch mutants, and embryos treated with a small-molecule inhibitor that impairs Notch signaling, displayed correlated increases in somitogenesis period and segment length, while there was no detectable change in arrest wavefront velocity. Combined, these findings suggested that segmentation clock period is increased in experimental conditions with impaired Delta-Notch signaling. Using a theoretical description of the segmentation clock as an array of coupled phase oscillators, the delay in the coupling and the autonomous frequency of individual cells were estimated from the direction and magnitude of the period changes. The mutants presented here are the first candidates for segmentation clock period mutants in any vertebrate. The nature of the molecular lesions in these mutants, all of which affect genes implicated in intercellular Delta-Notch signaling, suggests that communication between oscillating PSM cells is a key factor responsible for setting the period of the segmentation clock.

Oscillations are present at many different levels of biological organization. The cell cycle that directs the division of individual cells, the regular depolarization of neurons in the sinu-atrial node which underlies the regular beating of the heart, the circadian rhythms that govern the daily activity cycles of virtually all organisms, and the clocks that make entire populations of fireflies flash on and off in unison feature as prominent examples of biological clocks. During development, biological clocks regulate the patterning of growing tissues, as is the case in vertebrate somitogenesis, and potentially also in vertebrate limb outgrowth and axial segmentation of invertebrate embryos. During vertebrate segmentation, the embryonic axis is subdivided along its anterior-posterior axis into epithelial spheres of cells called somites. This rhythmic process is thought to be driven by a multicellular oscillatory gene network, the so-called segmentation clock. Oscillations of hairy and enhancer of split gene products have been proposed to constitute the core clockwork in individual cells, and these oscillators are coupled to each other by Delta-Notch intercellular signaling. The interaction of the segmentation clock with a posteriorly-moving arrest wavefront then translates the temporal information encoded by the clock into a spatial pattern of segments. In the framework of this Clock and Wavefront model, segment length is determined by both clock period and arrest wavefront velocity. How the period of the segmentation clock is regulated is presently unknown, and understanding the mechanism of period setting might yield insight into the nature and function of the segmentation clock. In this study, two different but complementary approaches were pursued to investigate how period is regulated in the zebrafish segmentation clock. First, it has been reported that zebrafish mind bomb (mib) mutant embryos form somites more slowly than their wt siblings, suggesting that Mib might be implicated in period setting. Mib is an E3 ubiquitin ligase required for ubiquitination and endocytosis of the Notch ligand Delta, and Notch signaling is impaired in mutants with defective Mib. It has been suggested that the mechanistic basis for the requirement of Delta endocytosis in Notch signaling is a need for Delta to enter a particular endocytic compartment, potentially a recycling endosome, in a ubiquitin-dependent manner, where its signaling ability might be established or amplified by an as yet unknown posttranslational modification. In the present study, Delta trafficking through the endocytic pathway was analyzed in the PSM of wt and mib embryos through colocalization studies with endocytic markers. The rationale of this approach was that if Delta gained access to a particular endocytic compartment through Mib-dependent endocytosis, the presence of Delta in this compartment would be expected to be reduced in mutants with defective Mib, thereby revealing the compartment’s identity. However, no qualitative changes in colocalization with different endocytic markers could be detected in mib mutants, and the methods available did not allow for quantification of colocalization in wt or mutant backgrounds. However, Delta colocalized with 13 markers of recycling endosomes, consistent with the hypothesis that these are functionally important in Notch signaling. More refined techniques will be necessary for a quantitative analysis of normal as compared to impaired Delta trafficking. A genetic approach to period regulation proved to be successful for the Drosophila circadian clock, where the identification of period mutants advanced the understanding of the clock’s genetic circuitry. This motivated a screen for period mutants of the segmentation clock, which was carried out by measuring somitogenesis period, segment length and arrest wavefront velocity in a pool of candidate mutants. A subset of Delta-Notch mutants, and embryos treated with a small-molecule inhibitor that impairs Notch signaling, displayed correlated increases in somitogenesis period and segment length, while there was no detectable change in arrest wavefront velocity. Combined, these findings suggested that segmentation clock period is increased in experimental conditions with impaired Delta-Notch signaling. Using a theoretical description of the segmentation clock as an array of coupled phase oscillators, the delay in the coupling and the autonomous frequency of individual cells were estimated from the direction and magnitude of the period changes. The mutants presented here are the first candidates for segmentation clock period mutants in any vertebrate. The nature of the molecular lesions in these mutants, all of which affect genes implicated in intercellular Delta-Notch signaling, suggests that communication between oscillating PSM cells is a key factor responsible for setting the period of the segmentation clock.

Un nouveau facteur de transcription, appartenant à la famille des protéines à domaine bHLH, a récemment été isolé dans notre laboratoire. Initialement appelé « clone bc8 » puis HRT1, ce facteur présentait des similitudes avec les protéines Hairy and Enhancer of split qui interviennent notamment dans le phénomène d’inhibition latérale lors de la formation du tissu neural. Des études d’hybridation in situ réalisées chez l'embryon de xénope ont suggéré un rôle important de XHRT1, la protéine HRT1 de xénope, dans le développement neural. Nous avons recherché les partenaires protéiques de XHRT1 par la technique du double-hybride afin de mieux comprendre son mécanisme d’action moléculaire dans la neurogenèse. Tout d’abord nous avons construit les outils appropriés pour l’élaboration du travail, à savoir, les clones de levures exprimant les appâts spécifiques des domaines de la protéine étudiée et la création d’une banque d’ADNc du xénope au stade de la neurulation. Ensuite, trois criblages ont été réalisés. Dans le premier cas, nous avons recherché les partenaires des domaines bHLH et Orange (bHLH-O). Le domaine bHLH est en effet responsable de la dimérisation de ce type de protéine. Le domaine Orange qui suit le domaine bHLH, pourrait participer dans le choix du partenaire d’hétérodimérisation. Nous avons isolé deux facteurs de type bHLH-Orange apparentés à HRT1, XHairy1 et XHairy2b et confirmé leur interaction avec XHRT1. Les domaines impliqués dans ces interactions sont les bHLH-O pour les trois facteurs. Ce même criblage nous a permis d’isoler un nouvel ADNc qui code une protéine sans domaine apparent connu actuellement. Nous avons montré que cette protéine reconnaissait spécifiquement le domaine Orange de HRT1 mais pas celui des autres facteurs de type bHLH-O. Elle a été baptisée BOIP pour Bc8 Orange Interacting Protein. Le rôle physiologique de cette interaction n’a pu être démontré. Nous avons établi que la protéine BOIP pouvait aussi s’homodimériser. Nous avons aussi déterminé son profil d’expression chez le xénope et la souris. Son transcrit est hautement présent dans les testicules adultes. La protéine pourrait donc jouer un rôle important dans la spermatogenèse. Les deux autres criblages, utilisant les domaines situés dans la partie C-terminale de XHRT1, ont apporté des nouveaux partenaires potentiels, mais ces interactions n’ont pu être confirmées dans un système indépendant. Enfin, en étudiant plus en détail les interactions entre XHRT1 et XHairy1 ou XHairy2b, nous avons mis à jour une possible fonction de spécificité dans le choix du partenaire dans la région C-terminale de HRT1. La formation de ces dimères pourrait jouer un rôle dans la formation du tube neural mais également dans d’autres différenciations tissulaires.

L'apparition de nouvelles structures telles que la crête neurale, les placodes et le mésoderme crânien a été essentielle pour l'émergence de la tête des vertébrées. Fait intéressant, le mésoderme de la tête des vertébrés n'est pas segmenté alors qu'il est supposé que le mésoderme de l'ancêtre de tous les chordés était totalement segmenté. De même le corps du le céphalochordé amphioxus est entièrement segmenté. Des travaux menés par l'équipe ont montré le rôle central du signal FGF dans la formation des somites les plus antérieures chez l'amphioxus. Afin de mieux comprendre le rôle de ce signal pour la formation de ces somites, nous avons réalisé une étude transcriptomique comparative par RNA-seq. Cette analyse a mis en évidence plusieurs gènes que sont impliqués dans la somitogenèse et la myogenèse et sous le contrôle du signal FGF. Nous avons pu montrer grâce à des analyses fonctionnelles que ER81/Erm/PEA3 et Six1/2 ont un rôle majeur dans la formation des somites les plus antérieures chez l'amphioxus. Inversement, Pax3/7 est impliqué dans la formation des somites postérieures. Cette cascade de régulation est semblable à celle observée lors de la somitogenèse pour les muscles du tronc chez les vertébrés, mais diverge de la cascade de gènes contrôlant la formation des muscles de la tête chez les vertébrés. Tous ces résultats supportent l'hypothèse selon laquelle le changement de fonction du signal FGF durant le développement précoce a été une étape clé pour la perte des somites antérieures, libérant ainsi les contraintes dans la partie antérieure de l'embryon et permettant dans un second temps l'acquisition des muscles de la tête chez l'ancêtre commun des vertébrés. / A central question in Evo-Devo is to understand the origin of the vertebrates’ head. The appearance of new structures such as the neural crest, placodes and a cranial mesoderm were essential for the appearance of the head in the vertebrates. Interestingly, it is supposed that the ancestor of all chordates was completely segmented. Remarkably, the cephalochordate amphioxus is completely segmented in the full length of its body as the hypothetical ancestor of all chordates. Moreover, it has been showed that the FGF signal plays a central role in the formation of the anterior-most somites of amphioxus. Thus, in order to understand the downstream signaling pathway under the control of the FGF signal for the formation of the anterior-most somites in amphioxus, we performed a comparative RNA-seq analysis. This analysis revealed several vertebrates orthologues genes playing roles in somitogenesis or myogenesis and under the control of the FGF signal. Furthermore, functional analysis revealed that ER81/Erm/PEA3 and Six1/2 plays majors roles in the formation of the anterior-most somites in amphioxus. Conversely, Pax3/7 is involved in the formation of the posterior somites. This regulatory cascade resembles that for the control of trunk somitogenesis in vertebrates and diverges from the gene cascades controlling the formation of the vertebrate head muscles. Altogether, our results strengthen the hypothesis that changes in the FGF function during early development were instrumental for the loss of anterior somites, releasing developmental constraints in the anterior part of the embryo and allowing a secondary acquisition of head muscles in the ancestor of vertebrates.

Le sujet de cette thèse est l'étude quantitative du rôle du morphogène Fgf8 durant la somitogenèse en utilisant des perturbations spatio-temporelles de sa concentration dans un embryon de poisson zèbre en développement. Mon objectif était d'élucider le rôle que joue Fgf8 dans le modèle du «clock and wavefront» de la somitogenèse . A cet effet, j'ai développé des moyens optiques afin de perturber rapidement sa concentration dans un embryon vivant par un éclairage approprié. J’ai montré que le blocage du Fgf8 endogène (en utilisant un morpholino contre Fgf8) ou sa surexpression (en utilisant une approche de photo-activation développée dans le laboratoire d'accueil) affectaient la taille des somites observées dans un embryon de poisson zèbre à 24 hpf. Pour comprendre la raison de ce changement de taille des somitesj’ai construit un système de vidéomicroscopie à intervalle régulier qui m'a permis de suivre l'évolution parallèle de 20-30 embryons de poisson zèbre en temps réel. Après avoir comparé la période de la segmentation, la vitesse d'allongement de la queue, la vitesse de racourcissement du PSM et la distribution spatiale de MAPK phosphorylé, mes résultats montrent que la sur-expression globale de Fgf8 induit un retard subtil dans la période de segmentation et ralentit la vitesse de déplacement du front d'onde, qui est la cause principale de la variation de taille des somites. / The subject of this thesis is the quantitative study of the role of Fgf8 in somitogenesis using spatio-temporal perturbations of its concentration in a developing zebrafish embryo. My goal was to elucidate the role that Fgf8 plays in the clock and wavefront model of somitogenesis. For that purpose, I have developed optical means to quickly perturb its concentration in a live embryo by an appropriate illumination. I have shown that blocking endogenous Fgf8 (using a morpholino against Fgf8) or inducing over-expression of Fgf8 (using a photo-activable approach developed in the host lab) affected the size of somites observed at 24 hpf in a zebrafish embryo. To address the reason for this change in somite size, I have built a time-lapse microscopy set-up that allowed me to monitor the parallel development of 20-30 zebrafish embryos in real time. After comparing the period of segmentation, the velocity of tail elongation, the speed of PSM shortening, and the MAPK pattern of phosphorylation (the target of Fgf8), my results show that global over-expression of Fgf8 induces a subtle delay in the segmentation period and slows down the posterior moving wavefront velocity, which is the major cause of the change in somite size.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22699号 / 医科博第114号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 影山 龍一郎, 教授 妻木 範行, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM