The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons.

We have identified a strong candidate cDNA for the mouse reeler gene. This 5 kb transcript encodes a 99.4 kD protein consisting of 881 amino acids and possessing two EGF-like motifs. We assayed two independent mutant alleles--'Jackson reeler', which has a deletion of the entire gene, and 'Orleans reeler' which exhibits a 220 bp deletion in the open reading frame, including the second EGF-like motif and resulting in a frame shift. In situ hybridization reveals that the transcript is detected exclusively in the pioneer neurons which guide neuronal cell migration along the radial array. Our findings offer an explanation for how the reeler mutant phenotype causes a disturbance of the complex architecture of the neuronal network.

JDP2 suppresses adipocyte differentiation by regulating histone acetylation.

Among the events that control cellular differentiation, the acetylation of histones plays a critical role in the regulation of transcription and the modification of chromatin. Jun dimerization protein 2 (JDP2), a member of the AP-1 family, is an inhibitor of such acetylation and contributes to the maintenance of chromatin structure. In an examination of Jdp2 'knock-out' (KO) mice, we observed elevated numbers of white adipocytes and significant accumulation of lipid in the adipose tissue in sections of scapulae. In addition, mouse embryo fibroblasts (MEFs) from Jdp2 KO mice were more susceptible to adipocyte differentiation in response to hormonal induction and members of the CCAAT/enhancer-binding proteins (C/EBP) gene family were expressed at levels higher than MEFs from wild-type mice. Furthermore, JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells.

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.

Strain specific sensitivity to diethylnitrosamine-induced carcinogenesis is maintained in hepatocytes of C3H/HeN in equilibrium with C57BL/6N chimeric mice.

The C3H/HeN (C3H) and C57BL/6N (C57) mouse strains are known, respectively, for their high and low susceptibility to both spontaneous and chemically induced hepatocarcinogenesis. The present study was aimed at elucidating whether this difference is dependent on intrinsic features of the target hepatocytes or in the in vivo milieu and associated growth promoting factors to which the cells are exposed. C3H in equilibrium with C57 chimeric mice were produced and given injections of diethylnitrosamine (20 microgram/g body weight) at the age of 15 days. The animals were sacrificed 6 or 9 months thereafter, and the numbers and sizes of altered cell lesions were scored. The clonal growth of both cell types was immunohistochemically confirmed using anti C3H-specific antigen antibodies. Quantitative assessment revealed C3H lesions in the chimera livers to be far larger (5:1) than those of C57 derivation and associated with more frequent malignant progression as was evident histologically. Furthermore, foamy change and hyalin body formation, which have been described as characteristics of C3H and C57BL hepatic tumors, respectively, were also featured as differentiative characteristics in lesions of both cell types in chimera mice. Thus, the results clearly demonstrated that the principal mechanism(s) underlying strain difference in diethylnitrosamine-initiated hepatocarcinogenesis exists in the target cells and is not milieu-dependent.

Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation.

Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.

Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras.

The potential contribution of cell-cell interactions and extracellular factors to cytoarchitectonic abnormalities in the cerebellum of the reeler mutant mouse was investigated by forming chimeras between the reeler and normal animals. The strain origin of Purkinje cells, granule cells and Golgi epithelial cells was immunohistologically identified with a strain-specific antibody. We analyzed 16 overt coat color chimeras, 10 reeler <--> C3H and 6 reeler <--> Balb/c. Abnormal behavioral traits of reeler were rescued in all chimeras. However, cerebellar histology was more affected in reeler <--> C3H chimeras than in reeler <--> Balb/c. Purkinje cells from the normal genotype occupy ectopic positions, and reeler genotype cells are arranged appropriately in the same chimeric cerebellum. We also obtained histologically normal chimeras with a significantly high contribution of the reeler genotype in Purkinje cells, Golgi epithelial cells and granule cells. These results clearly indicate that the abnormal cell positioning and cytoarchitecture of neurons and glia in the reeler is caused by a deficiency of extracellular environments, but is not determined cell-autonomously. The present data on chimeric mice suggest that Reelin is one of the important extracellular environmental factors that affects indirectly radial glial cells during cerebellar histogenesis.

Cell mixing during the early development of mouse aggregation chimera.

Two different inbred strain combinations of mouse aggregation chimeras - C3H/HeN (H-2k) x C57BL/6N (H-2b) and C3H/HeN x BALB/cA (H-2d) were used for cell mixing analysis at two points in time - 24 h after aggregation (just prior to transplantation into foster mothers) and 7.5 days post coitum (p.c.). The cell proportion of two H-2 haplotypes at the blastocyst stage was studied using a fluorescence-labeled monoclonal antibody recognizing a C3H-specific alloantigen - CSA (C3H strain-specific antigen) and laser scanning confocal microscopy. The 7.5-day-old chimeras were sectioned and subsequently processed by sensitive biotinylated antibody - avidin peroxidase immunohistochemical technique. Our results showed that 24 h after aggregation (blastocyst stage), there was equal cell mixing and no mouse strain used in the present study was dominant at this time. In 7.5-day-old C3H/HeN x BALB/cA chimeras, cells of both genotypes were intermingled, but the C3H/HeN strain was dominant in all cases. In contrast, the combination C3H/HeN x C57BL/6N clearly showed reduced numbers of C3H/HeN cells (CSA-positive) in 83% of the chimeras evaluated. Generally, CSA positive cells were found only in randomly distributed small distinct areas representing less than 20% of embryonal cells. Surprisingly, the extraembryonal ectoplacental cone was uniformly CSA positive in some C3H/HeN x C57BL/6N chimeras. Furthermore, in 36% of normally implanted chimeras of both strain combinations progressive degeneration was observed. We suggest that the cell mixing pattern as well as the absolute number of cells derived from each strain in the aggregation chimera can be affected by specific immune interactions involving H-2 haplotype combinations of the allogeneic fetus and the fully immunocompetent host organism, at later points in development.

Expression of paternal and maternal mitochondrial HSP70 family, hsc74, in preimplantation mouse embryos.

We have investigated the regulation of gene expression of a novel mitochondrial HSP70 family, hsc74 in preimplantation mouse embryos. We used a monoclonal antibody, anti-CSA, which reacts with only one of strain variants of the hsc74. By immunostaining with anti-CSA antibody, the hsc74 protein was constitutively detected in C3H embryos from 1-cell to blastocyst stage, but no signals were detectable in C57BL/6 embryos. To know the timing of paternal genome expression, we examined the expression of hsc74 in (C57BL/6 x C3H)F1 embryos. No positive signals were detectable in embryos before 8-cell stage. In early 8-cell stage weakly positive signals appeared in the peripheral region of the blastomeres. From late 8-cell stage, the protein was intensively detectable and was persistently expressed in all types of cells. We have also applied a sensitive methodology to distinguish genetic variants of hsc74 from C3H and C57BL/6 by reverse transcription polymerase chain reaction followed by single strand conformation polymorphism analysis. In (C57BL/6 x C3H)F1 embryos, the paternal transcripts were first detected in 4-cell embryos, while the maternal transcripts were constantly detectable. These results indicate that the transcripts and proteins of hsc74 were derived only from the maternal gene from 1-cell to 4-cell stages, and that from 4-cell stage the paternal gene is also transcribed, and the significant increase of the paternally derived protein occurred around late 8-cell stage.

Effect of tenascin-C deficiency on chemically induced dermatitis in the mouse.

Tenascin-C is a large extracellular matrix glycoprotein characterized by its spatiotemporal expression during embryogenesis, carcinogenesis, and wound healing. Many in vitro studies on tenascin-C have revealed its multifunctional properties; however, disruption of the tenascin-C gene did not reveal any obvious abnormalities during development, and its function in vivo remains unclear. Here, we investigated whether tenascin-C is involved in inflammatory dermatitis in adults by studying chemically induced dermatitis in tenascin-C knockout mice. An epicutaneous application of a hapten, 2,4-dinitrofluorobenzene, to the ear skin of BALB/CA mice resulted in inflammation and induced the expression of tenascin-C. In congenic tenascin-C knockout mice, the dermatitis occurred more severely than in wild-type mice; infiltration of polymorphonuclear cells in knockout mice persisted longer than in wild-type mice, and the elastosis-like disorganized extracellular matrix was also seen in the ear. These results suggest that tenascin-C plays a role in vivo in inflammatory responses in the skin, and that the genetic background has profound effects on the function of tenascin-C in mouse dermatitis.

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Loss of cortical and thalamic neuronal tenascin-C expression in a transgenic mouse expressing exon 1 of the human Huntington disease gene.

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD.

The mouse chimera during intrauterine stages: immunohistochemical analysis with the C3H strain-specific antibody.

Our objective was to establish an immunohistological method for analysis of chimerism in mouse chimeras at embryonic stages with an anti-C3H strain-specific antigen (CSA) antibody. We developed an effective new method to retain CSA antigenicity with good morphology of embryonic tissues by using microwave irradiation (MWI) for pre-fixation, 95% ethanol/1% acetic acid as post-fixative solution, and polyester wax as embedding material. We used a biotinylated mouse monoclonal anti-CSA antibody, peroxidase-avidin, and silver amplification. These procedures were successful in demonstrating the chimerisms in various tissues of C3H<-->Balb/c chimeras at different embryonic stages and postnatal days. In chimeras at Days 7 and 7.5 post coitum (p.c.), both genotypes were clearly identified and well intermingling in every embryonic tissue (embryonic ectoderm, mesoderm, extra-embryonic ectoderm, ectoplacental cone, amnion, and chorion). Chimerisms at Day 14.5 p.c. were also clearly observed in mesencephalon, neural retina, spinal cord, lung, kidney, and liver. We concluded that the present immunohistological procedures for analysis of chimerism during embryonic periods will give us insightful information about dynamic histological changes such as cell proliferation, migration, selection, and death during organogenesis.

Corneal wound healing in tenascin knockout mouse.

PURPOSE: Tenascin (TN) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has also been reported that TN is expressed in the embryonic cornea and during corneal wound healing. However, the role of TN in the cornea is not fully known. In this study, the role of TN in corneal wound healing was examined using the TN knockout (KO) mouse. METHODS: Two different injuries (a linear perforation wound and two 10-0 nylon suture wounds) were made separately on the corneas of both TNKO and congenic wild-type mice. The corneal wound healing was compared histologically, and the expression of TN and fibronectin (FN) on the injured cornea was examined immunohistochemically and by immunoblot analysis. RESULTS: Based on histologic analysis, there was no significant difference in the wound healing process between wild-type and TNKO mice in the linear incision experiment. However, the corneal stromata of TNKO mice were compressed prominently and devoid of migrating keratocytes in suture injury, which induced a more significant amount of TN than perforation wounds. Although FN expression on the sutured corneas of TNKO mice was upregulated during suture injury, the amount of FN protein was smaller than that of wild-type mice at the same time points after injury. CONCLUSIONS: In suture wounds, TN appears to enhance the amount of FN expression, and a lack of TN may impair stromal cell migration. TN plays a significant role in corneal wound healing, especially for wounds with mechanical stress.

Chromosomal localization of a gene responsible for vestibulocochlear defects of BUS/Idr mice: identification as an allele of waltzer.

Mice of the bustling mutant strain BUS/Idr have vestibulocochlear defects. bus/bus homozygotes, but not heterozygotes, are hyperactive and display an abnormal behavior such as circling, head bobbing and head tilting. To characterize BUS mice further, the auditory brain-stem response of the mutant was examined. In +/bus heterozygotes as well as control animals, the auditory brain-stem response was developmentally first recorded as early as 11 days of age and heterozygous and normal adults showed typical auditory brain-stem responses with five peaks in a threshold of 40-45 dB SPL. In contrast, bus/bus homozygotes showed no auditory brain-stem response at any age in response to stimuli up to 130 dB SPL, indicating that they are deaf throughout life. Linkage analysis revealed that the responsible gene, originally designated as bus, maps on chromosome 10, 1.09+/-0.9 cM distal to D10Mit127 and D10Mit59, and 0.72+/-0.51 cM proximal to three markers, D10Mit48, D10Mit112 and D10Mit258, at a site indistinguishable from that of the Albany waltzer, v(A/b). The results of allelism tests between BUS and Albany waltzer indicated that bus is allelic with v(Alb). From these data, we propose here that the bus mutation could represent another allele of waltzer, now designated v(bus).

A set of anti-crystallin monoclonal antibodies for detecting lens specificities: beta-crystallin as a specific marker for detecting lentoidogenesis in cultures of chicken lens epithelial cells.

Seven hybridoma lines which produced monoclonal antibodies against lens crystallins were established. They could detect alpha A-, alpha B-, gamma-, delta-, and several beta-crystallins of different species of animals with high avidities. Although immunohistological analysis of chicken and mouse lenses showed the typical distributions of each crystallin as have been reported so far, the ectopic expression of alpha B or delta-crystallin was observed in the brain or the kidney of chicken, respectively. Subsequently, crystallin production during the process of lentoidogenesis in cultures of chicken lens epithelial cells was examined with these antibodies. Western blot analysis revealed that beta-crystallins were not detected in cultured cells before lentoidogenesis, although all other crystallins were detected throughout the culture period. Immunostaining of cultures indicated clearly that expression of beta-crystallins was restricted to lentoid bodies. These data confirmed the lens fiber specificity of beta-crystallins as previously reported in the messenger RNA level. In addition, we found that small-size delta-crystallin (48 kDa) accumulated before the onset of lentoidogenesis. These results strongly suggest that the differentiated state of lens cells in vitro could be classified by examination of the expression pattern of crystallins. In addition, the anti-crystallin monoclonal antibodies produced in this study could be useful for detecting lens specificities.

Establishment of monoclonal antibody, gp21-34, against HTLV-II envelope protein (p20E).

A monoclonal antibody (MAb), gp21-34, specifically reactive with human T-lymphotropic virus type-II (HTLV-II) transmembranous envelope glycoprotein (p20E) was developed by immunization with a recombinant protein fused with thioredoxin moiety at the N-terminal portion. This MAb, which belongs to the IgG1 kappa subclass, reacted with HTLV-II infected cell lines (TON-1, C3-44, and Si-IIA) by IFA, but not with HTLV-I infected cell lines (TCL-Kan and MT-2). By Western blot analysis, this MAb reacted with p20E of HTLV-II lysates but not with HTLV-I lysates. Some epitope analyses with synthetic peptides were carried out to look for a plausible linear epitope in the C-terminal region of p20E.

Mutation induction by heavy-ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

Distribution of the C1473G polymorphism in tryptophan hydroxylase 2 gene in laboratory and wild mice.

The neurotransmitter serotonin is implicated in the regulation of various forms of behavior, including aggression, sexual behavior and stress response. The rate of brain serotonin synthesis is determined by the activity of neuronal-specific enzyme tryptophan hydroxylase 2. The missense C1473G substitution in mouse tryptophan hydroxylase 2 gene has been shown to lower the enzyme activity and brain serotonin level. Here, the C1473G polymorphism was investigated in 84 common laboratory inbred strains, 39 inbred and semi-inbred strains derived from wild ancestors (mostly from Eurasia) and in 75 wild mice trapped in different locations in Russia and Armenia. Among all the classical inbred strains studied, only substrains of BALB/c, A and DBA, as well as the IITES/Nga and NZW/NSlc strains were homozygous for the 1473G allele. In contrast to laboratory strains, the 1473G allele was not present in any of the samples from wild and wild-derived mice, although the wild mice varied substantially in the C1477T neutral substitution closely linked to the C1473G polymorphism. According to these results, the frequency of the 1473G allele in natural populations does not exceed 0.5%, and the C1473G polymorphism is in fact a rare mutation that is possibly eliminated by the forces of natural selection.

Suppression of cell-cycle progression by Jun dimerization protein-2 (JDP2) involves downregulation of cyclin-A2.

We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2-knockout (Jdp2KO) mice by targeting exon-1 to disrupt the site of initiation of transcription. The epidermal thickening of skin from the Jdp2KO mice after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) proceeded more rapidly than that of control mice, and more proliferating cells were found at the epidermis. Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation.