Molars and incisors: show your microarray IDs.

BACKGROUND: One of the key questions in developmental biology is how, from a relatively small number of conserved signaling pathways, is it possible to generate organs displaying a wide range of shapes, tissue organization, and function. The dentition and its distinct specific tooth types represent a valuable system to address the issues of differential molecular signatures. To identify such signatures, we performed a comparative transcriptomic analysis of developing murine lower incisors, mandibular molars and maxillary molars at the developmental cap stage (E14.5). RESULTS: 231 genes were identified as being differentially expressed between mandibular incisors and molars, with a fold change higher than 2 and a false discovery rate lower than 0.1, whereas only 96 genes were discovered as being differentially expressed between mandibular and maxillary molars. Numerous genes belonging to specific signaling pathways (the Hedgehog, Notch, Wnt, FGF, TGFβ/BMP, and retinoic acid pathways), and/or to the homeobox gene superfamily, were also uncovered when a less stringent fold change threshold was used. Differential expressions for 10 out of 12 (mandibular incisors versus molars) and 9 out of 10 selected genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of incisor versus molar differentially expressed genes revealed that 143 genes belonged to 9 networks with intermolecular connections. Networks with the highest significance scores were centered on the TNF/NFκB complex and the ERK1/2 kinases. Two networks ERK1/2 kinases and tretinoin were involved in differential molar morphogenesis. CONCLUSION: These data allowed us to build several regulatory networks that may distinguish incisor versus molar identity, and may be useful for further investigations of these tooth-specific ontogenetic programs. These programs may be dysregulated in transgenic animal models and related human diseases leading to dental anomalies.journal articleresearch support, non-u.s. gov't2013 Mar 262013 03 26importe

Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo

The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating patterning of prethalamus and ventral telencephalon

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3-/- corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3-/- VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development

Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells

A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement.

BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.journal articleresearch support, non-u.s. gov't2016 Feb2015 10 26importe

Effets de la régulation de Notch1 sur le devenir des cellules souches neurales et sur des lignées tumorales du système nerveux

Parmi les facteurs susceptibles d’influencer la production de neurones versus cellules gliales à partir de cellules souches neurales, nous avons étudié le rôle de la voie de signalisation intercellulaire Notch. En utilisant le modèle in vitro des neurosphères qui représentent la descendance clonale des cellules souches neurales, nous avons montré que la voie Notch agissait en deux étapes : (i) la voie Notch favorisait le lignage glial et réprimait le lignage neuronal ; (ii) Notch stimule la différenciation des astrocytes et inhibe celle des neurones et des oligodendrocytes. Nous avons sélectionné une petite molécule non peptidique, le tCFA15 qui favorise la production de neurones aux dépens des cellules gliales dans les neurosphères, un effet qui rappelle celui résultant de l’inactivation de la voie Notch. L’analyse moléculaire a montré que le tCFA15 diminuait spécifiquement l’expression de Notch1 et par conséquent l’expression de Hes5 ce qui confirme l’inactivation fonctionnelle de Notch1. Nous avons montré que le tCFA15 en plus de son action sur Notch1, diminue la phosphorylation de STAT3, suggérant une interaction entre les voies de signalisation Notch et STAT3. Nous avons montré par des expériences d’activation et d’inactivation de ces deux voies que STAT3 était en amont de Notch1. L’analyse moléculaire a confirmé que STAT3 contrôlait l’expression de Notch1. Le dysfonctionnement de la voie Notch est à l’origine de nombreuses pathologies qui vont de certaines maladies neurodégénératives à différents types de cancers. Notch exerce des effets opposés sur la différenciation des neurones, oligodendrocytes et astrocytes. Nous avons cherché à établir un parallèle entre cet effet physiologique et l’effet de la voie Notch sur les lignées tumorales du système nerveux. Nous avons montré que, même si dans tous les cas il provoque une diminution de Notch1, le tCFA15 conduit à des résultats opposés en ce qui concerne les neuroblastomes et les oligodendrogliomes d’une part, et les astrocytomes d’autre part. Les cellules souches neurales sont localisées dans des niches vasculaires. Nous avons étudié le rôle éventuel de la voie Notch dans les interactions réciproques entre les précurseurs des cellules endothéliales et les neurosphères. Nous avons montré que ces interactions existent in vitro et qu’elles passent par l’induction de l’expression de Dll4 dans les précurseurs des cellules endothéliales et dans les cellules des neurosphères. We are interested in the role of the intercellular Notch signaling pathway in the production of neurons versus glia from neural stem cells. Using the in vitro model system of neurospheres, which consists in the clonal progeny of neural stem cells, our group has shown that Notch signalling was acting in two steps. In a first step, Notch is acting as a switch promoting the glial lineage while it represses the neuronal lineage. In a second step, Notch is acting on the differentiation, and promotes astrocytic differentiation while it inhibits neuronal and oligodendroglial differentiation. We have selected a small non-peptidic molecule, tCFA15, which promotes neurons at the expense of glial cells in manner reminiscent of the inactivation of Notch. My work consisted first, in elucidating the mechanism of action of tCFA15 at the molecular level. We have shown that tCFA15 specifically decreases the expression of Notch1 gene, and subsequently of Hes5, a downstream target gene of Notch activation. In addition, we have shown that tCFA15 causes a diminution of the tyr705 phosphorylation of STAT3, thereby suggesting a link between both Notch and STAT3 signalings. Molecular investigations have shown that inhibiting STAT3 results in a decrease in Notch1 expression, thus confirming the gain and loss of functions experiments which placed STAT3 as an upstream regulator of Notch1. Notch dysfunction is at the origin of numerous pathologies, ranging from neurodegenerative diseases to cancers. We have shown that Notch has opposite effects on the differentiation of neurons, oligodendrocytes and astrocytes. We attempted to establish a parallel between those physiological effects and a putative effect of Notch on various tumour cell lines from the nervous system. Using tCFA15 as a pharmacological tool to modulate Notch function, we have shown that, although it consistently decreases Notch1 in most of all studied cancer cell lines, tCFA15 has an opposite effect on neuroblastoma and oligodendroglioma on the one hand and on astrocytoma, on the other hand. Neural stem cells are localised in vascular niches. We have examined the role of the Notch pathway in the reciprocal interactions between the endothelial precursors cells and the neurospheres. We have shown that those interactions do exist in vitro and that they are mediated by the reciprocal induction of the expression of Dll4 genes in both the endothelial precursors cells and neurospheres

Effects of Notch1 regulation on the cell fate of neural tem cells and on tumor cell lines of the nervous system

Parmi les facteurs susceptibles d'influencer la production de neurones versus cellules gliales à partir de cellules souches neurales, nous avons étudié le rôle de la voie de signalisation intercellulaire Notch.En utilisant le modèle in vitro des neurosphèWe are interested in the role of the intercellular Notch signaling pathway in the production of neurons versus glia from neural stem cells.Using the in vitro model system of neurospheres, which consists in the clonal progeny of neural stem cells, our gro

Effets de la régulation de Notch1 sur le devenir des cellules souches neurales et sur des lignées tumorales du système nerveux

Parmi les facteurs susceptibles d'influencer la production de neurones versus cellules gliales à partir de cellules souches neurales, nous avons étudié le rôle de la voie de signalisation intercellulaire Notch.En utilisant le modèle in vitro des neurosphères qui représentent la descendance clonale des cellules souches neurales, nous avons montré que la voie Notch agissait en deux étapes: (i) la voie Notch favorisait le lignage glial et réprimait le lignage neuronal; (ii) Notch stimule la différenciation des astrocytes et inhibe celle des neurones et des oligodendrocytes. Nous avons sélectionné une petite molécule non peptidique, le tCFA15 qui favorise la production de neurones aux dépens des cellules gliales dans les neurosphères, un effet qui rappelle celui résultant de l'inactivation de la voie Notch. L'analyse moléculaire a montré que le tCFA15 diminuait spécifiquement l'expression de Notch1 et par conséquent l'expression de Hes5 ce qui confirme l'inactivation fonctionnelle de Notch1. Nous avons montré que le tCFA15 en plus de son action sur Notch1, diminue la phosphorylation de STAT3, suggérant une interaction entre les voies de signalisation Notch et STAT3. Nous avons montré par des expériences d'activation et d'inactivation de ces deux voies que STAT3 était en amont de Notch1. L'analyse moléculaire a confirmé que STAT3 contrôlait l'expression de Notch1. Le dysfonctionnement de la voie Notch est à l'origine de nombreuses pathologies qui vont de certaines maladies neurodégénératives à différents types de cancers.Notch exerce des effets opposés sur la différenciation des neurones, oligodendrocytes et astrocytes. Nous avons cherché à établir un parallèle entre cet effet physiologique et l'effet de la voie Notch sur les lignées tumorales du système nerveux. Nous avons montré que, même si dans tous les cas il provoque une diminution de Notch1, le tCFA15 conduit à des résultats opposés en ce qui concerne les neuroblastomes et les oligodendrogliomes d'une part, et les astrocytomes d'autre part.Les cellules souches neurales sont localisées dans des niches vasculaires. Nous avons étudié le rôle éventuel de la voie Notch dans les interactions réciproques entre les précurseurs des cellules endothéliales et les neurosphères. Nous avons montré que ces interactions existent in vitro et qu'elles passent par l'induction de l'expression de Dll4 dans les précurseurs des cellules endothéliales et dans les cellules des neurosphères.We are interested in the role of the intercellular Notch signaling pathway in the production of neurons versus glia from neural stem cells.Using the in vitro model system of neurospheres, which consists in the clonal progeny of neural stem cells, our group has shown that Notch signalling was acting in two steps. In a first step, Notch is acting as a switch promoting the glial lineage while it represses the neuronal lineage. In a second step, Notch is acting on the differentiation, and promotes astrocytic differentiation while it inhibits neuronal and oligodendroglial differentiation.We have selected a small non-peptidic molecule, tCFA15, which promotes neurons at the expense of glial cells in manner reminiscent of the inactivation of Notch. My work consisted first, in elucidating the mechanism of action of tCFA15 at the molecular level. We have shown that tCFA15 specifically decreases the expression of Notch1 gene, and subsequently of Hes5, a downstream target gene of Notch activation. In addition, we have shown that tCFA15 causes a diminution of the tyr705 phosphorylation of STAT3, thereby suggesting a link between both Notch and STAT3 signalings. Molecular investigations have shown that inhibiting STAT3 results in a decrease in Notch1 expression, thus confirming the gain and loss of functions experiments which placed STAT3 as an upstream regulator of Notch1.Notch dysfunction is at the origin of numerous pathologies, ranging from neurodegenerative diseases to cancers. We have shown that Notch has opposite effects on the differentiation of neurons, oligodendrocytes and astrocytes. We attempted to establish a parallel between those physiological effects and a putative effect of Notch on various tumour cell lines from the nervous system. Using tCFA15 as a pharmacological tool to modulate Notch function, we have shown that, although it consistently decreases Notch1 in most of all studied cancer cell lines, tCFA15 has an opposite effect on neuroblastoma and oligodendroglioma on the one hand and on astrocytoma, on the other hand.Neural stem cells are localised in vascular niches. We have examined the role of the Notch pathway in the reciprocal interactions between the endothelial precursors cells and the neurospheres. We have shown that those interactions do exist in vitro and that they are mediated by the reciprocal induction of the expression of Dll4 genes in both the endothelial precursors cells and neurospheres.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Retinoic acid regulates olfactory progenitor cell fate and differentiation.

International audienceBACKGROUND: In order to fulfill their chemosensory function, olfactory neurons are in direct contact with the external environment and are therefore exposed to environmental aggressive factors. Olfaction is maintained through life because, unlike for other sensory neuroepithelia, olfactory neurons have a unique capacity to regenerate after trauma. The mechanisms that control the ontogenesis and regenerative ability of these neurons are not fully understood. Here, we used various experimental approaches in two model systems (chick and mouse) to assess the contribution of retinoic acid signaling in the induction of the olfactory epithelium, the generation and maintenance of progenitor populations, and the ontogenesis and differentiation of olfactory neurons. RESULTS: We show that retinoic acid signaling, although dispensable for initial induction of the olfactory placode, plays a key role in neurogenesis within this neuroepithelium. Retinoic acid depletion in the olfactory epithelium, both in chick and mouse models, results in a failure of progenitor cell maintenance and, consequently, differentiation of olfactory neurons is not sustained. Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid. CONCLUSIONS: Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells. Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium