Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

Food availability dictates the timing of parturition in insectivorous mouse-eared bats

Which of these two confounding factors, weather or food availability - that largely correlate and interact - controls the timing of parturition in insectivorous bats? To answer this question. we took advantage of a predator-prey system that offers a unique opportunity to perform natural experiments. The phenology of reproduction of two sibling bat species that inhabit the same colonial roosts, but exploit different feeding niches. was investigated. Myotis myotis feeds mainly on carabid beetles, a food source available from the end of hibernation onwards, whereas bush crickets, the main prey of M. blythii, are not available early in the season due to their successive instars; cockchafers are actually the sole possible alternative prey for M. blythii at that time of the year, but they occur every third year only, independently of local weather conditions. By comparing the species responses to the presence/absence of cockchafers, we could test the hypothesis that food availability, rather than climate. influences the timing of bat parturition. Our data show that Nt. blythii gave birth, on average. 10 d later than M. myotis in years without cockchafers, whilst parturition (1) was synchronous during cockchafer years, and (2) did not show much among-year time variation in M. myotis. This suggests that food availability is the chief factor regulating the timing of parturition in mouse-eared bats.

Modulation of the gap junction protein Connexin36 in neurons in a mouse model of transient focalcerebral ischemia

Intercellular communication is achieved at specialized regions of the plasma membrane by¦gap junctions. Gap junctions are transmembrane channels allowing direct contacts between¦the cytoplasms of neighboring cells. Each cell participates with one hemichannel, or¦connexon, made of six protein subunits named connexins. Thanks to these junctions, cells¦potentially share a pool of small molecules and metabolites, such as nucleotides, amino acids¦and second messengers.¦In an ischemic (i.e. non-perfused) territory of the brain, irreversible damage progresses over¦time from the centre of the most severe flow reduction to the periphery with less disturbed¦perfusion. Functionally impaired tissue can survive and recover if sufficient reperfusion is reestablished¦within a limited time period, which depends on various factors and mechanisms¦modulating the signaling pathways leading to cell death.¦Observations were made indicating the presence of electrical coupling between neurons which¦resist better to an ischemic insult. This electrical coupling is likely to be mediated by¦Connexin36 (Cx36), a neuron specific connexin isoform. It was demonstrated in the past that¦global ischemia induces a selective upregulation of Cx36 expression in regions with neurons¦that survive the insult whereas others undergo apoptosis and die. These observations raise the¦possibility that the neuronal gap junction Cx36 might play a role in the destiny of neurons¦after cerebral ischemia.¦The aim of this work was to characterize the regulation of Connexin36 in a mouse model of¦transient focal cerebral ischemia by immunofluorescence and Western blot analysis. Our¦immunofluorescence results suggest a specific increase in Cx36 in the penumbral region of¦the ischemic hemisphere.

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Identification of early metabolic markers of various degrees of ischemia in the mouse brain by localized H-1 magnetic resonance spectroscopy

Objectives: Magnetic resonance (MR) imaging and spectroscopy (MRS) allow the establishment of the anatomical evolution and neurochemical profiles of ischemic lesions. The aim of the present study was to identify markers of reversible and irreversible damage by comparing the effects of 10-mins middle cerebral artery occlusion (MCAO), mimicking a transient ischemic attack, with the effects of 30-mins MCAO, inducing a striatal lesion. Methods: ICR-CD1 mice were subjected to 10-mins (n = 11) or 30-mins (n = 9) endoluminal MCAO by filament technique at 0 h. The regional cerebral blood flow (CBF) was monitored in all animals by laser- Doppler flowmetry with a flexible probe fixed on the skull with < 20% of baseline CBF during ischemia and > 70% during reperfusion. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were acquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6 to 8 microliters). Six animals (sham group) underwent nearly identical procedures without MCAO. Results: The 10-mins MCAO induced no MR- or histologically detectable lesion in most of the mice and a small lesion in some of them. We thus had two groups with the same duration of ischemia but a different outcome, which could be compared to sham-operated mice and more severe ischemic mice (30-mins MCAO). Lactate increase, a hallmark of ischemic insult, was only detected significantly after 30-mins MCAO, whereas at 3 h post ischemia, glutamine was increased in all ischemic mice independently of duration and outcome. In contrast, glutamate, and even more so, N-acetyl-aspartate, decreased only in those mice exhibiting visible lesions on T2-weighted images at 24 h. Conclusions: These results suggest that an increased glutamine/glutamate ratio is a sensitive marker indicating the presence of an excitotoxic insult. Glutamate and NAA, on the other hand, appear to predict permanent neuronal damage. In conclusion, as early as 3 h post ischemia, it is possible to identify early metabolic markers manifesting the presence of a mild ischemic insult as well as the lesion outcome at 24 h.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human.

Local moderate magnetically induced hyperthermia using an implant formed in situ in a mouse tumor model.

Purpose: We investigate a new heat delivery technique for the local treatment of solid tumors. The technique involves injecting a formulation that solidifies to form an implant in situ. This implant entraps superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microbeads for magnetically induced moderate hyperthermia. Particle entrapment prevents phagocytosis and distant migration of SPIONs. The implant can be repeatedly heated by magnetic induction. Methods: We evaluated heating and treatment efficacies by means of thermometry and survival studies in nude mice carrying subcutaneous human colocarcinomas. At day 1, we injected the formulation into the tumor. At day 2, a single 20-min hyperthermia treatment was delivered by 141-kHz magnetic induction using field strengths of 9 to 12 mT under thermometry. Results: SPIONs embedded in silica microbeads were effectively confined within the implant at the injection site. Heat-induced necro-apoptosis was assessed by histology on day 3. On average, 12 mT resulted in tumor temperature of 47.8 degrees C, and over 70% tumor necrosis that correlated to the heat dose (AUC = 282 degrees C.min). In contrast, a 9-mT field strength induced tumoral temperature of 40 degrees C (AUC = 131 degrees C.min) without morphologically identifiable necrosis. Survival after treatment with 10.5 or 12 mT fields was significantly improved compared to non-implanted and implanted controls. Median survival times were 27 and 37 days versus 12 and 21 days respectively. Conclusion: Five of eleven mice (45%) of the 12 mT group survived one year without any tumor recurrence, holding promise for tumor therapy using magnetically induced moderate hyperthermia through injectable implants.

The fine-scale architecture of structural variants in 17 mouse genomes.

BACKGROUND: Accurate catalogs of structural variants (SVs) in mammalian genomes are necessary to elucidate the potential mechanisms that drive SV formation and to assess their functional impact. Next generation sequencing methods for SV detection are an advance on array-based methods, but are almost exclusively limited to four basic types: deletions, insertions, inversions and copy number gains. RESULTS: By visual inspection of 100 Mbp of genome to which next generation sequence data from 17 inbred mouse strains had been aligned, we identify and interpret 21 paired-end mapping patterns, which we validate by PCR. These paired-end mapping patterns reveal a greater diversity and complexity in SVs than previously recognized. In addition, Sanger-based sequence analysis of 4,176 breakpoints at 261 SV sites reveal additional complexity at approximately a quarter of structural variants analyzed. We find micro-deletions and micro-insertions at SV breakpoints, ranging from 1 to 107 bp, and SNPs that extend breakpoint micro-homology and may catalyze SV formation. CONCLUSIONS: An integrative approach using experimental analyses to train computational SV calling is essential for the accurate resolution of the architecture of SVs. We find considerable complexity in SV formation; about a quarter of SVs in the mouse are composed of a complex mixture of deletion, insertion, inversion and copy number gain. Computational methods can be adapted to identify most paired-end mapping patterns.