Jacky Bonaventure

Genotype-phenotype correlation in hereditary multiple exostoses

Hereditary multiple exostoses (HME) is a genetically heterogeneous autosomal dominant disorder characterised by the development of bony protuberances mainly located on the long bones. Three HME loci have been mapped to chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1and EXT2 genes encode glycosyltransferases involved in biosynthesis of heparan sulphate proteoglycans. Here we report on a clinical survey and mutation analysis of 42 HME French families and show that EXT1 andEXT2 accounted for more than 90% of HME cases in our series. Among them, 27/42 cases were accounted for byEXT1 (64%, four nonsense, 19 frameshift, three missense, and one splice site mutations) and 9/42 cases were accounted for by EXT2 (21%, four nonsense, two frameshift, two missense, and one splice site mutation). Overall, 31/36 mutations were expected to cause loss of protein function (86%). The most severe forms of the disease and malignant transformation of exostoses to chondrosarcomas were associated withEXT1 mutations. These findings provide the first genotype-phenotype correlation in HME and will, it is hoped, facilitate the clinical management of these patients.

Sex related expressivity of the phenotype in coronal craniosynostosis caused by the recurrent P250R FGFR3 mutation

A recurrent point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that converts proline 250 into arginine is commonly associated with coronal craniosynostosis and has allowed definition of a new syndrome on a molecular basis. Sixty-two patients with sporadic or familial forms of coronal craniosynostosis were investigated for the P250R FGFR3 mutation. It was identified in 20 probands originating from 27 unrelated families (74%), while only 6/35 sporadic cases (17%) harboured the mutation. In both familial and sporadic cases, females were significantly more severely affected than males. Hence, while 68% of females carrying the P250R mutation showed brachycephaly, only 35% of males had the same phenotype. In the most severe forms of the disease, the association of bicoronal craniosynostosis with hypertelorism and marked bulging of the temporal fossae were common hallmarks that might be helpful for clinical diagnosis. Taken together, these results indicate that the P250R FGFR3 mutation is mostly familial and is associated with a more severe phenotype in females than in males. The sex related severity of the condition points to the possible implication of modifier genes in this syndrome.

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.

Bypassing melanocyte senescence by β-catenin: A novel way to promote melanoma

The Wnt/beta-catenin signaling pathway plays a key role in several cellular functions during embryonic development and adult homeostasis. The deregulation of this pathway may lead to the development of cancer, including melanoma. Deregulation of the Wnt/beta-catenin pathway occurs through either the induction/repression of, or specific mutations in, various members of this signaling pathway; this results in the stabilization of beta-catenin and its translocation from the cytoplasm to the nucleus, where it regulates transcription. Although nuclear beta-catenin is clearly involved in malignant transformation, the mechanism by which it exerts its effects remains elusive. This review focuses on the molecular and cellular mechanisms that are driven by beta-catenin and lead to melanocyte transformation. In particular, we describe how beta-catenin induces melanocyte immortalization, a novel activity of this multifunction protein. Finally, we discuss how beta-catenin-induced immortalization can cooperate with MAPKinase pathways to produce melanoma.

The tyrosinase promoter is active in a subset of vagal neural crest cells during early development in mice.

Dear Sir, Tyrosinase (Tyr) is the key enzyme in melanin synthesis, involved in the conversion of tyrosine to dopaquinone. It was long thought to be specific to pigmented cells. This is certainly true in adults. However, during mouse development, the Tyr promoter is active in melanoblasts and cells along the entire neural tube (Tief et al., 1996). Transgenic mice producing the Cre recombinase under the control of the tyrosinase promoter (Tyr::Cre) were previously generated to allow Cre-mediated recombination of genes in melanocytes (Mc) (Delmas et al., 2003). The Cre protein is produced in classical and non-classical Mc during late embryogenesis in these mice (Yajima and Larue, 2008). Crossing Tyr::Cre mice with Rosa26flox reporter mice revealed recombination events in other non-pigmented tissues (Delmas et al., 2003; Tonks et al., 2003; Yajima et al., 2006). The finding of Tyr activity in cells along the neural tube indicated that Cre recombinase in Tyr::Cre mice is expressed in cells other than melanoblasts. Vagal neural crest cells (VNCC) can differentiate into various cells and tissues such as Mc, smooth muscle cells (Ms), neurons (N), glial cells and mesenchymal cells (Le Douarin and Kalcheim, 1999). At least two major events occurring after birth are associated with VNCC derivatives: modification of the blood stream after birth, requiring the closure of the ductus arteriosus (DA) and modification of the food supply at weaning, requiring the proper transit of solid feces including fiber into the intestine. The Tyr promoter described previously (Porter and Meyer, 1994) is active from E10.5 in NCC of the truncal region (Delmas et al., 2003). The examination of transverse sections obtained from Tyr::Cre ⁄ ; Rosa26flox ⁄ + and Dct::LacZ E10.5 embryos revealed that not only melanoblasts were defloxed (Figure S1), but also that these defloxed cells could be at the origin of a subset of DA and intestinal cells. First we confirmed the neural crest cell origin of the DA by classical molecular genetics cell lineage analysis (Figure S2). Based on the presence of X-gal-positive cells in the heart of Tyr::Cre ⁄ ; Rosa26flox ⁄ + embryos at E10.5, and given that cells forming the DA are of NCC origin, we analyzed the Tyr::Cre ⁄ ; Rosa26flox ⁄ + DA and the ligamentum arteriosum (LigA), which originates directly from the DA. E18.5 DA from Tyr::Cre ⁄ ; Rosa26flox ⁄ + were isolated and stained with X-gal. X-gal staining was clearly visible in whole mounted and transverse sections (Figure 1A,B). We estimated that approximately 16% of the cells stained positive for X-gal (=defloxed) and about 84% of the cells were X-gal negative (=floxed). Similar results were obtained with the Tyr::CreB mouse line, previously described (Delmas et al., 2003). These results suggested that the defloxing in these cells is independent of the site of integration of the transgene. Transverse cryosections of Tyr::Cre ⁄ ; Rosa26flox ⁄ + DA were subjected to immunofluorescence analysis using antibodies directed against smooth muscle actin (SMA). Most cells were SMA-positive (Figure 1C) confirming the smooth muscle nature of these cells. Consistent with this, Tyr::Cre ⁄ ; + ⁄ + and ⁄ ; Rosa26flox ⁄ + DA were also positive for SMA (data not shown). Finally, hearts from p28 mice were isolated and stained with X-gal. Besides a diffuse staining all around the heart, we observed an important stream of X-gal blue cells in the aorta and LigA of Tyr::Cre ⁄ ; Rosa26flox ⁄ + adult hearts (Figure 1D). As expected, the DA and the LigA of Tyr::Cre ⁄ ; + ⁄ + and ⁄ ; Rosa26flox ⁄ + control hearts stained negatively for X-gal (data not shown). We conclude that the Tyr promoter was active in a subset of Ms forming the DA ⁄ LigA. Moreover, these results demonstrate that a gene of interest can be targeted by the Cre-LoxP recombinase system in such cells using Tyr::Cre mice. Cre recombination in DA and LigA cells of Tyr::Cre ⁄ ; Rosa26flox ⁄ + mice was surprising because no Mc have been previously described in these structures. However, we and others have previously reported the presence

β-Catenin Inhibitor ICAT Modulates the Invasive Motility of Melanoma Cells

Inhibitor of β-catenin and TCF (ICAT) inhibits β-catenin transcriptional activity by competing with T-cell factor/lymphoid enhancer factor. We documented high ICAT levels in human melanoma cells, in which β-catenin signaling is frequently deregulated, finding a correlation with the capacity to form metastases in nude mice. Ectopic expression of ICAT in melanoma cells did not affect their proliferation but increased cell motility and Matrigel invasion of metastatic cells in a manner relying upon stable ICAT-β-catenin interaction. This effect was associated with conversion of an elongated/mesenchymal phenotype to a round/amoeboid phenotype in the absence of similar effects on elongated morphology of nonmetastatic melanoma cells. Transition from mesenchymal to amoeboid movement was associated with decreased levels of NEDD9 and activated Rac1, a positive regulator of mesenchymal movement. Ectopic ICAT promoted colonization of melanoma cells in the lungs of nude mice, suggesting an increase in metastatic potential. Together, our results showed that by downregulating Rac signaling in metastatic melanoma cells, ICAT increased their invasive motility by promoting a morphologic variation that facilitates a favorable adaptation to their microenvironment.

A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

Background Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.

Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans.

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation.

Structure-based mutational analysis of ICAT residues mediating negative regulation of β-catenin co-transcriptional activity

ICAT (Inhibitor of β-CAtenin and TCF) is a small acidic protein that negatively regulates β-catenin co-transcriptional activity by competing with TCF/LEF factors in their binding to β-catenin superhelical core. In melanoma cells, ICAT competes with LEF1 to negatively regulate the M-MITF and NEDD9 target genes. The structure of ICAT consists of two domains: the 3-helix bundle N-terminal domain binds to β-catenin Armadillo (Arm) repeats 10–12 and the C-terminal tail binds to Arm repeats 5–9. To elucidate the structural mechanisms governing ICAT/β-catenin interactions in melanoma cells, three ICAT residues Y15, K19 and V22 in the N-terminal domain, contacting hydrophobic β-catenin residue F660, were mutated and interaction was assessed by immunoprecipitation. Despite the moderate hydrophobicity of the contact, its removal completely abolished the interaction. In the ICAT C-terminal tail consensus sequence, neutralization of the electrostatic interactions between residues D66, E75 and β-catenin residues K435, K312, coupled to deletion of the hydrophobic contact between F71 and β-catenin R386, markedly reduced, but failed to abolish the ICAT-mediated negative regulation of M-MITF and NEDD9 promoters. We conclude that in melanoma cells, anchoring of ICAT N-terminal domain to β-catenin through the hook made by residue F660, trapped in the pincers formed by ICAT residues Y15 and V22, is crucial for stabilizing the ICAT/β-catenin complex. This is a prerequisite for binding of the consensus peptide to Arm repeats 5–9 and competition with LEF1. Differences between ICAT and LEF1 in their affinity for β-catenin may rely on the absence in ICAT of hydrophilic residues between D66 and F71.