Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression Profiling of Dll1 mutant mouse lines on different genetic background

ABSTRACT: The evolutionarily conserved Delta-Notch cell-cell signal-transduction pathway regulates the determination of various cell fates, which are important for the morphogenesis and development of numerous organs and tissues in many vertebrates and invertebrates. The Dll1 gene was mutated by homologous recombination in embryonic stem cells such that amino acids 2-116 were replaced with an in-frame fusion of the lacZ gene of E.coli. Mouse Dll1 genomic clones were isolated from a strain 129/SvPas library. The replacement vector was constructed by inserting a 4 kb ClaI/XhoI fragment, which encodes a part of the beta-galactosidase gene followed by the PGK-neo cassette, and a 2.6 kb XhoI/EcoRI fragment as the 3M-bM-^@M-^Y homologous region of the replacement vector into pKS+. This vector was linearized with SalI and ClaI, and a 4.1 kb SalI/NcoI as the 5M-bM-^@M-^Y homologous region plus an NcoI/ClaI fragment (800 bp) completing the b-galactosidase gene, were inserted, resulting in the complete replacement vector. This vector was linearized with SalI and electroporated in R1 embryonic stem cells. Correctly targeted clones were identified and verified by Southern blot analysis using external probes from the 3M-bM-^@M-^Y and 5M-bM-^@M-^Y region of the targeted area, and were injected into C57BL/6J embryo to obtain germline transmission. The two analysed Dll1 mutant mouse lines carry the same mutation but are on different genetic background: 1. Heterozygous F1-animals were backcrossed several times to the 129SV/J wild type; 2. Heterozygous F1-animals were outcrossed 11 generation to C3HeB/FeJ wild type Four organs (liver, spleen, thymus, brain) of two Dll1 mutant mouse lines on different genetic background carrying the same mutation were analysed by cDNA microarray technology. Experiment include 4-5 biological replicates for reference (wildtype) and mutant animals. Up to 4 technical replicates for each mutant mouse were performed. As reference pooled RNA was used. 50% of the chip hybridisations are dye sway experiments.

ORGANISM(S): Mus musculus

SUBMITTER: Marion Horsch

PROVIDER: E-GEOD-11867 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Dll1 haploinsufficiency in adult mice leads to a complex phenotype affecting metabolic and immunological processes.

Rubio-Aliaga Isabel I Przemeck Gerhard K H GK Fuchs Helmut H Gailus-Durner Valérie V Adler Thure T Hans Wolfgang W Horsch Marion M Rathkolb Birgit B Rozman Jan J Schrewe Anja A Wagner Sibylle S Hoelter Sabine M SM Becker Lore L Klopstock Thomas T Wurst Wolfgang W Wolf Eckhard E Klingenspor Martin M Ivandic Boris T BT Busch Dirk H DH Beckers Johannes J Hrabé de Angelis Martin M

PloS one 20090629 6

<h4>Background</h4>The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligand ...[more]

PMID: 19562077

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Project description:BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes. Four organs (liver, spleen, thymus, brain) of the Dll1 mutant mouse line analysed by cDNA microarray technology. Experiments include four biological replicates for reference (wildtype) and mutant animals. Two technical replicates for each mutant mouse were performed. As reference pooled RNA of the same organ was used. 50% of the chip hybridisations are dye sway experiments.

Project description:Previous studies have evaluated the role of miRNAs in the initiation and progression of cancer. MiR-34a was found to be downregulated in several tumors, including medulloblastoma. We here analysed the function of miR-34a in vivo by targeted transgenesis to generate mice with constitutive deletion of the miR-34a gene, which resulted in the absence of mir-34a in all analysed tissues. Nevertheless, these mice were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters performed by the German Mouse Clinic revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significant downregulation as compared to human cerebellum. Re-expression of mir-34a in human medulloblastoma cells in vitro reduced cell viability, cell proliferation and induced apoptosis. Among the targets downregulated by miR-34a in human medulloblastoma cells were NMYC and SIRT1. Activation of the Shh pathway by targeted overexpression of SmoA1 causes medulloblastoma in mice, which is dependent on the presence and upregulation of NMYC. Analysis of miR-34a in ND2:SmoA1-derived medulloblastomas revealed significant suppression of miR-34a compared to normal cerebellum. Crossbreeding these mice with miR-34a knockout mice significantly accelerated medulloblastoma growth in mice deficient for miR-34a. Interestingly, NMYC and SIRT1 were highly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastoma. Strategies aiming to re-express miR-34a in tumors could therefore represent an efficient therapy option. For genome-wide expression analysis total RNA from brain and thymus of three or four male miR34a and four control mice was isolated using RNeasy Midi kit (Qiagen, Hilden, Germany). The cDNA microarrays were generated, hybridized and analysed as described (Horsch et al 2009). Two chip hybridizations were performed with total RNA for each individual mutant mouse against a reference RNA pool of the same organ.

Project description:The alpha-fetoprotein (AFP) gene is a member of the albumin gene family and encodes a serum glycoprotein with an onco-fetal pattern of expression. AFP is produced in high concentrations during embryonic life by the hepatocytes and the visceral endoderm of the yolk sac and to a lesser extent by the developing gastrointestinal tract and kidney. Alpha-fetoprotein is the major serum fetal protein in mammals, with a concentration that reaches the order of several mg/ml. Its synthesis decreases dramatically shortly after birth to reach only trace amounts a few weeks later but can be restored during life when liver pathologies or some types of tumors develop (hepatitis, cirrhosis, hepatoma, teratocarcinoma, and some pancreatic and renal tumors). AFP is then mainly expressed by the adult liver. AFP produced by the embryo is secreted in the amniotic fluid and is able to cross the placental barrier to reach the maternal blood circulation, where its titer is used as a diagnostic marker to reveal developmental anomalies of the fetus. Abnormally high levels of AFP in the maternal serum indicates elevated risk for neural tube defects of the fetus such as spina bifida or anencephaly, whereas abnormally low levels indicates elevated risk for a Down’s syndrome. Measurements of the AFP levels in the maternal serum are undertaken at 14-22 weeks of each pregnancy and are part, along with unconjugated estradiol, human chorionic gonadotropin and inhibin A, of the quadruple test for antenatal Down’s syndrome screening. Abnormal AFP levels can also be indicative of other fetal pathologies. The reason why these pathologies induce abnormal levels of AFP is still not known. Homozygous Afp knock-out females (AFP KO) are sterile, due to anovulation. Ovaries contain mature follicles and are functional, but lack a proper signal from the hypothalamic-pituitarian axis (HPG) in order to ovulate. The expression profile of several genes in the HPG is impaired. Furthermore, AFP KO females are masculinized and defeminized. They exhibit a loss of female sexual behaviour (no lordosis) and a male pattern of tyrosine hydroxylase expressing neurons distribution in some sexual dimorphic areas of the brain. The phenotype of those mice is a consequence of an estrogen overexposure of their brains during the perinatal period. This phenotype can be reversed if these mice develop in an embryonic environment strongly reduced in estrogens. Homozygous AFP KO mice that developed in those conditions are fertile in adulthood (no further treatment needed), and exhibit proper sexual behaviour. cDNA microarray analysis of embryonic and adult liver tissue of AFP1 kO mice. Single mutant mice were analysed versus a pool of wt RNA in two technical replicates (only for adult tissue) including a dye swap experiment. In total 6 adult mutant male mice and 6 embryos (3 male and 3 female) were analysed

Project description:Col1a1 is located on mouse distal chromosome 11 (Celera 94607393-94622990bp; 15kb) and encodes type I procollagen that associates stoichiometrically with col1a2 in forming secreted type I mature collagen that self-assembles into a supramolecular fibrillar structure consisting of a protein family. Type I collagens are predominantly enriched in bone, cartilage, skin, tendons, and eye (i.e. in major fibrous tissues) making up the main extracellular matrix (ECM) component for support and scaffolding purposes. Col1a1 is known to be expressed in the embryo (E9.5; Northern dot blot), perioptic mesenchyme (E11), cornea (E11), skeleton (E14.5), brain meninges (E14.5), and cartilage (E14.5) at relatively early stages. Most evidence of type I collagen function is coupled to the latter stages of skeletal development, thus bypassing the major events of axis formation, tissue differentiation, and mesenchlymal-epithial interactions, for example, that are more symbolic of morphogens and growth factors. Members of the integrin cell surface receptor family of molecules mediate cell adhesion to ECM proteins such as collagen (mainly to type IV basement membrane collagen that forms into a polygonal meshwork), fibronectin, and laminin implicated in wound healing and inflammatory responses (e.g. Crohn disease, ulcerative colitis) in connective tissues. There is some evidence for protein-protein interactions between leukocyte-associated integrins and the interstitial matrix in promoting the migration and/or activation of extravasated leukocytes (e.g., T cells and monocytes) within the perivascular compartment, an ECM-rich environment. This region is surmised to be an important location where certain human leukocytes undergo differentiation (e.g. monocytes) and activation (neutrophils, monocytes, lymphocytes) upon extravascular migration. The von Willebrand factor (vWF), type C motif is found in various plasma proteins like complement factors, the integrins, collagen types, and other extracellular proteins. Thus, the majority of vWF-containing proteins are extracellular and a common feature appears to be involvement in multi protein complexes participating in numerous biological events (e.g. cell adhesion, migration, homing, pattern formation, and signal transduction). Three organs (lung, heart and bone) of 5 AGA002 mutant mice (age 2x 10 days and 3x 11 days) were analysed by cDNA microarray technology. As reference five mice, 11 days old were used 50% of the chip hybridisations are dye swap experiments.

Dataset Information

Expression Profiling of Dll1 mutant mouse lines on different genetic background

Publications

Dll1 haploinsufficiency in adult mice leads to a complex phenotype affecting metabolic and immunological processes.

Similar Datasets

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