Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human Filamin A (FLNA) diseases

<p>Abstract</p> <p>Background</p> <p>Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human <it>FLNA</it>/+ females, heterozygous for X-linked, filamin A gene (<it>FLNA</it>) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. <it>Flna</it>^{<it>Dilp2/+ </it>}mice, heterozygous for an X-linked filamin A (<it>Flna</it>) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}mice was affected in any way that might predict abnormal corneal epithelial maintenance.</p> <p>Results</p> <p>X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of <it>Flna</it>^{<it>Dilp2/+ </it>}and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, <it>LacZ </it>reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in <it>Flna</it>^{<it>Dilp2/+ </it>}corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in <it>Flna</it>^{<it>Dilp2/+ </it>}compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of <it>Flna</it>^{<it>Dilp2/+ </it>}than wild-type <it>Flna^+/+</it>X-inactivation mosaics.</p> <p>Conclusions</p> <p>Mosaic analysis identified no major effect of the mouse <it>Flna^Dilp2</it>mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.</p

The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

The maternal separation paradigm has been applied to C57BL/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology

A Dominant-Negative Mutation of Mouse Lmx1b Causes Glaucoma and Is Semi-lethal via LBD1-Mediated Dimerisation

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice

The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei

AbstractThe neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC2 receptor for VIP and PACAP (Vipr2−/−) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN

Forepaw phenotype of transgenic rescue mice.

<p>Pictures of the ventral and dorsal sides of forepaws from wild-type (WT), <i>Lmx1b^Icst</i>^/<i>Icst</i> (<i>Icst</i>/<i>Icst</i>) and <i>Lmx1b^KO/KO</i> (KO/KO) mice are shown and whether the BAC transgene is hemizygous or homozygous is indicated (BACx1 or BACx2). The ventral side of the paw is normal for all the rescue mice. The dorsal surface of all the homozygous mutant paws appears ventralised with pigmented footpads and no hair. The ages of the mice shown are as follows. Top two rows, P14; third row P26 and bottom row P35.</p

Genotyping results for <i>Lmx1b^Icst/+</i> intercross.

a<p>Test for significance using Chi-square test.</p

Forepaw skeletal phenotype of transgenic rescue mice.

<p>µCT scans of the ventral and dorsal sides from wild-type (WT), <i>Lmx1b^Icst</i>^/<i>Icst</i> (<i>Icst</i>/<i>Icst</i>) and <i>Lmx1b^KO/KO</i> (KO/KO) forepaws are shown and whether the BAC transgene is hemizygous or homozygous is indicated (BACx1 or BACx2). The ventral side of the skeleton appears normal for all the rescue mice (top panels). The dorsal surface appears completely ventralised for the <i>Lmx1b^Icst</i>^/<i>Icst</i> rescue mice homozygous for the transgene and for the <i>Lmx1b^KO/KO</i> rescue hemizygous for the BAC transgene. Arrows point to sesamoid bones that are a feature of the ventral surface. In the case of the <i>Lmx1b^KO/KO</i> homozygous for the BAC transgene the dorsal surface appears more normal although there are still aspects of ventral morphology seen, for example a sesamoid bone (arrowed). The images shown are from mice between four and five weeks of age except for <i>Lmx1b^KO/KO</i> hemizygous for the BAC transgene which was P26.</p

Homozygosity for the BAC transgene can rescue both the <i>Icst</i> and knockout lethal phenotypes.

<p>For each cross the progeny from two matings was counted.</p>a<p>all are homozygous for the transgenic BAC.</p>b<p>includes 1 taken for analysis before weaning.</p>c<p>includes 3 taken for analysis before weaning. There were 2 found dead on P0.</p>d<p>includes 5 taken for analysis before weaning. There were 4 that did not survive to weaning.</p>e<p>includes 2 taken for analysis before weaning. There was 1 found dead on P1.</p>f<p>includes 4 taken for analysis before weaning. There were 3 that that did not survive to weaning.</p>g<p>Test for significance using Chi-square test.</p