Gene Trap Insertion Research Articles

A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression invivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 invivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the β-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of β-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings invivo.

Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function.

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

The role of the innate immune response regulatory gene ABCF1 in mammalian embryogenesis and development

ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis and arthritis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2). To study ABCF1 expression and function in development and disease, we used a single gene trap insertion in the Abcf1 gene in murine embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous Abcf1 promoter by following the expression of a β-galactosidase reporter gene. From the ES cells, heterozygous mice (Abcf1+/-) were produced. No live born Abcf1-/- progeny were ever generated, and the lethality was not mouse strain-specific. Thus, we have determined that Abcf1 is an essential gene in development. Abcf1-/- mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while Abcf1+/- mice appeared developmentally normal. Abcf1+/- mice were fertile and showed no significant differences in their anatomy when compared with their wild type littermates. The Abcf1 promoter was found to be active in all organs in adult mice, but varies in levels of expression in specific cell types within tissues. Furthermore, we observed high promoter activity in the blastocysts and embryos. Overall, Abcf1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types.

Generation of a multipurpose Prdm16 mouse allele by targeted gene trapping.

ABSTRACTGene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific gene functions.

Loss of SynDIG1 Reduces Excitatory Synapse Maturation But Not Formation In Vivo.

Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro. Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1-deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo.

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

Deletion of Inpp5a causes ataxia and cerebellar degeneration in mice

The progressive and permanent loss of cerebellar Purkinje cells (PC) is a hallmark of many inherited ataxias. Mutations in several genes involved in the regulation of Ca(2+) release from intracellular stores by the second messenger IP3 have been associated with PC dysfunction or death. While much is known about the defects in production and response to IP3, less is known about the defects in breakdown of the IP3 second messenger. A mutation in Inpp4a of the pathway is associated with a severe, early-onset PC degeneration in the mouse model weeble. The step preceding the removal of the 4-phosphate is the removal of the 5-phosphate by Inpp5a. Gene expression analysis was performed on an Inpp5a (Gt(OST50073)Lex) mouse generated by gene trap insertion using quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blot. Phenotypic analyses were performed using rotarod, β-galactosidase staining, and phosphatase activity assay. Statistical significance was calculated. The deletion of Inpp5a causes an early-onset yet slowly progressive PC degeneration and ataxia. Homozygous mutants (90%) exhibit perinatal lethality; surviving homozygotes show locomotor instability at P16. A consistent pattern of PC loss in the cerebellum is initially detectable by weaning and widespread by P60. Phosphatase activity toward phosphoinositol substrates is reduced in the mutant relative to littermates. The ataxic phenotype and characteristics neurodegeneration of the Inpp5a (Gt(OST50073)Lex) mouse indicate a crucial role for Inpp5a in PC survival. The identification of the molecular basis of the selective PC survival will be important in defining a neuroprotective gene applicable to establishing a disease mechanism.

Analysis of Additional Targeted Lman1 Alleles in the Mouse Suggests a Dose Dependence for FV/FVIII Secretion and Novel Role for an LMAN1-Dependent Cargo in Thrombopoiesis

Combined deficiency of coagulation factors V and VIII (F5F8D) is a human autosomal recessive bleeding disorder caused by mutations in LMAN1 or MCFD2, which encode the components of the only known cargo-receptor complex in the mammalian secretory pathway. The LMAN1/MCFD2 complex facilitates secretion of coagulation factors V (FV) and VIII (FVIII) to the plasma. F5F8D patients exhibit FV and FVIII levels that are ~10-15% of normal. The relationship between Lman1 expression levels and the corresponding secretion of LMAN1-dependent cargos has not been characterized, and additional functions of LMAN1 remain to be determined. We previously described Lman1 mutant mice carrying a gene-trap insertion in intron 10 (Lman1gt1). Lman1gt1/gt1 mice have ~50% of normal FV and FVIII activity levels, and are therefore less severely affected than human F5F8D patients. This difference could be explained by low levels of residual LMAN1 expression, or by differences in FV and FVIII secretion between mice and humans. Lman1gt1/gt1 mice also exhibit a partially penetrant, perinatal lethality that is restricted to certain mouse genetic strains. We generated a second, independent LMAN1-deficient mouse with no Lman1 gene expression (Lman1-/-), as well as hypomorphic LMAN1-deficient mice (Lman1cgt/cgt) that retain ~7% of wildtype Lman1 expression levels on a C57BL/6J genetic background. Plasma FV and FVIII levels in Lman1-/- and Lman1cgt/cgtmice were measured by functional coagulation assays and compared to wildtype (WT) and Lman1gt1/gt1 mice. A dose-response relationship was observed, with Lman1-/- mice exhibiting FV and FVIII levels that are ~50% of normal (compared to ~10-15% in human patients), while hypomorphic Lman1cgt/cgtmice demonstrate an intermediate reduction in FV and FVIII (~70% of WT levels). A similar result was demonstrated for alpha-1 antitrypsin, another known LMAN1-dependent cargo. Taken together with the presence of two null LMAN1 alleles in nearly all reported LMAN1 F5F8D patients, these results suggest that hypomorphic LMAN1 mutations in humans may also result in milder reductions in plasma FV and FVIII that may have been missed clinically, though still contributing to FV and FVIII level variation and to bleeding susceptibility. The partially penetrant, strain-specific, perinatal lethality previously reported in Lman1gt1/gt1 mice was also confirmed in mice homozygous for the independently generated Lman1- allele on a pure C57BL/6J genetic background (p < 0.007), thereby ruling out a passenger gene effect as the cause of the originally reported Lman1gt1/gt1 lethality. In contrast, Lman1cgt/cgt mice on the same background are viable and observed in the expected Mendelian ratios (p > 0.19). Lman1-/- lethality was not observed on a mixed C57BL/6J and 129S1/SvImJ background (p > 0.79). F5F8D is not associated with fetal loss in humans, nor would fetal loss be expected from moderate deficiency of FV and FVIII. These results thus suggest a critical role for additional, as yet unknown, LMAN1 cargo(s). Finally, adult Lman1-/- mice exhibit a mild thrombocytopenia compared to WT controls (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004). In contrast, Lman1+/-, Lman1cgt/cgt, and Mcfd2-/- mice all exhibit normal platelet counts indistinguishable from WT controls. No Lman1-/- platelet morphologic abnormalities are evident by standard or transmission electron microscopy, with normal appearance of megakaryocyte number and morphology on bone marrow examination. Although thrombocytopenia has not been noted previously in human F5F8D patients, these results suggest a novel role for an LMAN1-dependent secretory cargo in thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

GLUT4 defects in adipose tissue are early signs of metabolic alterations in Alms1GT/GT, a mouse model for obesity and insulin resistance.

Dysregulation of signaling pathways in adipose tissue leading to insulin resistance can contribute to the development of obesity-related metabolic disorders. Alström Syndrome, a recessive ciliopathy, caused by mutations in ALMS1, is characterized by progressive metabolic alterations such as childhood obesity, hyperinsulinemia, and type 2 diabetes. Here we investigated the role of Alms1 disruption in AT expansion and insulin responsiveness in a murine model for Alström Syndrome. A gene trap insertion in Alms1 on the insulin sensitive C57BL6/Ei genetic background leads to early hyperinsulinemia and a progressive increase in body weight. At 6 weeks of age, before the onset of the metabolic disease, the mutant mice had enlarged fat depots with hypertrophic adipocytes, but without signs of inflammation. Expression of lipogenic enzymes was increased. Pre-adipocytes isolated from mutant animals demonstrated normal adipogenic differentiation but gave rise to mature adipocytes with reduced insulin-stimulated glucose uptake. Assessment of whole body glucose homeostasis revealed glucose intolerance. Insulin stimulation resulted in proper AKT phosphorylation in adipose tissue. However, the total amount of glucose transporter 4 (SLC4A2) and its translocation to the plasma membrane were reduced in mutant adipose depots compared to wildtype littermates. Alterations in insulin stimulated trafficking of glucose transporter 4 are an early sign of metabolic dysfunction in Alström mutant mice, providing a possible explanation for the reduced glucose uptake and the compensatory hyperinsulinemia. The metabolic signaling deficits either reside downstream or are independent of AKT activation and suggest a role for ALMS1 in GLUT4 trafficking. Alström mutant mice represent an interesting model for the development of metabolic disease in which adipose tissue with a reduced glucose uptake can expand by de novo lipogenesis to an obese state.

The Mmachc gene is required for pre-implantation embryogenesis in the mouse

Patients with mutations in MMACHC have the autosomal recessive disease of cobalamin metabolism known as cblC. These patients are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin and consequently have elevated homocysteine and methylmalonic acid in blood and urine. In addition, some cblC patients have structural abnormalities, including congenital heart defects. MMACHC is conserved in the mouse and shows tissue and stage-specific expression pattern in midgestation stage embryos. To create a mouse model of cblC we generated a line of mice with a gene-trap insertion in intron 1 of the Mmachc gene, (MmachcGt(AZ0348)Wtsi). Heterozygous mice show a 50% reduction of MMACHC protein, and have significantly higher levels of homocysteine and methylmalonic acid in their blood. The MmachcGt allele was inherited with a transmission ratio distortion in matings with heterozygous animals. Furthermore, homozygous MmachcGt embryos were not found after embryonic day 3.5 and these embryos were unable to generate giant cells in outgrowth assays. Our findings confirm that cblC is modeled in mice with reduced levels of Mmachc and suggest an early requirement for Mmachc in mouse development.

Disparate SEC23B Deficient Phenotypes in Humans and Mice

Abstract 974SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Mutations in Lyar and p53 are synergistically lethal in female mice

Ly-1 antibody reactive clone (LYAR) is a nucleolar zinc finger protein that has been implicated in cell growth, self-renewal of embryonic stem cells, and medulloblastoma. To test whether LYAR is critical for cell growth and development, we generated Lyar mutant mice. Mice carrying the mutant Lyar(gt) allele were generated from embryonic stem cells that contained a gene-trap insertion in the Lyar gene. Phenotypic analyses were performed on Lyar mutant mice and mouse embryonic fibroblasts. Lyar(gt/gt) mice were crossed to mice lacking the p53 tumor suppressor protein and Lyar/p53 compound mutants scored for external abnormalities. Lyar(gt/gt) homozygotes are viable, fertile, and indistinguishable from wild type littermates. However, the growth of Lyar(+/gt) and Lyar(gt/gt) mouse embryonic fibroblasts (MEFs) was impaired, coincident with an increase in the steady-state level of p53 and a key p53 effector of growth arrest, p21, suggesting that a cellular stress response is triggered in the absence of a wild type level of LYAR. Remarkably, the majority of Lyar(+/gt) and Lyar(gt/gt) female mice lacking p53 mice failed to survive. The neural tube defect (NTD) exencephaly was observed in ≈26% and ≈61% of female Lyar(+/gt;) p53(-/-) and Lyar(gt/gt;) p53(-/-) embryos, respectively. Lyar/p53 mutant mice represent a new digenic model of NTDs. Furthermore, these studies identify Lyar as a novel candidate gene for a role in human NTDs. These results provide new data to support the idea that loss of a p53-mediated developmental checkpoint may increase the risk of NTDs owing to some germline mutations.

The Role of Dlc1 Isoform 2 in K-Ras2G12D Induced Thymic Cancer

The Deleted in liver cancer one (Dlc1) tumor suppressor gene encodes a RhoGTPase activating protein (RhoGAP). The Dlc1 gene has multiple transcriptional isoforms and we have previously established a mouse strain containing a gene trap (gt) insertion, which specifically reduces the expression of the 6.1 kb isoform (isoform 2). This gene trapped allele when homozygous results in embryonic lethality and the heterozygous gene trapped mice do not show an increased incidence of cancers, suggesting that cooperating oncogenic changes may be required for transformation. In the present work, we have studied the in vivo cooperation between oncogenic K-Ras2 and Dlc1 genes in tumourigenesis. We have observed an increase in invasive thymic cancers, including both thymomas and lymphomas, resulting in significantly shortened life spans in mice heterozygous for the gt Dlc1 allele and an inducible LSL-K-Ras2G12D allele compared with the LSL-K-Ras2G12D only mice. The heterozygous mice showed a high degree of metastasis in the lung. We have found tumour specific selective hypermethylation of the Dlc1 isoform 2 promoter and reduction of the corresponding protein expression in thymic lymphoma (TL) and thymic epithelial carcinoma (TEC) derived from the thymic tumours. The Dlc1 deficient thymic lymphoma cell lines exhibited increased trans-endothelial cell migration. TEC cell lines also exhibited increased stress fiber formation and Rho activity. Introduction of the three Dlc1 isoforms tagged with GFP into these cells resulted in different morphological changes. These results suggest that loss of expression of only isoform 2 may be sufficient for the development of thymic tumors and metastasis.

A Screen for Genes Expressed in the Olfactory Organs of Drosophila melanogaster Identifies Genes Involved in Olfactory Behaviour

BackgroundFor insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.Methodology/Principal FindingsWe have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.Conclusions/SignificanceWe identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.

TET2 is essential for survival and hematopoietic stem cell homeostasis

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2(trap)) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2(trap/trap) mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2(trap/trap) HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2(trap/trap) HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

Lmo4 in the Basolateral Complex of the Amygdala Modulates Fear Learning

Pavlovian fear conditioning is an associative learning paradigm in which mice learn to associate a neutral conditioned stimulus with an aversive unconditioned stimulus. In this study, we demonstrate a novel role for the transcriptional regulator Lmo4 in fear learning. LMO4 is predominantly expressed in pyramidal projection neurons of the basolateral complex of the amygdala (BLC). Mice heterozygous for a genetrap insertion in the Lmo4 locus (Lmo4gt/+), which express 50% less Lmo4 than their wild type (WT) counterparts display enhanced freezing to both the context and the cue in which they received the aversive stimulus. Small-hairpin RNA-mediated knockdown of Lmo4 in the BLC, but not the dentate gyrus region of the hippocampus recapitulated this enhanced conditioning phenotype, suggesting an adult- and brain region-specific role for Lmo4 in fear learning. Immunohistochemical analyses revealed an increase in the number of c-Fos positive puncta in the BLC of Lmo4gt/+ mice in comparison to their WT counterparts after fear conditioning. Lastly, we measured anxiety-like behavior in Lmo4gt/+ mice and in mice with BLC-specific downregulation of Lmo4 using the elevated plus maze, open field, and light/dark box tests. Global or BLC-specific knockdown of Lmo4 did not significantly affect anxiety-like behavior. These results suggest a selective role for LMO4 in the BLC in modulating learned but not unlearned fear.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

Neurons and a Subset of Interstitial Cells of Cajal in the Enteric Nervous System Highly Express Stam2 Gene

Signal transducing adaptor molecule 2 (STAM2) is a phosphotyrosine protein, which is a member of the endosomal sorting complex required for transport (ESCRT-0) and is involved in the sorting process of the mono-ubiquitinated endosomal cargo for degradation in the lysosome. Analysis of gene trap mice carrying lacZ in frame with Stam2 revealed beta-galactosidase activity in the enteric nervous system (both in the myenteric and submucosal plexus) throughout the digestive tract. STAM2 immunostaining confirmed that the observed beta-galactosidase activity coincided with high Stam2 expression. To identify cell types with high Stam2 expression, STAM2 immunostaining was colocalized with the neuronal markers microtubule-associated protein 2 and protein gene product 9.5 and with c-kit as a marker for interstitial cells of Cajal (ICCs). STAM2 and c-kit positive cells comprised a subset of ICCs in the enteric nervous system. Qualitative and quantitative analysis of the morphology of the enteric nervous system in the homozygous mice carrying gene trap insertion in the Stam2 gene did not reveal phenotype changes; therefore, STAM2 function in the digestive tube remains elusive.

The Trithorax Group Protein Ash1l Is An Essential Epigenetic Regulator of Adult Hematopoietic Stem Cell Maintenance

Abstract 387The Trithorax family of epigenetic regulators is intimately linked to normal and malignant hematopoiesis. While substantial work indicates that Mixed lineage leukemia (Mll) is required for hematopoietic stem cell (HSC) homeostasis, the functions of many other Trithorax family members have not been evaluated. We have discovered that the mammalian Trithorax group gene absent, small, or homeotic 1-like (ash1l) is required for the maintenance of adult, but not fetal HSCs. Mice homozygous for a gene trap insertion into the first intron of ash1l (GT/GT) had a ca. 90% reduction in ash1l transcripts. These animals had normal numbers of phenotypically defined fetal liver HSCs (CD150+CD48−Lin−Sca-1hic-Kithi cells), but a 10-fold reduction in adult bone marrow HSCs already apparent by 6 weeks after birth. GT/GT bone marrow HSC depletion began in the first three weeks of life, the period during which HSCs turn off their fetal homeostasis program and enter a state of increased quiescence in the bone marrow. Cell cycle analysis revealed that GT/GT HSCs had an increased cycling fraction with a profound reduction in quiescent HSCs in the G0 phase of the cell cycle. This suggested that ash1l is essential to establish and/or maintain a quiescent population of adult-type HSCs. Furthermore, competitive and non-competitive transplantation assays showed that both fetal liver and adult bone marrow were incapable of providing long-term reconstitution in lethally irradiated recipients, indicating that neither compartment could sustain long-term HSC activity after reaching the recipient’s bone marrow. To understand this profound HSC defect, we next sought to analyze the in vivo function of Ash1l. Like MLL, Ash1l is a SET domain-containing histone methyltransferase. Although MLL functions as an H3K4 methyltransferase, the in vivo specificity of the Ash1l SET domain has not been described. Transcriptional analysis of GT/GT HSCs indicated that expression of Hoxa9, a known target of MLL, was reduced by 50%. This suggested that Hoxa9 could be a useful locus at which to evaluate the biochemical activity of Ash1l by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). ChIP-qPCR revealed that GT/GT bone marrow cells had reduced H3K36 dimethylation but preserved H3K4 trimethylation at the Hoxa9 locus. This was consistent with previous in vitro analysis of Ash1l SET domain activity and computer predictions showing a high degree of conservation between Ash1l and SET2, an H3K36 methyltransferase. Thus, Ash1l and MLL are both required for Hoxa9 expression although their enzymatic activities differ, suggesting that Ash1l and MLL may act cooperatively at Hoxa9 and other target loci. To evaluate the functional consequences of this interaction, we studied mice deficient for ash1l and lacking Menin, a factor required for proper MLL targeting to target loci. Strikingly, these animals rapidly progressed to hematopoietic failure with a complete obliteration of the hematopoietic stem and progenitor compartment, a phenotype not observed in either genetic background independently. These data indicate that Ash1l and MLL/menin work cooperatively in hematopoiesis. Together, our findings reveal an essential function of ash1l in the maintenance of adult HSCs. Furthermore, they indicate that Ash1l functions in vivo as an H3K36 methyltransferase and suggest that different Trithorax family members can coregulate target genes by providing distinct activating histone marks. Future work will reveal the full extent and mechanisms of these cooperative effects in normal and malignant hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis

The kinase VRK1 has been implicated in mitotic and meiotic progression in invertebrate species, but whether it mediates these events during mammalian gametogenesis is not completely understood. Previous work has demonstrated a role for mammalian VRK1 in proliferation of male spermatogonia, yet whether VRK1 plays a role in meiotic progression, as seen in Drosophila, has not been determined. Here, we have established a mouse strain bearing a gene trap insertion in the VRK1 locus that disrupts Vrk1 expression. In addition to the male proliferation defects, we find that reduction of VRK1 activity causes a delay in meiotic progression during oogenesis, results in the presence of lagging chromosomes during formation of the metaphase plate, and ultimately leads to the failure of oocytes to be fertilized. The activity of at least one phosphorylation substrate of VRK1, p53, is not required for these defects. These results are consistent with previously defined functions of VRK1 in meiotic progression in Drosophila oogenesis, and indicate a conserved role for VRK1 in coordinating proper chromosomal configuration in female meiosis.