FMR1: A gene with three faces

The FMR1 gene is involved in three different syndromes, the fragile X syndrome (FXS), premature ovarian insufficiency (POI) and the fragile X-associated tremor/ataxia syndrome (FXTAS) at older age. Fragile X syndrome is caused by an expansion of a CGG repeat above 200 units in the FMR1 gene resulting in the absence of the FMR1 mRNA and protein. The FMR1 protein is proposed to act as a regulator of mRNA transport and of translation of target mRNAs at the synapse. FXS is seen as a loss of function disorder. POI and FXTAS are found in individuals with an expanded repeat between 50 and 200 CGGs and are associated with increased FMR1 mRNA levels. The presence of elevated FMR1 mRNA in FXTAS suggests that FXTAS may represent a toxic RNA gain-of-function effect. The molecular basis of POI is yet unknown. The role of the FMR1 gene in these disorders is discussed.     

Targeting the G-quadruplex-forming region near the P1 promoter in the human BCL-2 gene with the cationic porphyrin TMPyP4 and with the complementary C-rich strand

The B-cell lymphoma-2 (bcl-2) gene contains a region that has been implicated in the regulation of bcl-2 gene expression. This region can form G-quadruplex structures in solution [J.X. Dai, T.S. Dexheimer, D. Chen, M. Carver, A. Ambrus, R.A. Jones, D.Z. Yang, An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution, J. Am. Chem. Soc. 128 (2006) 1096–1098.]. Here, we examined the acid–base and conformational equilibria of this G-quadruplex-forming region (BCL2G), as well as its interaction with both the porphyrin TMPyP4 and with the complementary C-rich strand. We used molecular absorption and circular dichroism techniques, in tandem with multivariate analysis tools. The results revealed the formation of an interaction complex BCL2G:TMPyP4 with a stoichiometry of 1:2 and an equilibrium constant equal to 5.0 (±2.3) × 10¹³ M⁻². Addition of the complementary C-rich strand to BCL2G induces the predominant formation of the Watson–Crick double-helix with an equilibrium constant equal to 10^7.7 M⁻¹ (at pH 7.1). Finally, the pH-induced formation of quadruplex structures from the Watson–Crick double-helix is characterized.     

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the <Emphasis Type="Italic">fmr1</Emphasis> gene

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.Edited by D. Tautz     

Prenatal diagnosis of foetuses with congenital abnormalities and duplication of the MECP2 region

MECP2 duplication results in a well-recognised syndrome in 100% of affected male children; this syndrome is characterised by severe neurodevelopmental disabilities and recurrent infections. However, no sonographic findings have been reported for affected foetuses, and prenatal molecular diagnosis has not been possible for this disease due to lack of prenatal clinical presentation. In this study, we identified a small duplication comprising the MECP2 and L1CAM genes in the Xq28 region in a patient from a family with severe X-linked mental retardation and in a prenatal foetus with brain structural abnormalities. Using high-resolution chromosome microarray analysis (CMA) to screen 108 foetuses with congenital structural abnormalities, we identified additional three foetuses with the MECP2 duplication. Our study indicates that ventriculomegaly, hydrocephalus, agenesis of the corpus callosum, choroid plexus cysts, foetal growth restriction and hydronephrosis might be common ultrasound findings in prenatal foetuses with the MECP2 duplication and provides the first set of prenatal cases with MECP2 duplication, the ultrasonographic phenotype described in these patients will help to recognise the foetuses with possible MECP2 duplication and prompt the appropriate molecular testing.     

Ectopic Pax2 expression in chick ventral optic cup phenocopies loss of Pax2 expression

Pax2 is essential for the development of the urogenital system, neural tube, otic vesicle, optic cup and optic tract [Dressler, G.R., Deutsch, U., et al., 1990. PAX2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109 (4), 787-795; Nornes, H.O., Dressler, G.R., et al., 1990. Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109 (4), 797-809; Eccles, M.R., Wallis, L.J., et al., 1992. Expression of the PAX2 gene in human fetal kidney and Wilms’ tumor. Cell Growth Differ 3 (5), 279-289]. Within the visual system, a loss-of-function leads to lack of choroid fissure closure (known as a coloboma), a loss of optic nerve astrocytes, and anomalous axonal pathfinding at the optic chiasm [Favor, J., Sandulache, R., et al., 1996. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl. Acad. Sci. U. S. A. 93 (24), 13870-13875; Torres, M., Gomez-Pardo, E., et al., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122 (11), 3381-3391]. This study is directed at determining the effects of ectopic Pax2 expression in the chick ventral optic cup past the normal developmental period when Pax2 is found. In ovo electroporation of Pax2 into the chick ventral optic cup results in the formation of colobomas, a condition typically associated with a loss of Pax2 expression. While the overexpression of Pax2 appears to phenocopy a loss of Pax2, the mechanism of the failure of choroid fissure closure is associated with a cell fate switch from ventral retina and retinal pigmented epithelium (RPE) to an astrocyte fate. Further, ectopic expression of Pax2 in RPE appears to have non-cell autonomous effects on adjacent RPE, creating an ectopic neural retina in place of the RPE.     

Influence of Four Nematodes on Root and Shoot Growth Parameters in Grape

Two grape cultivars, susceptible French Colombard and tolerant Rubired, and four nematodes, Meloidogyne incognita, Pratylenchus vulnus, Tylenchulus semipenetrans, and Xiphinema index, were used to quantify the equilibrium between root (R) and shoot (S) growth. Root and shoot growth of French Colombard was retarded by M. incognita, P. vulnus, and X. index but not by T. semipenetrans. Although the root growth of Rubired was limited by all the nematodes, the shoot growth was limited only by X. index. The R:S ratios of Rubired were higher than those of French Colombard. The reduced R:S ratios of Rubired were primarily an expression of reduction in root systems without an equal reduction in shoot growth, whereas in French Colombard the reduced R:S ratios were due to a reduction in both shoot growth and root growth and to a greater reduction in root growth than shoot growth. All nematodes reproduced equally well on both cultivars. Both foliage and root growth of French Colombard were significantly reduced by M. incognita and P. vulnus. Nematodes reduced the shoot length by reducing the internode length. Accumulative R:S ratios in inoculated plants were significantly smaller than those in controls in all nematode treatments but not at individual harvest dates. Bud break was delayed by X. index and was initiated earlier by P. vulnus and M. incognita. All buds in nematode treatments were less vigorous than in controls.     

Drosophila modeling of heritable neurodevelopmental disorders

Heritable neurodevelopmental disorders are multifaceted disease conditions encompassing a wide range of symptoms including intellectual disability, cognitive dysfunction, autism and myriad other behavioral impairments. In cases where single, causative genetic defects have been identified, such as Angelman syndrome, Rett syndrome, Neurofibromatosis Type 1 and Fragile X syndrome, the classical Drosophila genetic system has provided fruitful disease models. Recent Drosophila studies have advanced our understanding of UBE3A, MECP2, NF1 and FMR1 function, respectively, in genetic, biochemical, anatomical, physiological and behavioral contexts. Investigations in Drosophila continue to provide the essential mechanistic understanding required to facilitate the conception of rational therapeutic treatments.     

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.Key words: Drosophila, Fragile X, autism, social behavior, novel assay     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

A Novel Function for Fragile X Mental Retardation Protein in Translational Activation

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.     

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1^−/− mice lack any obvious limb or skeletal defects, Sost^−/− mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost^−/−; Sostdc1^−/− mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost^−/− and Sost^−/−; Sostdc1^−/− mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.     

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France)

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the “glaseri-group”. The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.