We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair1. In addition, antigens present within necrotic cells can sometimes provoke a specific immune response2-4 and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection5, 6. In the mouse, the CD8α+ subset of dendritic cells (DC) phagocytoses dead cell remnants and crossprimes CD8+ T cells against cell-associated antigens7. Here, we show that CD8α+ DC utilise CLEC9A (DNGR-1), a recently-characterised C-type lectin8-10, to recognise a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair uptake of necrotic cell material by CD8α+ DC but specifically reduces crosspresentation of dead cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue within its intracellular tail that allows recruitment and activation of the tyrosine kinase Syk, which is also essential for crosspresentation of dead cell-associated antigens. Thus, CLEC9A functions as a Syk-coupled C-type lectin receptor to mediate sensing of necrosis by the principal DC subset involved in regulating crosspriming to cell-associated antigens.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Using homologous recombination, mice lacking cyclin Dl were generated by replacing most of the first exon of the Cyl-1 gene with sequences encoding neomycin resistance. Cyl-1~'~ mice were viable and fertile but consistently smaller than their heterozygous or wild-type littermates. The nullizygous animals also showed two distinctive abnormalities: a severe retinopathy caused by impaired development of all layers of the retina and, in the mammary gland during pregnancy, a marked reduction in acinar development accompanied by a failure to lactate. Approximately 50% of animals also had a malformation of the jaw that manifested itself as a misalignment of the incisor teeth. Mouse embryo fibroblasts isolated from 14 day nullizygous, heterozygous, or wild-type embryos and grown under standard conditions showed similar cell-cycle and growth characteristics. Thus although cyclin Dl kinase activity may facilitate G, progression, it is not essential for the development of most tissues and organs, and only a few specialized cell lineages are demonstrably sensitive to its absence.

Following immunogenic challenge, infiltrating and dividing lymphocytes significantly increase lymph node (LN) cellularity leading to organ expansion1,2. Here we report that the physical elasticity of LNs is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells (DCs). We show that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-kinase. Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunised mice augments LN expansion. In contrast, the latter is significantly constrained in mice selectively lacking CLEC-2 expression in DCs. Thus, the same DCs that initiate immunity by presenting antigens to T lymphocytes3 also initiate remodeling of LNs by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid LN expansion driven by lymphocyte influx and proliferation that is the critical hallmark of adaptive immunity.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 335 exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3 mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of genetargeted Trex1؊/؊ mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3 termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1 ؊/؊ mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.Two distinct nuclear exonucleases account for the major part of the total exonucleolytic activity on DNA observed in mammalian cell extracts (24, 25). They were identified as a 3Ј35Ј exonuclease acting preferentially on single-stranded DNA and a 5Ј33Ј exonuclease specific for double-stranded DNA that could remove a single-stranded 5Ј overhang as an oligonucleotide. These nuclear enzymes were designated DNase III and DNase IV, as they are distinct from the pancreatic and macrophage lysosomal DNA endonucleases DNase I and DNase II. DNase IV was later renamed flap endonuclease 1 (FEN1) (23), and its main function is processing displaced 5Ј single strands that arise during lagging-strand DNA replication, as well as during DNA repair, recombination, and triplet repeat expansion. The elimination of Fen1 activity leads to early embryonic lethality in mice, consistent with an essential role of the enzyme in DNA replication (20). In contrast, DNase III is expressed at similar levels in nonproliferating and proliferating tissues; it was isolated as the major nuclear 3Ј35Ј DNA exonuclease from the adult rabbit liver (14) and also from the calf thymus and human myoblasts, where it was designated TREX1 (37, 38).The human TREX1/DNase III cDNA (14, 29) shares amino acid sequence homology with the Escherichia coli DnaQ/MutD editing subunit of the replicative DNA polymerase III holoenzyme, which can increase the fidelity of an exonuclease-deficient mammalian DNA polymerase in vitro (36). The TREX1/ DNase III cDNA encodes a nonprocessive 3Ј35Ј DNA-specific exonuclease, with a preference for single-stranded DNA or mispaired 3Ј termini; like the native protein isolated from mammalian cells, it forms homodimers (14, 29, 30). Since two of the major mammalian nuclear DNA polymerases, Pol␣ and Pol␤, do not have an intrinsic 3Ј exonuclease function, it was proposed that TREX1/DNase III may serve to edit mismatched deoxyribonucleotides during lagging-strand DNA synthesis or gap filling in DNA base excision repair, which are conducted by Pol␣ and Pol␤, respectively...