Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain

International audienceBACKGROUND: Neurons arise in very specific regions of the neural tube, controlled by components of the Notch signalling pathway, proneural genes, and other bHLH transcription factors. How these specific neuronal areas in the brain are generated during development is just beginning to be elucidated. Notably, the critical role of proneural genes during differentiation of the neuronal populations that give rise to the early axon scaffold in the developing brain is not understood. The regulation of their downstream effectors remains poorly defined. RESULTS: This study provides the first overview of the spatiotemporal expression of proneural genes in the neuronal populations of the early axon scaffold in both chick and mouse. Overexpression studies and mutant mice have identified a number of specific neuronal genes that are targets of proneural transcription factors in these neuronal populations. CONCLUSION: Together, these results improve our understanding of the molecular mechanisms involved in differentiation of the first neuronal populations in the brain

Etude du rôle de la voie NOTCH dans la régulation de Shh au cours du développement du cerveau antérieur chez les vertébrés

Holoprosencephaly (HPE) is a developmental pathology defined by a lack of forebrain cleavage along the midline and characterized by phenotypic heterogeneity and incomplete penetrance. The morphogen SHH is the main molecule involved in the onset of HPE. The NOTCH pathway has also been implicated in the occurrence of HPE in patients. In this study, I showed that NOTCH pathway activation was necessary to maintain Shh expression in the ventral forebrain. Furthermore, I demonstrated the implication of NOTCH pathway in early neurogenesis in the hypothalamus. Finally, a hypomorphic mouse model for both SHH and NOTCH pathways has shown the appearance of pituitary gland dysplasia associated with basisphenoid bone malformations reminiscent of abnormalities observed in HPE patients. This work has allowed us to generate an animal model reproducing an HPE phenotype based on the partial inactivation of two molecular signaling pathways. This result reinforces the hypothesis of a multigenic transmission for this complexe pathology that affects midline development.L’holoprosencéphalie (HPE) est une pathologie du développement définie par un défaut de clivage du cerveau antérieur le long de la ligne médiane. Elle est caractérisée par une hétérogénéité phénotypique et une pénétrance incomplète. Le morphogène SHH est la principale molécule impliquée dans l’apparition de l’HPE. La voie NOTCH a été également impliquée dans l’apparition d'HPE chez les patients. Dans cette étude, j’ai montré que c’est en maintenant l’expression de Shh dans le cerveau ventral que NOTCH contribue à l’apparition de l’HPE. J’ai également montré que la voie NOTCH était impliquée dans la neurogenèse précoce au niveau de l’hypothalamus. Enfin, des mutants souris hypomorphes pour la voie SHH et la voie NOTCH présentent des anomalies discrètes de la ligne médiane correspondant à une dysplasie de la glande hypophysaire et une malformation de l’os basisphénoïde. Les mêmes anomalies sont observées chez les patients HPE. Ce travail nous a donc permis de générer un modèle animal reproduisant une forme d'HPE qui repose sur la double inactivation partielle de deux voies de signalisation moléculaire. Ce résultat renforce l'hypothèse d'une transmission multigénique pour cette pathologie complexe du développement de la ligne médiane

Study of the role of NOTCH pathway in Shh regulation during vertebrate brain development

L’holoprosencéphalie (HPE) est une pathologie du développement définie par un défaut de clivage du cerveau antérieur le long de la ligne médiane. Elle est caractérisée par une hétérogénéité phénotypique et une pénétrance incomplète. Le morphogène SHH est la principale molécule impliquée dans l’apparition de l’HPE. La voie NOTCH a été également impliquée dans l’apparition d'HPE chez les patients. Dans cette étude, j’ai montré que c’est en maintenant l’expression de Shh dans le cerveau ventral que NOTCH contribue à l’apparition de l’HPE. J’ai également montré que la voie NOTCH était impliquée dans la neurogenèse précoce au niveau de l’hypothalamus. Enfin, des mutants souris hypomorphes pour la voie SHH et la voie NOTCH présentent des anomalies discrètes de la ligne médiane correspondant à une dysplasie de la glande hypophysaire et une malformation de l’os basisphénoïde. Les mêmes anomalies sont observées chez les patients HPE. Ce travail nous a donc permis de générer un modèle animal reproduisant une forme d'HPE qui repose sur la double inactivation partielle de deux voies de signalisation moléculaire. Ce résultat renforce l'hypothèse d'une transmission multigénique pour cette pathologie complexe du développement de la ligne médiane.Holoprosencephaly (HPE) is a developmental pathology defined by a lack of forebrain cleavage along the midline and characterized by phenotypic heterogeneity and incomplete penetrance. The morphogen SHH is the main molecule involved in the onset of HPE. The NOTCH pathway has also been implicated in the occurrence of HPE in patients. In this study, I showed that NOTCH pathway activation was necessary to maintain Shh expression in the ventral forebrain. Furthermore, I demonstrated the implication of NOTCH pathway in early neurogenesis in the hypothalamus. Finally, a hypomorphic mouse model for both SHH and NOTCH pathways has shown the appearance of pituitary gland dysplasia associated with basisphenoid bone malformations reminiscent of abnormalities observed in HPE patients. This work has allowed us to generate an animal model reproducing an HPE phenotype based on the partial inactivation of two molecular signaling pathways. This result reinforces the hypothesis of a multigenic transmission for this complexe pathology that affects midline development

Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus.

International audienceThe vertebrate embryonic prosencephalon gives rise to the hypothalamus, which plays essential roles in sensory information processing as well as control of physiological homeostasis and behavior. While patterning of the hypothalamus has received much attention, initial neurogenesis in the developing hypothalamus has mostly been neglected. The first differentiating progenitor cells of the hypothalamus will give rise to neurons that form the nucleus of the tract of the postoptic commissure (nTPOC) and the nucleus of the mammillotegmental tract (nMTT). The formation of these neuronal populations has to be highly controlled both spatially and temporally as these tracts will form part of the ventral longitudinal tract (VLT) and act as a scaffold for later, follower axons. This review will cumulate and summarize the existing data available describing initial neurogenesis in the vertebrate hypothalamus. It is well-known that the Notch signaling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. It has only recently been proposed that loss of Notch signaling in the developing chick embryo causes an increase in the number of neurons in the hypothalamus, highlighting an early function of the Notch pathway during hypothalamus formation. Further analysis in the chick and mouse hypothalamus confirms the expression of Notch components and Ascl1 before the appearance of the first differentiated neurons. Many newly identified proneural target genes were also found to be expressed during neuronal differentiation in the hypothalamus. Given the critical role that hypothalamic neural circuitry plays in maintaining homeostasis, it is particularly important to establish the targets downstream of this Notch/proneural network

Additional file 1: Figure S1. of Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain

Ascl1 overexpression did not cause ectopic expression of Pax6. (a, b, n = 3) Brain was dissected, flatmounted and in lateral view. There was no upregulation of Pax6 when the embryo was electroporated with the pAscl1 plasmid, which confirmed the specificity of the plasmid. The un-transfected side also showed normal expression of Pax6. For abbreviations see Table 1. (PDF 486 kb

Reduced phenotypic expression in genetic hemochromatosis with time role of exposure to nongenetic modifiers

International audienceBackground & aims - Genetic hemochromatosis is mainly related to the homozygous p.Cys282Tyr (C282Y) mutation in the HFE gene, which causes hepcidin deficiency. Its low penetrance suggests the involvement of cofactors that modulate its expression. We aimed to describe the evolution of disease presentation and of non-genetic factors liable to impact hepcidin production in the long term. Methods - Clinical symptoms, markers of iron load, and risk factors according to the year of diagnosis were recorded over 30 years in a cohort of adult C282Y homozygotes. A total of 2,050 patients (1,460 probands [804 males and 656 females] and 542 relatives [244 males and 346 females]) were studied. Results - Over time: (i) the proband-to-relative ratio remained roughly stable; (ii) the gender ratio tended towards equilibrium among probands; (iii) age at diagnosis did not change among males and increased among females; (iv) the frequency of diabetes and hepatic fibrosis steadily decreased while that of chronic fatigue and distal joint symptoms remained stable; (v) transferrin saturation, serum ferritin and the amount of iron removed decreased; and (vi) the prevalence of excessive alcohol consumption decreased while that of patients who were overweight increased. Tobacco smoking was associated with increased transferrin saturation. Conclusion - Genetic testing did not alter the age at diagnosis, which contrasts with the dramatic decrease in iron load in both genders. Tobacco smoking could be involved in the extent of iron loading. Besides HFE testing, which enables the diagnosis of minor forms of the disease, the reduction of alcohol consumption and the increased frequency of overweight patients may have played a role in the decreased long-term iron load, as these factors are likely to improve hepcidin production. Lay summary - Genetic hemochromatosis is an inherited disorder that leads to progressive iron overload in the body. It results in chronic fatigue and in potential liver (cirrhosis), pancreas (diabetes) and joint (arthritis) damage in adulthood. The present study showed that tobacco smoking may aggravate iron loading, but that hemochromatosis has become less and less severe over the last 30 years despite patients being older at diagnosis, likely because of the protective effects of lower alcohol consumption and of increased weight in the French population

Array-CGH diagnosis in ovarian failure: identification of new molecular actors for ovarian physiology

International audienceBackground: Ovarian failure (OF) is considered premature if it occurs before the age of 40. This study investigates the genetic aetiology underlying OF in women under the age of 40 years.Methods: We conducted an experimental prospective study performing all genome microarrays in 60 patients younger than 40 years presenting an OF revealed by a decrease of circulating Anti-Mullerian Hormone (AMH) and leading to an oocyte donation program.Results: We identified nine significant copy number variations (CNVs) including candidate genes potentially implicated in reproductive function. These genes are principally involved in cell division and chromosome segregation (SYCE1, CLASP1, CENP-A, CDC16), in ciliary development and/or function (RSPH1, KIF24), are linked with known gonadal genes or expressed in female genital tract (CSMD1, SEMA6D, KIAA1324).Conclusions: Our data strengthen the idea that microarrays should be used in combination with karyotype for aetiological assessment of patients with OF. This analysis may have a therapeutic impact as the identification of new molecular actors for gonadal development or ovarian physiology is useful for the prediction of an ovarian reserve decline and makes possible preventive fertility preservation

Mutational Landscape of DDR2 Gene in Lung Squamous Cell Carcinoma Using Next-generation Sequencing

International audienceBackground - Lung cancer represents the leading cause of cancer-related death worldwide. Despite great advances in lung cancer management with the recent emergence of molecular targeted therapies for non-squamous non-small-cell lung cancer, no dramatic improvements have been achieved in lung squamous cell carcinoma (SCC). Mutations in discoidin domain receptor 2 (DDR2) gene were recently identified as promising molecular targets in this histology. The aim of this study is to describe the DDR2 mutational landscape of lung SCC and investigate the associated clinical factors. Methods - Next-generation sequencing of the DDR2 gene was performed on 271 samples of lung SCC. Patients followed in our institution from January 2011 to August 2014 were retrospectively selected for data collection. Other driver gene alterations (EGFR, KRAS, BRAF, HER2, and PI3KCA) were analyzed using pyrosequencing. Results - A total of 11 patients harboring a DDR2 mutation was detected among the 271 sequenced lung SCC samples (4%). We describe 10 unreported mutations, comprising a novel DDR2 exon 7 splice mutant. DDR2 mutations were not mutually exclusive with other driver gene alterations. One hundred thirty-six patients were included for clinical comparison and logistic regression analysis. No difference was detected between DDR2-mutant and DDR2 wild-type lung SCC regarding clinical characteristics or survival. Conclusion - DDR2 mutations were observed in 4% of cases of lung SCC of European descent. DDR2-mutated tumors can exhibit other driver gene alterations. No clinical characteristics were significantly associated with DDR2 mutation

Mutational Landscape of DDR2 Gene in Lung Squamous Cell Carcinoma Using Next-generation Sequencing

International audienceBackground - Lung cancer represents the leading cause of cancer-related death worldwide. Despite great advances in lung cancer management with the recent emergence of molecular targeted therapies for non-squamous non-small-cell lung cancer, no dramatic improvements have been achieved in lung squamous cell carcinoma (SCC). Mutations in discoidin domain receptor 2 (DDR2) gene were recently identified as promising molecular targets in this histology. The aim of this study is to describe the DDR2 mutational landscape of lung SCC and investigate the associated clinical factors. Methods - Next-generation sequencing of the DDR2 gene was performed on 271 samples of lung SCC. Patients followed in our institution from January 2011 to August 2014 were retrospectively selected for data collection. Other driver gene alterations (EGFR, KRAS, BRAF, HER2, and PI3KCA) were analyzed using pyrosequencing. Results - A total of 11 patients harboring a DDR2 mutation was detected among the 271 sequenced lung SCC samples (4%). We describe 10 unreported mutations, comprising a novel DDR2 exon 7 splice mutant. DDR2 mutations were not mutually exclusive with other driver gene alterations. One hundred thirty-six patients were included for clinical comparison and logistic regression analysis. No difference was detected between DDR2-mutant and DDR2 wild-type lung SCC regarding clinical characteristics or survival. Conclusion - DDR2 mutations were observed in 4% of cases of lung SCC of European descent. DDR2-mutated tumors can exhibit other driver gene alterations. No clinical characteristics were significantly associated with DDR2 mutation

Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings.

International audienceHoloprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis