CARACTERISATION DU RESEAU DE SIGNALISATION IMPLIQUE DANS LA MAINTENANCE ET LA PROLIFERATION DES CELLULES SOUCHES DE LA RETINE DU XENOPE

In contrast to the adult mammals, the retina of amphibians shows continuous growth during adulthood through active neural stem cells localized in the defined niche called ciliary marginal zone (CMZ). This model offers an exceptional tool to study in vivo the molecular mechanisms involved in the maintenance and proliferation of neural stem cells during post-embryonic stages. In this order, the identification and the characterization of the signaling pathways acting in biological retinal stem cell niche is an essential step.My PhD research was divided in two main parts: the study of the interaction between the Wnt and Hedgehog pathways within the CMZ and the functional study of Yap, the downstream effector of the Hippo pathway in this model. By using genetic and pharmacological tools, the first part of this project demonstrated an unexpected antagonism between the Wnt and the Hedgehog signaling in the CMZ that regulates proliferative activity of retinal stem and progenitor cells. In this article, we propose a model in which an antagonistic interplay of Wnt and Hedgehog pathways may regulate the balance proliferation/differentiation in the post-embryonic retina. Second, gain and loss of function experiments of Yap have shown that this factor plays a key role in the regulation of temporal replication of DNA retinal stem cells. Indeed, inhibition of Yap leads to strong reduction of the S-phase length during the cell cycle associated with genomic instability. c-Myc and p53-p21 overactivation seems to be involved in this phenotype. This work also allowed us to identify a novel YAP partner, the transcriptional factor PKNOX1. We indeed propose a model in which the YAP/PKNOX1 complex may be required for the successful convening of the replication phase on stem cells, essential for the maintenance of genome integrity on the cells and their progeny.Contrairement aux mammifères adultes, la rétine des amphibiens possède la particularité de croître durant toute la vie de l'animal grâce à l'activité continue d'une population de cellules souches localisée au sein d'une niche bien délimitée, la zone marginale ciliaire (ZMC). Ce modèle offre ainsi la possibilité d'étudier in vivo les mécanismes moléculaires à l'origine du maintien et de la prolifération des cellules souches neurales à des stades post-embryonnaires. Dans ce but, l'identification et la caractérisation des différentes voies de signalisation présentes au sein de la niche biologique des cellules souches rétiniennes est une première étape indispensable. Mon projet de thèse a été divisé en deux objectifs principaux: l'étude des interactions entre les voies Wnt et Hedgehog au sein de la ZMC chez le xénope et la réalisation de l'étude fonctionnelle de Yap, l'effecteur principal de la voie de signalisation Hippo dans ce modèle. Par des approches génétiques et pharmacologiques, la première partie de ce projet a permis de mettre en évidence un antagonisme inattendu entre les signaux Wnt et Hedgehog au sein de la ZMC qui régule l'activité proliférative des cellules souches et des progéniteurs rétiniens. Ce travail nous a conduit à proposer un modèle dans lequel ces deux voies réguleraient la balance prolifération/différenciation dans la rétine post-embryonnaire. Dans un deuxième temps, les expériences de gain et de perte de fonction du gène Yap ont montré que ce dernier joue un rôle essentiel dans la régulation du programme temporel de la phase de réplication de l'ADN des cellules souches rétiniennes. En effet, l'inhibition de Yap entraîne une importante réduction de la durée de la phase S du cycle cellulaire associée à une instabilité génomique. Une surexpression de c-Myc et de la voie p53-p21 semble impliquée dans ce phénotype. Nos travaux nous ont également permis d'identifier un nouveau partenaire de YAP, le facteur de transcription PKNOX1. L'ensemble de ces données nous a ainsi conduit à proposer un modèle selon lequel le complexe YAP/PKNOX1 pourrait être nécessaire au bon déroulement de la phase de réplication des cellules souches, indispensable à la maintenance de l'intégrité du génome de ces cellules et de leur descendance

Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS

CHARACTERIZATION OF THE SIGNALING NETWORK INVOLVED IN THE MAINTENANCE AND PROLIFERATION OF XENOPUS RETINAL STEM CELLS

Contrairement aux mammifères adultes, la rétine des amphibiens possède la particularité de croître durant toute la vie de l'animal grâce à l'activité continue d'une population de cellules souches localisée au sein d'une niche bien délimitée, la zone marginale ciliaire (ZMC). Ce modèle offre ainsi la possibilité d'étudier in vivo les mécanismes moléculaires à l'origine du maintien et de la prolifération des cellules souches neurales à des stades post-embryonnaires. Dans ce but, l'identification et la caractérisation des différentes voies de signalisation présentes au sein de la niche biologique des cellules souches rétiniennes est une première étape indispensable. Mon projet de thèse a été divisé en deux objectifs principaux: l'étude des interactions entre les voies Wnt et Hedgehog au sein de la ZMC chez le xénope et la réalisation de l'étude fonctionnelle de Yap, l'effecteur principal de la voie de signalisation Hippo dans ce modèle. Par des approches génétiques et pharmacologiques, la première partie de ce projet a permis de mettre en évidence un antagonisme inattendu entre les signaux Wnt et Hedgehog au sein de la ZMC qui régule l'activité proliférative des cellules souches et des progéniteurs rétiniens. Ce travail nous a conduit à proposer un modèle dans lequel ces deux voies réguleraient la balance prolifération/différenciation dans la rétine post-embryonnaire. Dans un deuxième temps, les expériences de gain et de perte de fonction du gène Yap ont montré que ce dernier joue un rôle essentiel dans la régulation du programme temporel de la phase de réplication de l'ADN des cellules souches rétiniennes. En effet, l'inhibition de Yap entraîne une importante réduction de la durée de la phase S du cycle cellulaire associée à une instabilité génomique. Une surexpression de c-Myc et de la voie p53-p21 semble impliquée dans ce phénotype. Nos travaux nous ont également permis d'identifier un nouveau partenaire de YAP, le facteur de transcription PKNOX1. L'ensemble de ces données nous a ainsi conduit à proposer un modèle selon lequel le complexe YAP/PKNOX1 pourrait être nécessaire au bon déroulement de la phase de réplication des cellules souches, indispensable à la maintenance de l'intégrité du génome de ces cellules et de leur descendance.In contrast to the adult mammals, the retina of amphibians shows continuous growth during adulthood through active neural stem cells localized in the defined niche called ciliary marginal zone (CMZ). This model offers an exceptional tool to study in vivo the molecular mechanisms involved in the maintenance and proliferation of neural stem cells during post-embryonic stages. In this order, the identification and the characterization of the signaling pathways acting in biological retinal stem cell niche is an essential step.My PhD research was divided in two main parts: the study of the interaction between the Wnt and Hedgehog pathways within the CMZ and the functional study of Yap, the downstream effector of the Hippo pathway in this model. By using genetic and pharmacological tools, the first part of this project demonstrated an unexpected antagonism between the Wnt and the Hedgehog signaling in the CMZ that regulates proliferative activity of retinal stem and progenitor cells. In this article, we propose a model in which an antagonistic interplay of Wnt and Hedgehog pathways may regulate the balance proliferation/differentiation in the post-embryonic retina. Second, gain and loss of function experiments of Yap have shown that this factor plays a key role in the regulation of temporal replication of DNA retinal stem cells. Indeed, inhibition of Yap leads to strong reduction of the S-phase length during the cell cycle associated with genomic instability. c-Myc and p53-p21 overactivation seems to be involved in this phenotype. This work also allowed us to identify a novel YAP partner, the transcriptional factor PKNOX1. We indeed propose a model in which the YAP/PKNOX1 complex may be required for the successful convening of the replication phase on stem cells, essential for the maintenance of genome integrity on the cells and their progeny

Neurog2 Deficiency Uncovers a Critical Period of Cell Fate Plasticity and Vulnerability among Neural-Crest-Derived Somatosensory Progenitors.

Functionally distinct classes of dorsal root ganglia (DRG) somatosensory neurons arise from neural crest cells (NCCs) in two successive phases of differentiation assumed to be respectively and independently controlled by the proneural genes Neurog2 and Neurog1. However, the precise role of Neurog2 during this process remains unclear, notably because no neuronal loss has been reported hitherto in Neurog2-/- mutants. Here, we show that at trunk levels, Neurog2 deficiency impairs the production of subsets of all DRG neuron subtypes. We establish that this phenotype is highly dynamic and reflects multiple defects in NCC-derived progenitors, including somatosensory-to-melanocyte fate switch, apoptosis, and delayed differentiation which alters neuronal identity, all occurring during a narrow time window when Neurog2 temporarily controls onset of Neurog1 expression and neurogenesis. Collectively, these findings uncover a critical period of cell fate plasticity and vulnerability among somatosensory progenitors and establish that Neurog2 function in the developing DRG is broader than initially envisaged.info:eu-repo/semantics/publishe

Loss of G9a does not phenocopy the requirement for Prdm12 in the development of the nociceptive neuron lineage.

Prdm12 is an epigenetic regulator expressed in developing and mature nociceptive neurons, playing a key role in their specification during neurogenesis and modulating pain sensation at adulthood. In vitro studies suggested that Prdm12 recruits the methyltransferase G9a through its zinc finger domains to regulate target gene expression, but how Prdm12 interacts with G9a and whether G9a plays a role in Prdm12's functional properties in sensory ganglia remain unknown. Here we report that Prdm12-G9a interaction is likely direct and that it involves the SET domain of G9a. We show that both proteins are largely co-expressed in dorsal root ganglia during early murine development, opening the possibility that G9a plays a role in DRG and may act as a mediator of Prdm12's function in the development of nociceptive sensory neurons. To test this hypothesis, we conditionally inactivated G9a in neural crest using a Wnt1-Cre transgenic mouse line. We found that the specific loss of G9a in the neural crest lineage does not lead to dorsal root ganglia hypoplasia due to the loss of somatic nociceptive neurons nor to the ectopic expression of the visceral determinant Phox2b as observed upon Prdm12 ablation. These findings suggest that Prdm12 function in the initiation of the nociceptive lineage does not critically involves its interaction with G9a.info:eu-repo/semantics/publishe

The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish

AbstractLymphangiogenesis, the formation of lymphatic vessels is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in zebrafish. We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of a proper trunk lymphatic network and leading to edema development. We show that Sorbs1 is probably not part of the Vegfc signaling, but instead might interacts with the BMP pathways. Mechanistically, we show that Sorbs1 controls FAK/Src signaling to impact on Rac1 and RhoA GTPases-regulated cytoskeleton processes. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contact rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. Our data thus establish Sorbs1 as an important regulator of lymphangiogenesis distinct from the Vegfc signaling axis, increasing our understanding of context-specific vascular and lymphatic development.</jats:p