Genomic imprinting is the phenomenon whereby a subset of genes is differentially expressed according to parental origin. Imprinted genes tend to occur in clusters, and microRNAs are associated with the majority of well-defined clusters of imprinted genes. We show here that two microRNAs, miR-296 and miR-298 , are part of the imprinted Gnas/GNAS clusters in both mice and humans. Both microRNAs show imprinted expression and are expressed from the paternally derived allele, but not the maternal allele. They arise from a long, noncoding antisense transcript, Nespas , with a promoter more than 27 kb away. Nespas had been shown previously to act in cis to regulate imprinted gene expression within the Gnas cluster. Using microarrays and luciferase assays, IKBKE , involved in many signaling pathways, and Tmed9 , a protein transporter, were verified as new targets of miR-296. Thus, Nespas has two clear functions: as a cis -acting regulator within an imprinted gene cluster and as a precursor of microRNAs that modulate gene expression in trans . Furthermore, imprinted microRNAs, including miR-296 and miR-298, impose a parental specific modulation of gene expression of their target genes.

Background Vertebrates show clear asymmetry in left-right (L-R) patterning of their organs and associated vasculature. During mammalian development a cilia driven leftwards flow of liquid leads to the left-sided expression of Nodal , which in turn activates asymmetric expression of the transcription factor Pitx2 . While Pitx2 asymmetry drives many aspects of asymmetric morphogenesis, it is clear from published data that additional asymmetrically expressed loci must exist. Results A L-R expression screen identified the cytoskeletally-associated gene, actin binding lim protein 1 ( Ablim1 ), as asymmetrically expressed in both the node and left lateral plate mesoderm (LPM). LPM expression closely mirrors that of Nodal . Significantly, Ablim1 LPM asymmetry was detected in the absence of detectable Nodal . In the node, Ablim1 was initially expressed symmetrically across the entire structure, resolving to give a peri-nodal ring at the headfold stage in a flow and Pkd2 -dependent manner. The peri-nodal ring of Ablim1 expression became asymmetric by the mid-headfold stage, showing stronger right than left-sided expression. Node asymmetry became more apparent as development proceeded; expression retreated in an anticlockwise direction, disappearing first from the left anterior node. Indeed, at early somite stages Ablim1 shows a unique asymmetric expression pattern, in the left lateral plate and to the right side of the node. Conclusion Left LPM Ablim1 is expressed in the absence of detectable LPM Nodal , clearly revealing existence of a Pitx2 and Nodal -independent left-sided signal in mammals. At the node, a previously unrecognised action of early nodal flow and Pkd2 activity, within the pit of the node, influences gene expression in a symmetric manner. Subsequent Ablim1 expression in the peri-nodal ring reveals a very early indication of L-R asymmetry. Ablim1 expression analysis at the node acts as an indicator of nodal flow. Together these results make Ablim1 a candidate for controlling aspects of L-R identity and patterning.

Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor alpha (ER alpha(-/-)) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgtb1, Lrp1, Ostn, Timp, Mmp, Ctgf Postn and Irs1, BMP and DLX5). The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ER alpha(-/-) and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ER alpha(-/-) mice, the number of genes differentially regulated by loading was markedly reduced at all time points.These data indicate that in resident bone cells. both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone. (C) 2009 Elsevier Inc. All rights reserved.

An increase in the expression of stretch/stress response elements in fast and slow muscles has been previously described in a transcriptional profiling of KY deficient muscles. Here, we have characterized the induction of this titin-based family of signalling proteins in ky/ky muscles at the protein level. Changes in expression of MLP, MARP2 and Xin have been related to the onset of dystrophic and adaptive changes that operate in ky/ky muscles. Our results indicate that induction of this set of genes is an early consequence of the interference caused by the absence of the KY protein. A search of muscle profiles of mouse models revealed such molecular hallmark only in muscles subjected to a single bout of eccentric contractions and specific titin mutants. Based on the role of this family as titin-based stress response molecules, it is suggested that titin structural/signalling instability is common to ky and titin mouse mutants and eccentric contractions.

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways. Sustained expression of SHP leads to the depletion of hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver. The mechanism responsible for this phenotype includes SHP-mediated direct repression of downstream target genes and the bile acid sensor FXR alpha, and an indirect activation of PPAR gamma and SREBP-1c genes. We present evidence for the role of altered chromatin configurations in defining distinct gene-specific mechanisms by which SHP mediates differential transcriptional repression. The multiplicity of genes under its control suggests that SHP is a pleiotropic regulator of diverse metabolic pathways.

Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression H3 on the DNA-methylated maternal allele, whereas histone H3 on the unmethylated allele has lysine-4 methylation and is acetylated. The same modifications were detected on the KvDMR1, the ICR that regulates a large imprinted domain (the Kcnq1 domain) on chromosome 7 comprising several genes that are essential for extra-embryonic development. The KvDMR1 ICR has DNA methylation on the maternal allele, and produces a non-coding transcript (Lit1) from the paternal allele. We explored whether the paternal repression in cis, mediated by the ICR, involves the acquisition of specific histone modifications. Histone methylation and acetylation were analysed along the domain. Our data show that on the paternal chromosome, specific histone H3 modifications (including K27 trimethylation and K9 di-methylation) become established along the imprinted domain during pre-implantation development. These paternal modifications are maintained in an extra-embryonic-lineage-specific manner. is imprinted at most of its choroid leptomeninges of the brain. Igf2 also frequently undergoes loss of imprinting in some Fraser syndrome (FS) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects; other defects occur more rarely. The condition is genetically heterogeneous with two disease loci identified, genes at both encoding a protein with similarity to the sea urchin extracellular matrix (ECM) blastocoelar protein ECM3 . The domain structure of FRAS1 and FRAS2 suggests a structural role within the extracellular matrix as well as in cell signalling. Mouse blebbed mutants provide a model for FS, with mutations in five blebbed loci giving similar phenotypes to FS. Mutations in FRAS1 , and Fras1 in the bl mouse, have been identified that result in the premature termination of the protein. A missense mutation has been identified at a second locus, FRAS2 , which has homology to a gene closely linked to the my (myelencephalic blebs) locus. Loss of the cytoplasmic multi-PDZ domain protein glutamate receptor interacting protein 1 (GRIP1) results in a phenotype similar to that of FS. In the eye blebs mouse we have detected a deletion covering two cod-ing exons that disrupts the ( Grip1 ) gene, resulting in a premature termination. These mouse models for FS should provide important insights into the development of epithelial structures, as well as eye and kidney development. Sensory deprivation is associated with a mutation in the rat chaperonin delta subunit The study involves characterization of a mutant mouse line first identified in the biochemical screen Programme. GENA line 328 mutants display significantly lowered linkage phenome database and may further our understanding of hypophosphatasia in humans. Ocular coloboma is the incomplete closure of the embryonic optic fissure resulting in abnormal eye development affecting the iris, retina, choroid, optic nerve or eyelids. The molecular mechanisms orches-trating this fundamental process are not understood; however, disease genes can give clues to the molecular events leading to optic fissure closure. Approximately, 1 : 10 000 births are affected and OMIM lists 69 dis-eases with an ocular coloboma phenotype. To date 24 chromosomal loci have been mapped, 11 chromosomal aberrations verified and 17 of the genes identified. To identify genes involved in coloboma we have been investigating families from our Genetic Ophthalmology in London Database (GOLD), and searching for the causative genes. A family that had ocular coloboma segregating with Hirschsprung disease and high myopia were studied and we identified a missense mutation in the ZFHX1B gene. This gene is a transcriptional repressor which is expressed in a spatiotemporal manner commensurate with closure of the optic fissure. A second family was identified that segregated ocular coloboma with oto-dental dysplasia. We are currently investigating a candidate gene for this phenotype. These studies suggest there is a complex interaction of at least 35 genes that are involved in optic fissure closure in man. Mechanisms regulating the initiation of neural tube closure The differentiation of murine embryonic stem (ES) cells in vitro is a valuable model of embryonic development. To identify genes that may be involved in promoting differentiation a day 5–day 3 subtracted embryoid body cDNA library was generated. We selected a novel homeobox gene ( Ehox ) from the subtracted library and analysed its function using loss and gain of function strategies in ES cells. Over-expressing Ehox sense or antisense transcripts in ES cells provided evidence that Ehox plays a role in the initial differentiation of a precursor cell type. In vivo , whole-mount in situ hybridization analyses have shown that Ehox is expressed in the trophoblast lineage and in pharyngeal pouches 2 and 3. These expression patterns suggest two distinct roles for Ehox during embryogenesis: (1) within the stem cell/pre-cursor compartments of the developing placenta and (2) during development of the pharyngeal pouches and thymus. Work Leukaemia control We generated a cDNA library of embryonic chick ventral spinal cord enriched for sequences up-regulated between E5 and E7.5, looking for genetic markers for glial lineages and compartments of ventricular neuroepithelium. We screened the library for differentially expressed transcripts, examining expression of 128 candidates by in situ hybridization, and sequenced those of greatest interest. Three cDNA fragments were analysed in more detail: (1) cDNA for KIAA1102, a recently identified transmembrane protein with a LIM domain on C-terminal. KIAA1102 is expressed in the ventral half of neuroepithelium complementary to Mash1. (2) cDNA for an axon-guidance molecule Semaphorin-3a, which we found to be involved in the compartmentalization of the spinal cord. Overlap between Sema3a and Nkx2.2 de-termines the domain that generates first oligodendrocyte progenitors. (3) cDNA for chick receptor tyrosine phosphatase zeta/beta (cRPTP f / b ). cRPTP f / b is expressed throughout the ventricular zone and in scattered cells outside it. cRPTP f / b is down-regulated in neurons or oligodendrocyte progenitors. The majority of scattered cRPTP f / b + cells co-expressed glutamine synthetase and fibroblast growth factor receptor-3 , indicating that the cells are astrocyte precursors. Our data suggest that alternatively-spliced isoforms of cRPTP f / b , including phosphacan, are not present in the developing chick spinal cord. X-gal staining of neonatal hearts from knockout animals shows Nat2 strongly expressed in the crux of the heart, closely associated with the atrioventricular node, and in the valves of the heart. Immuno-histochemical studies of human embryonic hearts show a similar timing and pattern of expression of the equivalent human NAT isoenzyme. The familial obesity protein BBS4 targets PCM1 to the pericentriolar region and is required for microtubule progression proteins causing Bardet-Biedl (BBS), a multisystemic disorder of genetic and Here we localizes the centriolar satellites of cen-trosomes and basal bodies of primary cilia, and that functions as an adaptor of the p150 glued subunit of the dynein transport machinery to recruit PCM1 (Pericentriolar Matrix 1 protein) and associated to the satellites. BBS4 silencing induces PCM1 mislocalization and concomitant de-anchoring of centrosomal microtubules, the nuclear abnormalities observed in early human development. The sprouty ( spry ) gene was identified in Drosophila as a regulator of branching morphogenesis of the trachea. SPROUTY proteins act as general, intracellular feedback inhibitors of ras/MAPK signalling downstream of growth factor receptors. In order to investigate the role of the first mammalian homologue of this family, Sprouty1 , during embryogenesis, the Sprouty1 flat-tened cyst-like irides but were otherwise normal. These striking differences provide a further warning of the importance of standardizing the genetic background for phenotype comparisons of different mutants in an allelic series. The six types of retinal neuron and Muller glial cells derive from retinal progenitor cells (RPCs) of the embryonic optic cup. A full complement of retinal neurons is born by postnatal day 11 in the mouse when neurogenesis is complete. Little is known about how RPCs regulate the timing of the decision to con-tinue dividing or to differentiate. In mice lacking the Chx10 homeobox gene, proliferation of RPCs is reduced during development and adult mice have microphthalmia. In this study we investigate the hypothesis that Chx10 plays a critical role in regulating the number of RPCs. We compared the distribution of RPCs in the postnatal Chx10 null and wild-type retina, using incorporation of the thymidine analogue BrdU to label dividing cells. We report that lack of Chx10 causes a significant number of cycling cells to persist in the adult neural retina. Immuno-staining with nestin and neuronal marker b 3-tubulin indicates that the BrdU-labelled cells can give rise to neurons within the adult retina. This is the first report of neurogenesis in the mature mammalian central retina. More dividing cells were also detected in the mutant ciliary epithelium (CE) than in the wild-type CE (which is known to harbour cells with stem cell properties).

Microarrays allow monitoring of gene expression for tens of thousands of genes in parallel and are being used routinely to generate huge amounts of valuable data. Handling and analysis of such data are becoming major bottlenecks in the utilization of the technology. To enable the researcher to interpret the results postanalysis, we have developed a laboratory information management system for microarrays (LIMaS) with an n-tier Java front-end and relational database to record and manage large-scale expression data preanalysis. This system enables the laboratory to replace the paper trail with an efficient and fully customizable interface giving it the ability to adapt to any working practice, e.g., handling many resources used to form many products (chaining of resources). The ability to define sets of activities, resources, and workflows makes LIMaS MIAME-supportive.

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.