dcc orchestrates the development of the prefrontal cortex during adolescence and is altered in psychiatric patients.

Adolescence is a period of heightened susceptibility to psychiatric disorders of medial prefrontal cortex (mPFC) dysfunction and cognitive impairment. mPFC dopamine (DA) projections reach maturity only in early adulthood, when their control over cognition becomes fully functional. The mechanisms governing this protracted and unique development are unknown. Here we identify dcc as the first DA neuron gene to regulate mPFC connectivity during adolescence and dissect the mechanisms involved. Reduction or loss of dcc from DA neurons by Cre-lox recombination increased mPFC DA innervation. Underlying this was the presence of ectopic DA fibers that normally innervate non-cortical targets. Altered DA input changed the anatomy and electrophysiology of mPFC circuits, leading to enhanced cognitive flexibility. All phenotypes only emerged in adulthood. Using viral Cre, we demonstrated that dcc organizes mPFC wiring specifically during adolescence. Variations in DCC may determine differential predisposition to mPFC disorders in humans. Indeed, DCC expression is elevated in brains of antidepressant-free subjects who committed suicide.

Constitutive CD27/CD70 interaction induces expansion of effector-type T cells and results in IFNgamma-mediated B cell depletion.

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T cell expansion. Normally, tightly regulated expression of CD70 ensures the transient availability of this costimulatory signal. Mice expressing constitutive CD70 on B cells had higher peripheral T cell numbers that showed increased differentiation toward effector-type T cells. B cell numbers in CD70 transgenic (TG) mice progressively decreased in primary and secondary lymphoid organs. This B cell depletion was caused by CD27-induced production of IFNgamma in T cells. We conclude that apart from its role in controlling the size of the activated T cell pool, CD27 ligation contributes to immunity by facilitating effector T cell differentiation.

Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice.

CDKN2A (INK4a/ARF) is frequently disrupted in various types of human cancer, and germline mutations of this locus can confer susceptibility to melanoma and other tumours. However, because CDKN2A encodes two distinct cell cycle inhibitory proteins, p16INK4a and p14ARF (p19Arf in mice), the mechanism of tumour suppression by CDKN2A has remained controversial. Genetic disruption of Cdkn2a(p19Arf) (hereafter Arf) alone predisposes mice to tumorigenesis, demonstrating that Arf is a tumour-suppressor gene in mice. We mutated mice specifically in Cdkn2a(p16Ink4a) (hereafter Ink4a). Here we demonstrate that these mice, designated Ink4a*/*, do not show a significant predisposition to spontaneous tumour formation within 17 months. Embryo fibroblasts derived from them proliferate normally, are mortal, and are not transformed by oncogenic HRAS. The very mild phenotype of the Ink4a*/* mice implies that the very strong phenotypes of the original Ink4a/ArfDelta2,3 mice were primarily or solely due to loss of Arf. However, Ink4a*/Delta2,3 mice that are deficient for Ink4a and heterozygous for Arf spontaneously develop a wide spectrum of tumours, including melanoma. Treatment of these mice with the carcinogen 7,12-dimethylbenzanthracene (DMBA) results in an increased incidence of melanoma, with frequent metastases. Our results show that, in the mouse, Ink4a is a tumour-suppressor gene that, when lost, can recapitulate the tumour predisposition seen in humans.

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance.

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.

A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.

A mouse model with a targeted mutation in the 3' end of the endogenous Brca1 gene, Brca1(1700T), was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca1(1700T) mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca1(1700T) mutation is embryonic lethal in homozygous state. However, homozygous Brca1(1700T) embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1(null) models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5-10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function.

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.

Mice with a targeted disruption of the Fanconi anemia homolog Fanca.

Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer predisposition. Bone marrow failure resulting in pancytopenia is the main cause of death of FA patients. Diagnosis of FA is based on their cellular hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic complementation experiments suggest the involvement of at least eight genes in FA. The gene for complementation group A (FANCA) is defective in the majority of FA patients. We show here that mice deficient of FANCA: are viable and have no detectable developmental abnormalities. The hematological parameters showed a slightly decreased platelet count and a slightly increased erythrocyte mean cell volume in mice at young age, but this did not progress to anemia. Consistent with the clinical phenotype of FA patients, both male and female mice showed hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the knock-out mice exhibited spontaneous chromosomal instability and were hyper-responsive to the clastogenic effect of the crosslinker mitomycin C.

PIM1 reconstitutes thymus cellularity in interleukin 7- and common gamma chain-mutant mice and permits thymocyte maturation in Rag- but not CD3gamma-deficient mice.

The majority of lymphomas induced in Rag-deficient mice by Moloney murine leukemia virus (MoMuLV) infection express the CD4 and/or CD8 markers, indicating that proviral insertions cause activation of genes affecting the development from CD4(-)8(-) pro-T cells into CD4(+)8(+) pre-T cells. Similar to MoMuLV wild-type tumors, 50% of CD4(+)8(+) Rag-deficient tumors carry a provirus near the Pim1 protooncogene. To study the function of PIM proteins in T cell development in a more controlled setting, a Pim1 transgene was crossed into mice deficient in either cytokine or T cell receptor (TCR) signal transduction pathways. Pim1 reconstitutes thymic cellularity in interleukin (IL)-7- and common gamma chain-deficient mice. In Pim1-transgenic Rag-deficient mice but notably not in CD3gamma-deficient mice, we observed slow expansion of the CD4(+)8(+) thymic compartment to almost normal size. Based on these results, we propose that PIM1 functions as an efficient effector of the IL-7 pathway, thereby enabling Rag-deficient pro-T cells to bypass the pre-TCR-controlled checkpoint in T cell development.

Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells.

Signaling through the pre-TCR is essential for early T cell development and is severely impaired in mice lacking the CD3 gamma chain of the pre-TCR. We here address the molecular mechanisms underlying this defect. Impaired pre-TCR signaling is shown to be associated with a profound increase in the number of apoptotic CD4- CD8- (DN) thymocytes. Introduction of p53 deficiency into CD3 gamma-deficient mice completely reverses the cell survival defect in CD3 gamma-deficient DN thymocytes and rescues the block in pre-T cell differentiation. In addition, the CD4+ CD8+ (DP) compartment is expanded to its normal size. These findings suggest that the pre-TCR regulates progression through the DNA-damage checkpoint of the DN to DP transition by inactivating p53.

Identification and characterization of collaborating oncogenes in compound mutant mice.

We have used proviral tagging in tumor-prone transgenic mice to identify collaborating oncogenes and genes contributing to tumor progression. This has yielded a series of oncogenes that could be assigned to different complementation groups in transformation: the myc, Pim, Bmi1, and Frat1 complementation groups. Frat1 is involved in tumor progression and appears to function in the Wnt signaling pathway. Overexpression of Fratl confers a growth advantage to transplanted tumor cells in vivo and to cells grown in vitro at high density. Frat1 might exert its activity by impairing the kinase activity of Gsk3beta, which is involved in the degradation of beta-catenin. Pim genes appear to act in tumor initiation and show strong synergism with myc in lymphomagenesis. Overexpression of Pim1 can also overcome some of the proliferative defects caused by defective interleukin signaling supporting a role of Pim1 in cell proliferation. We have applied proviral tagging in compound mutant Emu-myc/Pim1-/-/Pim2-/- mice to identify genes that can complement for the loss of Pim1 and Pim2 and, therefore, are able to synergize with c-myc in lymphomagenesis. A number of new as well as known genes have been found by this "complementation tagging." The latter included c-kit, Tp12, and cyclin D2, suggesting that Pim kinases might act upstream of or parallel to these known proto-oncogenes.

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.

Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.

The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Immunological defects in mice with a targeted disruption in Bcl-3.

The proto-oncogene bcl-3 is a member of the IkappaB family. The Bcl-3 protein is known to interact specifically with the p50 and p52 subunits of NFkappaB. However, the function of this interaction is not well understood. To determine the in vivo role of Bcl-3, mice were generated that lack the bcl-3 gene, Bcl 3(-/-). Here we report that Bcl 3(-/-) mice appear developmentally normal, but exhibit severe defects in humoral immune responses and protection from in vivo pathogenic challenges. Relative to wild-type mice, Bcl 3(-/-) mice are unable to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae. This phenotype is similar to that observed in the p50(-/-) mice and the cross between the Bcl-3(-/-) and p50(-/-) mice generates animals with an enhanced phenotype. In accordance with the observed defects in their immune response, the Bcl 3(-/-) mice have normal immunoglobulin levels before and after immunization, but fail to produce antigen-specific antibodies. Additionally, spleens from Bcl-3(-/-) mice are abnormal and void of germinal centers. In contrast, the p50(-/-) mice have normal germinal centers. We propose that in in vivo, Bcl-3 can function independently of p50.

Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice.

Integrins are heterodimeric transmembrane glycoproteins which are engaged in a variety of cellular functions, such as adhesion, migration and differentiation1. The integrin alpha 6 beta 4 is expressed on squamous epithelia, on subsets of endothelial cells, immature thymocytes and on Schwann cells and fibroblasts in the peripheral nervous system. In stratified epithelia, alpha 6 beta 4 is concentrated in specialised adhesion structures, called hemidesmosomes, which are implicated in the stable attachment of the basal cells to the underlying basement membrane by connecting the intermediate filaments with the extracellular matrix. The nature of the interactions between the various hemidesmosomal proteins, that lead to the formation of hemidesmosome is poorly understood. To study the contribution of the integrin alpha 6 beta 4 in hemidesmosome formation and their anchoring properties, we inactivated the beta 4 gene in mice by targeted gene disruption. Homozygous beta 4 null mice died shortly after birth and displayed extensive detachment of the epidermis and other squamous epithelia. The dramatically reduced adhesive properties of the skin was accompanied by the absence of hemidesmosomes at the basal surface of keratinocytes. No evidence was found for impaired T-cell development, nor for defects in myelination in the peripheral nervous system.

Expression analysis of the individual bovine beta-, alpha s2- and kappa-casein genes in transgenic mice.

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5'- and 8 kb of 3'-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5'- and 1.5 kb and 19 kb of 3'-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.

Expression of human lactoferrin in milk of transgenic mice.

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.

Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia.

Apolipoprotein (apo) E3-Leiden, described in a large Dutch family, is associated with a dominantly inherited form of familial dysbetalipoproteinemia. To study the effect of the APOE*3-Leiden mutation in vivo, transgenic mice were generated using a genomic 27-kilobase DNA construct isolated from the APOE*3-Leiden proband. This construct carried the APOE gene, the APOC1 gene, and all known regulatory elements including an element that mediates liver expression. Three strains were generated that showed human APOE and APOC1 expression. All strains had significantly elevated levels of total plasma cholesterol and triglycerides on a regular diet. When mice of one strain were fed a semisynthetic cholesterol-rich diet, total plasma cholesterol and triglyceride levels increased dramatically. This increase was observed mainly in the very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-sized fractions. In cholesterol-fed mice, the apoE3-Leiden protein became equally distributed between the VLDL/LDL and HDL-sized fractions, while in mice kept on a regular diet, apoE3-Leiden protein was mainly associated with HDL-sized fractions. The presence of hyperlipoproteinemia in the APOE*3-Leiden-expressing transgenic mice supports our finding that the apoE3-Leiden variant behaves like a dominant trait in the expression of familial dysbetalipoproteinemia. ApoE3-Leiden transgenic mice may serve as a model to elucidate additional factors involved in the metabolism of apoE containing remnant lipoproteins in general and the etiology of familial dysbetalipoproteinemia in particular.

Efficient generation of functional transgenes by homologous recombination in murine zygotes.

To assess the feasibility of generating functional transgenes directly via homologous recombination between microinjected DNA fragments, three overlapping genomic DNA fragments, together constituting the human serum albumin (hSA) gene, were coinjected into murine zygotes. The resulting transgenic mice were analyzed for structure and expression of the transgene. All transgenic mice carried recombined hSA DNA fragments and 74% contained a reconstituted hSA gene. HSA expression could be detected in liver and serum in most (72%) of these animals. Only correctly sized hSA transcripts were observed. Transgenic hSA could not be distinguished from the human serum-derived protein by radioimmunoassay or Western blotting. The high frequency and accuracy of homologous recombination in murine zygotes reported here allows the efficient generation of relatively large transgenes.

Generation of transgenic dairy cattle using 'in vitro' embryo production.

We have combined gene transfer, by microinjection, with 'in vitro' embryo production technology, enabling us to carry out non-surgical transfer, to recipient cows, of microinjected embryos that have been cultured from immature oocytes. Using this approach, we have established 21 pregnancies from which 19 calves were born. Southern blot analysis proved that in two cases the microinjected DNA had been integrated in the host genome.