Evolution de la régulation des gènes Dlx au cours de la morphogenèse

La morphogenèse des mâchoires est un processus complexe impliquant la migration et la différenciation des cellules de la crête neurale (CCNCs) ainsi que des cellules mésodermiques qui vont coloniser le premier arc branchial (AB1). Avant d'atteindre leur destination finale, les CCNCs ne sont pas encore engagées dans des voies morphogénétiques spécifiques. Des signalisations moléculaires et des interactions cellulaires vont avoir lieu au cours de la colonisation de l'AB1 permettant l'engagement des CCNCs vers une voie de signalisation donnée. La voie de signalisation à l'Edn1 est nécessaire et suffisante pour induire l'identité de la mâchoire inférieure; cependant le mécanisme par lequel la spécification a lieu n'est pas totalement élucidé. De plus, l'absorption d'acide rétinoïque (AR) ou de son précurseur, la vitamine A, durant la grossesse est associée à une augmentation de l'incidence des lésions craniofaciales. Au début de ma thèse, l'origine de ces effets tératogènes était énigmatique car la transduction de la signalisation à l'AR n'est pas active dans les CCNCs. Dans les deux premières études, nous avons pu montrer, chez la souris et Xenopus laevis, que l'action de l'AR s exerce au niveau des signalisations Edn1 et Fgf8 provenant des épitheliums de l'AB1. L'AR va réduire l'expression de ces deux signaux moléculaires de façon graduelle selon le temps d administration, la dose utilisée et la durée du traitement. L Edn1 et Fgf8 induisent l'activation des gènes Dlx dans les CCNCs, permettant le déclenchement de programmes morphogénétiques donnant naissance aux différents éléments squelettiques constituant les mâchoires. Ainsi, un bref traitement à l'AR provoque des malformations craniofaciales différents selon l age exact de l embryon lors de l administration. L action tératogène de l AR au niveau craniofacial s exerce dans une fenêtre temporelle réduite correspondant à la colonisation de l'AB1 par les CCNCs. La troisième étude ouvre de nouvelles perspectives sur la dynamique spatio-temporelle et le dosage génique du processus permettant la morphogenèse des mâchoires proposant ainsi un lien entre les altérations de ce processus et l'origine de malformations humaines appelées "syndromes du premier arc". Cette thèse a permis de contribuer à cinq autres études impliquant la fonction et la régulation de Dlx5 et Dlx6 dans l ostéogenèse, le développement des membres et du tubercule génital et la différenciation des cellules de Leydig.Jaw morphogenesis is a complex process involving the migration and differentiation of cephalic neural crest cells (CNCCs) and mesodermal cells to the first pharyngeal arch (PA1). Before reaching their destination CCNCs are not yet engaged in specific morphogenetic pathways. Molecular signalling and cellular interactions occurring during PA1 colonization determine the genesis of facial structures. Endothelin-1 (Edn1) signaling is necessary and sufficient to specify lower jaw identity; the mechanism through which this specification takes place is not yet completely elucidated. Intake of retinoic acid (RA) or of its precursor, vitamin A, during early pregnancy is associated with increased incidence of craniofacial lesions. Before my thesis, the origin of these teratogenic effects was enigmatic as in cranial neural crest cells (CNCCs), the RA-transduction pathway is not active. In the first two articles we show that, in mouse and Xenopus laevis, RA acts on the signalling epithelium of PA1 reducing the expression of Edn1 and Fgf8. Depending on the time of administration and on the dose and length of treatment, RA reduces progressively the expression of these two molecular signals. Edn1 and Fgf8 are instrumental in activating Dlx genes in incoming CNCCs, thereby triggering the morphogenetic programs, which specify different jaw elements. We report that, RA treatments provokes dramatically different craniofacial malformations when administered at slightly different developmental times within a narrow temporal interval corresponding to the colonization of the 1st PA by CNCCs. The third article provides new insight into the spatio-temporal and gene-dosage dynamics of the jaw morphogenetic process proposing a link between the alteration of this process and the origin of human malformation collectively known as "First Arch Syndromes". This thesis contributed also to five other studies on the role and regulation of Dlx5 and Dlx6 during osteogenesis, limb development, genital tubercle morphogenesis and Leydig cells differentiation.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Combined function of <i>HoxA</i> and <i>HoxB</i> clusters in neural crest cells

The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity.</p

Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain.

International audienceEmbryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments

Spatio-temporal dynamics of gene expression of the Edn1-Dlx5/6 pathway during development of the lower jaw

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Tumor Necrosis Factor-Alpha-Elicited Stimulation of Gamma-Secretase Is Mediated by C-Jun N-Terminal Kinase-Dependent Phosphorylation of Presenilin and Nicastrin

gamma-Secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1, and Pen-2, and it catalyzes the final proteolytic step in the processing of amyloid precursor protein to generate amyloid- beta. Our previous results showed that tumor necrosis factor-alpha ( TNF- alpha) can potently stimulate gamma-secretase activity through a c-Jun N- terminal kinase (JNK)-dependent pathway. Here, we demonstrate that TNF- alpha triggers JNK-dependent serine/threonine phosphorylation of PS1 and NCT to stimulate gamma-secretase activity. Blocking of JNK activity with a potent JNK inhibitor (SP600125) reduces TNF-alpha-triggered phosphorylation of PS1 and NCT. Consistent with this, we show that activated JNKs can be copurified with gamma- secretase complexes and that active recombinant JNK2 can promote the phosphorylation of PS1 and NCT in vitro. Using site-directed mutagenesis and a synthetic peptide, we clearly show that the Ser(319)Thr(320) motif in PS1 is an important JNK phosphorylation site that is critical for the TNF-alpha-elicited regulation of gamma-secretase. This JNK phosphorylation of PS1 at Ser(319) Thr(320) enhances the stability of the PS1 C-terminal fragment that is necessary for gamma-secretase activity. Together, our findings strongly suggest that JNK is a critical intracellular mediator of TNF-alpha- elicited regulation of gamma-secretase and governs the pivotal step in the assembly of functional gamma-secretase

Positive Regulation of Steroidogenic Acute Regulatory Protein Gene Expression through the Interaction between Dlx and GATA-4 for Testicular Steroidogenesis

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BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations