Cells In Mutant Mice Research Articles

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.

An in-frame deletion in peripheral myelin protein-22 gene causes hypomyelination and cell death of the schwann cells in the new Trembler mutant mice

Cloning and sequencing of the peripheral myelin protein-22 cDNA and genomic DNA from newly found Trembler mice revealed an in-frame deletion including exon IV which codes for the second (TM2) and a part of third (TM3) transmembrane domain of peripheral myelin protein-22. This mutation was distinct from those in both other allelic Trembler and Trembler-J mice, which carry point mutations within the putative transmembrane spanning regions of peripheral myelin protein-22. Inheritance was autosomal dominant. The affected mice revealed an abnormal gait, which appeared at 15–20 days of age, followed by motor and sensory ataxia, which remained throughout life. Most of the affected mice could survive more than one year. One of the most notable pathological phenotypes was a giant vacuolar formation in the sciatic nerve of homozygotes. They vary in size within the cytoplasm of Schwann cells, which failed to assemble myelin at any ages studied. Heterozygotes showed normal myelination during the early postnatal stages, followed by a segmental demyelination at an advanced stage. Vacuolar formation was not so frequent as in the homozygotes. These results suggest that the missing of transmembrane spanning region (TM2 and TM3) of peripheral myelin protein-22 may disturb a dual biological function of peripheral myelin protein-22, leading to a dysmyelination of axons and to a vacuolar formation within the cytoplasm of the Schwann cells. The latter phenotype is discussed in conjunction with the disruption of an intracellular transport system and subsequent cell death.

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.

302 Multiple climbing fiber innervation of cerebellar purkinje cells in mutant mice lacking the phospholipase C-β4 (PLC β4)

302 Multiple climbing fiber innervation of cerebellar purkinje cells in mutant mice lacking the phospholipase C-β4 (PLC β4)

Altered intracellular localization of the glutamate receptor channel δ2 subunit in weaver and reeler Purkinje cells

The glutamate receptor (GluR) channel δ2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR δ2 mRNA is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR δ2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR δ2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR δ2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells form synapses with parallel fibers, GluR δ2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR δ2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR δ2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR δ2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers.

Incomplete regression of multiple climbing fibre innervation of cerebellar Purkinje cells in mGluR1 mutant mice

Recent reports have suggested the existence of a causal relationship between impaired regression of multiple climbing fibre innervation and impaired motor coordination in protein kinase C gamma subunit (PKC gamma) mutant mice. In the present patch-clamp study, performed in thin cerebellar slices prepared from adult mutant mice deficient in metabotropic glutamate receptors of the mGluR1 subtype, only 15% of Purkinje cells remained multiply innervated by climbing fibres, but motor coordination was largely impaired in these animals. The present results do not preclude the existence of a causal relationship between impairement of regression of multiple innervation during development and improper motor coordination in the adult.

Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta.

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

Expression pattern of integrin beta 1 subunit in Purkinje cells of rat and cerebellar mutant mice.

We found that integrin beta 1 subunit (INT beta 1)-immunoreactive Purkinje cells first appeared caudally at postnatal day (PD) 6 of rat and most Purkinje cells gradually became positive by PD 12. The expression of INT beta 1 was then suppressed in some of these cells, so that the positive Purkinje cells in the adult were organized into parasagittal bands interposed by negative cells throughout the vermis and hemispheres. When Purkinje cells were deprived of their climbing fiber innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the subsequent patterning of INT beta 1-positive Purkinje cells was not changed. In both reeler and weaver mice, the INT beta 1-positive parasagittal bands were observed, however, the Purkinje cells in the staggerer mice did not express INT beta 1 at any stage. These data suggest that the expression of INT beta 1 in Purkinje cells is genetically programmed in the developing cerebellum, and that the afferent synaptic inputs by climbing and parallel fibers are not prerequisites for INT beta 1 expression in Purkinje cells. Therefore, the unique distribution patterns of INT beta 1-positive Purkinje cells provides a new marker for postnatal development of rodent cerebella.

Phagosome Number and Distribution in Retinal Pigment Epithelial Cells of Vitiligo Mutant Mice

The vitiligo mutant mouse has a disorder affecting the interaction of retinal pigment epithelium (RPE) and photoreceptor cells of the neural retina. Among the phenotypic features are patches of hyper- and hypopigmentation in the embryonic RPE, increased RPE cell production neonatally, and a later onset of progressive photoreceptor cell degeneration that continues for more than one year until all photoreceptor cells are gone. Failure of RPE microvilli to intertwine with rod outer segments (ROS) at any age, the accumulation of ROS membranous fragments in the subretinal space, and a relatively early retinal separation from the RPE suggested analysis of whether RPE phagocytosis might be impaired. Post-natal day 23 (P23) and P36 mutant and congenic control wild-type mice were kept in darkness overnight and eyes were examined by light and transmission electron microscopy 0.5hr before, 1.5hr after and 10.5hr after lights turned on at 0700hr. At these ages ROS have not yet degenerated, though they are shorter than normal and somewhat misoriented. The number of phagosomes per RPE cell was markedly reduced in mutants compared to controls at both ages and all time points. Nonetheless, the highest counts were obtained 1.5hr after the lights turned on in mutant and control specimens. In the mutant eyes, the proportion of phagosomes in the microvillous zone of the RPE cells was consistently lower than in any other cellular compartment. Phagosome distribution in the apical and basal zones of the RPE cell cytoplasm was within normal limits. Macrophage-like cells become numerous in the subretinal space at older ages, but were already present at P23 and P36, and contained phagosomes in their cytoplasm. The hypothesis is proposed that binding of ROS to RPE cells might be defective in vitiligo mice, in contrast to the rdyrat, where the work of others indicates that binding is normal and the subsequent ingestion of phagosomes is impaired.

Pregnancy‐Associated Uterine Granulated Metrial Gland Cells in Mutant and Transgenic Mice

Granulated metrial gland (GMG) cells are pregnancy-specific uterine lymphocytes found in rodents. The lineage relationships of GMG cells are incompletely defined, although genetic and immunophenotyping studies suggest membership in the natural killer (NK) cell lineage. Pregnancy-specific functions have been postulated for GMG cells, but no successful depletion of these cells has been achieved that would permit assessment of any critical roles that might influence gestational outcome. Routine histological methods for light microscopy were used to assess implantation sites from wild-type mice and mice of the following genotypes: tgE26, mi/mi, and p53 knockout. GMG cells are transient, histamine-negative cells found in the pregnant uteri of most mice. Pregnancies in the NK and T-cell-deficient tgE26 were characterized by infrequent GMG cells, very small placentae, and an elevated rate of fetal and perinatal mortality. In term placentae of mi/mi with dystocia. GMG cells were found in a new location along the plane of placental separation, and they appeared degranulated. A normal life-history was observed for GMG cells in p53 knockout mice. Pregnancies in mutant and transgenic mice indicate that GMG cells are natural killer cells and have critical roles in placental development and pregnancy success. The disappearance of GMG cells at term is independent of p53 gene expression.

Phenotype and function of CD4+ T cells in mice lacking invariant chain.

Mice bearing a targeted disruption of the gene encoding the invariant chain (Ii) have provided a deeper understanding of the critical role that Ii plays in the assembly, transport, and peptide occupancy of MHC class II molecules. In this study, we have investigated the consequence of the altered class II expression in Ii-deficient (Ii zero) mice on the phenotype and function of their CD4+ T cells. As seen by others, these mice show a sharp reduction in the CD4+8- subset in the thymus and periphery. Furthermore, a large proportion of the peripheral CD4+, but not CD8+, T cells in Ii zero mice exhibit decreased surface TCR-alpha beta levels and express markers of T cell activation. Although the CD4+ T cells respond to mitogens, anti-CD3 mAbs, and alloantigens, they respond poorly to the bacterial superantigen staphylococcal enterotoxin B and do not respond to peptide and protein Ags. Analysis of reciprocal radiation bone marrow chimeras constructed using Ii zero and wild-type mice indicate that the lack of Ii in the thymus is responsible for the phenotype of the CD4+ T cells in mutant mice. These results suggest that the altered thymic peptide repertoire displayed by class II molecules in the absence of Ii has a dramatic impact on the selection of CD4+ T cells and their phenotype in the periphery.

Protective Role of CD40 in Leishmania major Infection at Two Distinct Phases of Cell-Mediated Immunity

CD40-deficient mice are susceptible to Leishmania major infection while their wild-type littermates can resolve the infection. Upon stimulation with L. major antigens, draining lymph node T cells of the mutant mice and the susceptible mice, BALB/c, secrete comparable amounts of IL-4. The mutant mice produce less IFNγ than wild-type mice. The expression of IL-12 p40 mRNA was significantly lower in L. major antigen-stimulated cells of mutant mice than those of wild-type or BALB/c mice. In normal mice, engagement of CD40 activates macrophages to a leishmanicidal state in vitro in the presence of IFNγ. The results suggest that the CD40–CD40 ligand interaction plays an important role in two critical steps of cell-mediated immunity to L. major infection: the generation of a Th1 response and activation of macrophages to a leishmanicidal state.

MGluR1 mutant mice as a tool to study calcium signalling and multiple innervation in the cerebellum

The present study reports that calcium signalling through voltage-gated calcium channels and release from internal stores is impaired in Purkinje cells of mutant mice lacking in GluR1 receptors and that the absence of these receptors also leads to an incomplete regression of multiple innervation in the cerebellum of these animals.

Responses of flocculus and vestibular nuclei neurons in Weaver mutant mice (B6CBA wv/wv) to combined head and body rotation.

The responses of vestibular nuclei (Vn) neurons and floccular Purkinje (P) cells to natural stimulation of the horizontal canals were recorded in paralyzed Weaver mutant mice. The Weaver mice suffer from an almost complete postnatal degeneration of granule cells and a portion of the P cells (Sidman et al. 1965). Parallel fibers are never elaborated (Bradley and Berry 1978). Recording sites were localized by means of small, iontophoretically applied HRP markings. Phase and sensitivity were analyzed by a Fourier analysis and a "best sine fitting" program. As in the normal "control" mice (Grüsser-Cornehls et al. 1995), the "simple spike" discharges of Vn and P cells in Weaver mutant mice are modulated sinusoidally upon sinusoidal stimulation. The neuronal response amplitude at fundamental frequency (determined from peristimulus time histograms, PSTHs increased with frequency (0.05-0.5 Hz) for both Vn and floccular neurons. The stimulus frequency/response amplitude and sensitivity (re velocity) curves for floccular neurons are distinctly lower in magnitude than those of Vn neurons (P < 0.01). In our sample of neurons, the Vn neurons curves of the mutants display a remarkable be behavior: the mean value curve of type I neurons is shifted upward, indicating a loss of inhibition but that of type II, downward, demonstrating a downregulation in comparison with the control values. The difference between the two curves is statistically significant (P < 0.001). The mean value curve of all mutant Vn neurons depends on the different fractions of type I and type II neurons in the sample investigated. In our investigations, the mean value curves of both type I and type II neurons also exceed those of the normal controls. The phase shift relative to head angular velocity in the midfrequency range in Vn neurons was very similar to that in normal controls, but the phase advance in the range of 0.3-0.5 Hz was somewhat larger and the SD larger over the whole range tested. Concerning the phase relationship for floccular neurons, a major difference occurred in contrast to the normal controls: the phase lead and phase lag varied from neurons to neuron, in individual neurons from frequency to frequency, and in some neurons distinctly from trial to trail. It is hypothesized that an intact mossy fiber-granule cell-parallel fiber system plays an important role in an orderly information flow, transmitted through the P-cell axons, and that the morphological disruption has implications for target cell activity. There is a strong suggestion that the diverse behavior of type I and type II neurons in the Vn may have implications for the poor motor performance in Weaver mutant mice.

Protein phosphatase mRNA expression in Purkinje cells of staggerer and reeler mutant mice.

We used in situ hybridization to search for the expression of three protein phosphatase (PP) mRNAs in Purkinje cells of normal mice, and staggerer (sg/sg) and reeler (rl/rl) mutant mice, two strains with known Purkinje cell disorders. The expression of the mRNAs was comparable in the normal and rl/rl Purkinje cells, but considerably reduced in the sg/sg. We interpret this finding as indicating: (a) the staggerer mutant gene may directly affect the phosphatase component of the protein phosphorylation-dephosphorylation cycle in the sg/sg Purkinje cells; or (b) the reduced mRNA expression may be a secondary phenomenon, resulting from abnormal Purkinje cell function due to the lack of synaptic input.

Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ52-JH region of the immunoglobulin heavy chain locus.

We investigated gene rearrangements in the mutant IgH locus of a mouse strain generated by insertion of a rearranged heavy chain variable region gene (VT15) into the DQ52-JH region through gene targeting. In more than half of the B cells of heterozygous mutant mice, the mutant IgH locus was silenced by the rearrangement of an endogenous DH or DH and VH gene to the inserted VT15 gene. In these cases, a functional VHDHJH gene was present on the wild-type allele. The silencing rearrangement appeared to be mediated by recombination signal sequence (RSS)-like elements present in the "recipient" VT15 gene. Among the many such elements on the inserted VT15 gene, which apparently met the requirement for an RSS with respect to nucleotide sequence, only two were observed in the actual rearrangements. This indicates that targeting of the recombination machinery involves sequences in addition to the RSS motifs as they have been characterized so far. In homozygous mutant mice, most B cells appeared to carry the intact VT15 gene on both mutant IgH alleles, although single-cell polymerase chain reaction revealed that silencing rearrangements occurred frequently in B cell progenitors in the bone marrow. This observation indicates that once silencing rearrangements are initiated in a cell, they involve both VT15 genes in most cases, reminiscent of normal DH-JH rearrangement. B cells which did not initiate such rearrangements develop to populate the peripheral B cell compartment.

Age-dependent reduction of Bcl-2 expression in peripheral T cells of lpr and gld mutant mice.

Autoimmune-prone lpr and gld mice carry defects in the apoptosis-mediating cell surface molecule Fas and its ligand, respectively. These mice develop lymphadenopathy because of an age-related accumulation of nonmalignant CD4- CD8- T cells in the peripheral lymphoid organs, suggesting a role for Fas-mediated apoptosis in peripheral T cell homeostasis. However, these accumulating cells are more susceptible to apoptosis ex vivo than peripheral T cells from control mice. To investigate the influence of additional regulatory elements on defects in the Fas-mediated apoptosis pathway, we analyzed the expression of Bcl-2 protein, a repressor of apoptosis, in T cells of lpr and gld mice. The expression levels of Bcl-2 in peripheral T cells of aged lpr and gld mice were significantly reduced when compared with their normal counterparts. Bcl-2 expression decreased with age in peripheral T cells, but not in thymocytes, suggesting that down-regulation of Bcl-2 protein occurs in the periphery. Analysis of T cell subsets indicated that CD4+ and CD4- CD8- T cells expressed significantly reduced levels of Bcl-2, whereas CD8+ cells maintain high levels of Bcl-2 expression. However, all peripheral T cell subsets including CD8+ cells were susceptible to glucocorticoid-induced apoptosis, indicating that there is no direct correlation between the levels of Bcl-2 expression and susceptibility to glucocorticoid-induced apoptosis. These studies suggest the presence of complex regulatory mechanisms for lymphocyte apoptosis and survival.

Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta.

In mice lacking the interleukin-2 receptor beta chain (IL-2R beta), T cells were shown to be spontaneously activated, resulting in exhaustive differentiation of B cells into plasma cells and the appearance of high serum concentrations of immunoglobulins G1 and E as well as autoantibodies that cause hemolytic anemia. Marked infiltrative granulocytopoiesis was also apparent, and the animals died after about 12 weeks. Depletion of CD4+ T cells in mutant mice rescued B cells without reversion of granulocyte abnormalities. T cells did not proliferate in response to polyclonal activators, nor could antigen-specific immune responses be elicited. Thus, IL-2R beta is required to keep the activation programs of T cells under control, to maintain homeostasis, and to prevent autoimmunity.

Positive selection of self- and alloreactive CD8+ T cells in Tap-1 mutant mice.

Mice with a homozygous deletion in their Tap-1 gene (-/- mice) express very low levels of cell membrane major histocompatibility complex class I molecules and have < 1% peripheral CD8+ T cells. We show that these -/- mice but not their +/- littermates display strong primary syngeneic anti-H-2Kb and -Db-specific responses mediated by CD8+ T cells. These responses are augmented by in vivo priming. Further, -/- mice primed in vivo with H-2d alloantigens generate an anti-H-2d response which appears nearly as strong as that found in +/- littermates. Both -/- anti-H-2b and anti-H-2d T cells do not recognize target cells from Tap-1 -/- animals or Tap-2-deficient RMA-S cells. Thus, some CD8+ anti-self and alloreactive T cells can be selected in the absence of Tap proteins.

Peripheral T cells in mice lacking p56lck do not express significant antiviral effector functions.

Mutant mice lacking p56lck, the T cell-specific protein tyrosine kinase, have a profound thymic atrophy and possess only an immature thymocyte population. Only 5 to 10% of normal levels of mature T cells (TCR-alpha beta+, CD4+, or CD8+) are present in these mice. These T cells, but also B cells of mutant mice, can be activated by mitogens, and T cells proliferate upon stimulation with IL-2 or mAb against the TCR. Thus, it appears that selected T cells can mature without lck and that they may have circumvented the need for this enzyme. This study evaluated the capacity of these mice to generate antiviral immune responses: 1) mutant mice failed to generate virus-specific CTL after acute infection with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus. 2) LCMV was not cleared from the spleen on day 10 after infection. 3) Infection of LCMV into footpads of mutant mice did not induce a T cell-dependent swelling reaction. 4) Intracerebral inoculation with LCMV did not induce in mutant mice any clinical signs of sickness. 5) Vesicular stomatitis virus (VSV)-primed mutant mice could not eliminate nucleoprotein of VSV recombinant vaccinia virus upon intracerebral infection. 6) VSV infection of mutant mice elicited normal, specific T cell-independent IgM responses, but no significant switch to IgG. These results show that, despite the presence of in vitro activatable CD8+ and CD4+ T cells in the mutant mice, no biologically relevant functions can be detected. Therefore, these data indicate that p56lck is directly or indirectly crucially involved in in vivo Ag-induced T cell responses.