An Application of Molecular Genotyping in Mice

Microsatellite markers are simple sequence repeats within the mammalian genome that can be used for identifying disease loci, mapping genes of interest as well as studying segregation patterns related to meiotic nondisjunction. Different strains of mice have variable CA repeat lengths and PCR based methods can be used to identify them, thus allowing for specific genotypes to be assigned. Molecular genotyping offers such identification at any developmental stage, which allows for a broad range of anomalies to be studied. We studied chromosomal segregation in relation to nondisjunction in early-gestation mouse embryos using molecular genotyping. Information on the parental origin as well as the number of chromosomes a given progeny carried was obtained in our analysis

A long noncoding RNA influences the choice of the X chromosome to be inactivated

X chromosome inactivation (XCI) is the process of silencing one of the X chromosomes in cells of the female mammal which ensures dosage compensation between the sexes. Although theoretically random in somatic tissues, the choice of which X chromosome is chosen to be inactivated can be biased in mice by genetic element(s) associated with the so-called X-controlling element (Xce). Although the Xce was first described and genetically localized nearly 40 y ago, its mode of action remains elusive. In the approach presented here, we identify a single long noncoding RNA (lncRNA) within the Xce locus, Lppnx, which may be the driving factor in the choice of which X chromosome will be inactivated in the developing female mouse embryo. Comparing weak and strong Xce alleles we show that Lppnx modulates the expression of Xist lncRNA, one of the key factors in XCI, by controlling the occupancy of pluripotency factors at Intron1 of Xist. This effect is counteracted by enhanced binding of Rex1 in DxPas34, another key element in XCI regulating the activity of Tsix lncRNA, the main antagonist of Xist, in the strong but not in the weak Xce allele. These results suggest that the different susceptibility for XCI observed in weak and strong Xce alleles results from differential transcription factor binding of Xist Intron 1 and DxPas34, and that Lppnx represents a decisive factor in explaining the action of the Xce

New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism, but not in suckling

The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism

Occurrence of testicular microlithiasis in androgen insensitive hypogonadal mice

<b>Background</b>: Testicular microliths are calcifications found within the seminiferous tubules. In humans, testicular microlithiasis (TM) has an unknown etiology but may be significantly associated with testicular germ cell tumors. Factors inducing microlith development may also, therefore, act as susceptibility factors for malignant testicular conditions. Studies to identify the mechanisms of microlith development have been hampered by the lack of suitable animal models for TM.<BR/> <b>Methods</b>: This was an observational study of the testicular phenotype of different mouse models. The mouse models were: cryptorchid mice, mice lacking androgen receptors (ARs) on the Sertoli cells (SCARKO), mice with a ubiquitous loss of androgen ARs (ARKO), hypogonadal (hpg) mice which lack circulating gonadotrophins, and hpg mice crossed with SCARKO (hpg.SCARKO) and ARKO (hpg.ARKO) mice.<BR/> <b>Results</b>: Microscopic TM was seen in 94% of hpg.ARKO mice (n=16) and the mean number of microliths per testis was 81 +/- 54. Occasional small microliths were seen in 36% (n=11) of hpg testes (mean 2 +/- 0.5 per testis) and 30% (n=10) of hpg.SCARKO testes (mean 8 +/- 6 per testis). No microliths were seen in cryptorchid, ARKO or SCARKO mice. There was no significant effect of FSH or androgen on TM in hpg.ARKO mice.<BR/> <b>Conclusions</b>: We have identified a mouse model of TM and show that lack of endocrine stimulation is a cause of TM. Importantly, this model will provide a means with which to identify the mechanisms of TM development and the underlying changes in protein and gene expression

Nondisjunction and transmission ratio distortion ofChromosome 2 in a (2.8) Robertsonian translocation mouse strain

Aneuploidy results from nondisjunction of chromosomes in meiosis and is the leading cause of developmental disabilities and mental retardation in humans. Therefore, understanding aspects of chromosome segregation in a genetic model is of value. Mice heterozygous for a (2.8) Robertsonian translocation were intercrossed with chromosomally normal mice and Chromosome 2 was genotyped for number and parental origin in 836 individuals at 8.5 dpc. The frequency of nondisjunction of this Robertsonian chromosome is 1.58%. Trisomy of Chromosome 2 with two maternally derived chromosomes is the most developmentally successful aneuploid karyotype at 8.5 dpc. Trisomy of Chromosome 2 with two paternally derived chromosomes is developmentally delayed and less frequent than the converse. Individuals with maternal or paternal uniparental disomy of Chromosome 2 were not detected at 8.5 dpc. Nondisjunction events were distributed randomly across litters, i.e., no evidence for clustering was found. Transmission ratio distortion is frequently observed in Robertsonian chromosomes and a bias against the transmission of the (2.8) Chromosome was detected. Interestingly, this was observed for female and male transmitting parents

Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function

The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome

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

Transcriptome-Wide Identification of Novel Imprinted Genes in Neonatal Mouse Brain

Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting

RNF12 Activates Xist and Is Essential for X Chromosome Inactivation

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12−/− knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI

Necdin Controls Proliferation of White Adipocyte Progenitor Cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development