Transient exposure of neonatal mice to neuregulin-1 results in hyperdopaminergic states in adulthood: implication in neurodevelopmental hypothesis for schizophrenia.

Neuregulin-1 (NRG1) is implicated in the etiology or pathology of schizophrenia, although its biological roles in this illness are not fully understood. Human midbrain dopaminergic neurons highly express NRG1 receptors (ErbB4). To test its neuropathological role in the neurodevelopmental hypothesis of schizophrenia, we administered type-1 NRG1 protein to neonatal mice and evaluated the immediate and subsequent effects on dopaminergic neurons and their associated behaviors. Peripheral NRG1 administration activated midbrain ErbB4 and elevated the expression, phosphorylation and enzyme activity of tyrosine hydroxylase (TH), which ultimately increased dopamine levels. The hyperdopaminergic state was sustained in the medial prefrontal cortex after puberty. There were marked increases in dopaminergic terminals and TH levels. In agreement, higher amounts of dopamine were released from this brain region of NRG1-treated mice following high potassium stimulation. Furthermore, NRG1-treated mice exhibited behavioral impairments in prepulse inhibition, latent inhibition, social behaviors and hypersensitivity to methamphetamine. However, there were no gross abnormalities in brain structures or other phenotypic features of neurons and glial cells. Collectively, our findings provide novel insights into neurotrophic contribution of NRG1 to dopaminergic maldevelopment and schizophrenia pathogenesis.

Multi-step lymphomagenesis deduced from DNA changes in thymic lymphomas and atrophic thymuses at various times after gamma-irradiation.

Whole-body gamma-irradiation to mice causes thymic atrophy where a population of precancerous cells with mutation can be found. Thus, clonal growth and DNA changes at Bcl11b, Ikaros, Pten, Notch1 and Myc were examined in not only thymic lymphomas but also in atrophic thymuses at various times after irradiation. Clonal expansion was detected from the distinct patterns of rearrangements at the TCRbeta receptor locus in a fraction of atrophic thymuses at as early as 30 days after irradiation. This expansion may be in part owing to the rearranged TCRbeta signaling because the transfer of bone marrow cells with the rearrangement and the wild-type locus into severe-combined immunodeficiency mice showed preferential growth of the rearranged thymocytes in atrophic thymus. Loss of heterozygosity (LOH) at Bcl11b and trisomy of Myc were found at high frequencies in both lymphomas and atrophic thymuses, and in contrast, LOH at Ikaros and Pten were rare in atrophic thymuses but prevalent in lymphomas. Notch1 activation was detected in lymphomas and in atrophic thymuses only at a late stage. Similar patterns of DNA changes were found in atrophic thymuses induced in Bcl11b(+/-) mice. These results suggest the order of genetic changes during lymphomagenesis, Bcl11b and Myc being at the early stage; whereas Ikaros, Pten and Notch1 at the late stage.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages.

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b(-/-) newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b(-/-) thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Formation of the transcription initiation complex on mammalian rDNA.

Steps for the formation of transcription initiation complex on the human rRNA gene (rDNA) in vitro were analyzed with partially purified transcription factors and RNA polymerase I. The reaction requires at least two factors besides RNA polymerase I for maximal efficiency. Preincubation and short-pulse analyses of the accurate transcripts revealed the following steps. First, the species-dependent factor, designated TFID, bound to the rDNA template, forming a preinitiation complex (PIC-1) which was resistant to a moderate concentration (0.015 to 0.02%) of Sarkosyl. Other factors, designated TFIA and RNA polymerase I, were then added to convert it to the final preinitiation complex PIC-3. This complex incorporated the first two nucleoside triphosphates of the starting site to complete the initiation complex (IC), which was resistant to a high concentration (0.2%) of Sarkosyl. Binding of TFID was rate limiting in the overall initiation reaction in vitro. Together with the kinetics of incorporation, the results are interpreted to mean that TFID, one bound, remains complexed with rDNA together with TFIA as the PIC-2 for many rounds of transcription by RNA polymerase I. Thus, the formation of PIC-2 may be a prerequisite for the stable opening of rDNA for transcription in vivo.

Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter.

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.

A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.

Structural determinant of the species-specific transcription of the mouse rRNA gene promoter.

Mammalian ribosomal DNA (rDNA) transcription has a certain species specificity such that, both in vivo and in vitro, human rDNA cannot be transcribed by mouse machinery and vice versa. This is due to a species-dependent transcription factor, TFID (Y. Mishima, I. Financsek, R. Kominami, and M. Muramatsu, Nucleic Acids Res. 10:6659-6670, 1982). On the basis of the information obtained from 5' and 3' substitution mutants, we prepared a chimeric gene in which the mouse sequence from positions -32 to -14 was inserted into the corresponding location of the human rDNA promoter. The chimeric gene could be transcribed by mouse extracts nearly as efficiently as the wild-type mouse promoter. The chimeric gene could also sequester transcription factor TFID at an efficiency similar to that for the mouse promoter. Partially purified mouse TFID that could not protect the human rDNA promoter against DNase I produced a clear footprint on this chimeric gene that was similar to that on mouse rDNA promoter. The basic structure of the mouse rDNA core promoter is discussed in relation to the interaction with TFID.

Association of minisatellite instability with c-myc amplification and K-ras mutation in methylcholanthrene-induced mouse sarcomas.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Allelic losses and metastatic ability of mouse tumor cell clones derived from a heterozygous mouse.

Some chromosomal or subchromosomal deletions observed in a variety of cancer cells may be related with metastatic properties of the tumor cells. Thus, relationship between allelic losses and metastatic ability is here investigated of ten in vitro clones obtained from 505 cells that are derived from a fibrosarcoma induced in a F1 mouse between C3H and C57BL strains. Allelic loss was examined using 61 microsatellite markers spanning all autosomes and deletions of various loci were observed in all of the clones. One clone showed a metastatic ability higher than the parental 505 cells and the other clones. This particular clone specifically lost a chromosome 16 derived from C57BL, suggesting an association of this chromosome deletion with the metastatic ability. Consistently, examination of metastatic nodules derived from 505 cells and one in vitro clone showed that the cells with deletion of the C57BL chromosome increased in number during metastasis. These results suggest that mouse chromosome 16 may harbor a gene(s) involved in metastasis.

Allelic loss mapping and physical delineation of a region harboring a putative thymic lymphoma suppressor gene on mouse chromosome 12.

Our previous allelic loss analysis of gamma-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains mapped the Tlsr4 region exhibiting a high frequency of allelic loss (62%) to a 2.9 cM interval between the markers D12Mit53 and D12Mit279 on mouse chromosome 12. To narrow further the interval harboring a putative tumor suppressor gene, a high-density scan has been carried out for informative 361 thymic lymphomas. Construction of a physical map of Tlsr4 with 3 YAC and 15 BAC clones and isolation of YAC- and BAC-derived polymorphic probes lead to fine allelic loss mapping. Three successive polymorphic sites within one BAC exhibit the retention of both alleles in seven, one and four lymphomas, suggesting that a common region of allelic loss for Tlsr4 exists within the BAC region. Pulsed-field gel electrophoresis of NotI digests of this and other clones determines that the commonly lost region is a 35 kb interval with a NotI site. NotI sites are frequently associated with coding regions, and our preliminary sequencing has identified ESTs in the region. Thus, the present study facilitates the identification of genes in the Tlsr4 region that would lead to isolation of a novel tumor suppressor gene.

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 gamma-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(-/+) and 13% for p53(+/+), suggesting co-operative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

Allele-specific inactivation of the alpha4 integrin gene expression in fibrosarcoma cell lines and relevance for spontaneous metastasis.

Deregulated expression of genes is found in cancer cells, which may affect malignant properties, but it is unclear whether such modulation occurs allele-specifically. This study shows that the gene coding alpha4 integrin, a cell adhesion molecule, underwent allelic inactivation in a series of heterozygous murine fibrosarcoma cell lines (MST lines) with different metastatic potentials. P4 cells expressed the alpha4 integrin gene from one allele at a level comparable to that of the primary MST1 tumor, whereas the descendent lines of P4 exhibited decreased expression of both alleles. No allelic loss of DNA was observed in these cells. Other four clones derived from P4 and five clones from a different tumor also showed such two-step inactivation. Intriguingly, the loss of expression was correlated with the acquisition of spontaneous, but not artificial, metastatic ability. This is consistent with the previous result of inverse relation between the expression of alpha4/beta1 integrin and the invasive potential of B16 melanoma cells. Analysis of DNA methylation and chromatin state of the alpha4 integrin gene failed to provide a clue to difference between the two alleles in the cell lines. These results suggest that the allelic inactivation is a process giving loss of function to one allele, although the mechanism is unclear.

Homozygous deletions and point mutations of the Ikaros gene in gamma-ray-induced mouse thymic lymphomas.

Our previous genome-wide analysis of allelic loss for thymic lymphomas that were induced by gamma-irradiation in F1 hybrid mice between BALB/c and MSM strains suggested the centromeric region on chromosome 11 as a site harboring a tumor suppressor gene. Interestingly, to this region the mouse Ikaros gene was mapped which was postulated to participate in oncogenic process from the study of Ikaros knockout mice. Here we show fine allelic loss mapping in the vicinity of Ikaros in 191 lymphomas, indicating that the critical region of allelic loss was centered at the Ikaros locus. PCR analysis revealed that nine lymphomas failed to give PCR-amplification for either of two exon primer pairs, indicative of homozygous deletion. Six and five mutations were detected in the N-terminal zinc finger domain and the activation domain of Ikaros, respectively, and six of the eleven were frameshift or nonsense mutations that resulted in truncation of Ikaros protein. The results strongly suggest a direct role for Ikaros in development of mouse thymic lymphomas. This provides the experimental basis for further analysis of Ikaros mutations in human cancer.

Genetic loci controlling susceptibility to gamma-ray-induced thymic lymphoma.

BALB/c is a susceptible strain for the development of gamma-ray induced mouse thymic lymphoma whereas MSM shows resistance. Association analysis of 220 backcross mice between the two strains using 67 markers was carried out to identify loci involved in the control of susceptibility. The genotype of mice with lymphoma showed excess heterozygosity relative to MSM homozygosity at D2Mit15 and D4Mit12 and was skewed toward MSM-derived alleles at D5Mit5. The P values in Mantel-Cox test were 0.0048 (D2Mit15), 0.0034 (D4Mit12) and 0.0048 (D5Mit5), suggesting association at the three loci in the susceptibility. Cooperative effect on lymphomagenesis was also observed among the three loci. To obtain independent evidence for linkage at D4Mit12, we made partially congenic mice in which a D4Mit12 region in BALB/c was replaced by MSM-derived homolog. Examination for the lymphoma susceptibility in 78 progeny of the congenic mice confirmed the effect of the locus near D4Mit12 (P=0.0037). The result, together with the linkage analysis, shows that the locus near D4Mit12 is regarded as a confirmed linkage but the other two loci as marginally suggestive.

Amplified ribosomal spacer sequence: structure and evolutionary origin.

A novel class of repeated sequences consisting of tandem arrays of ribosomal spacer sequence has been discovered in a mouse genome. Comparison to normal ribosomal DNA reveals that one repeat unit consists of two separate parts of spacer sequence. This amplified spacer sequence has a pseudogene-like structure but is distinct from the previously reported pseudogenes and orphons in regions lacking coding sequences. So far the amplified spacer sequence has been found only in the BALB/c mouse genome but not in ten other laboratory strains and several wild-type mouse stocks. Surprisingly, a part of the amplified spacer sequence unit had a higher homology to the corresponding part of the ribosomal DNA sequence of Mus musculus molossinus, a Japanese wild-type mouse, than to the corresponding part of the rDNA of the BALB/c mouse. These findings suggest that the amplified spacer sequence of the BALB/c mouse might have partly originated in M. m. molossinus or in a related subspecies.

Structure of the core promoter of human and mouse ribosomal RNA gene. Asymmetry of species-specific transcription.

In vitro transcription of the ribosomal RNA gene (rDNA) shows a remarkable species specificity such that human and mouse rDNA cannot use heterologous extracts of each other. The region that is responsible for this specificity has been studied using human-mouse chimeric genes and characteristic structures of both core promoters are presented. When the mouse sequence is substituted by the corresponding human sequence from upstream, the promoter activity in the mouse extract begins to decline at nucleotide -32 or -30, decreasing gradually and is lost completely at -19. A similar gradual decrease was noted for the 3' side substitution, which started at nucleotide -14 and was completed when up to the nucleotide -22 mouse position was replaced by the corresponding sequence from human. Thus, in the mouse rDNA core promoter, the sequence that is involved in species specificity resides only in a stretch encompassing the non-conserved region between the distal conserved sequence (DCS) and the proximal conserved sequence (PCS), plus two altered nucleotides in the PCS. When human rDNA is transcribed with human cell extract, the mouse sequence cannot substitute for the human sequence within the region from nucleotide -43 to +17 without affecting promoter activity significantly. This asymmetry of species specificity is due to the presence of nucleotides -43, +1 and +17, which are sensitive to change in only the human core promoter. The difference in the 5' border is ascribed to the species specificity of a transcription factor TFID, which recognizes this region. But the large difference of the 3' border is apparently due to another factor, possibly RNA polymerase I itself, because this region is not recognized by TFID in either human or mouse. Mammalian rDNA core promoter appears to consist of a tandem mosaic in which three evolutionarily conserved sequences alternate with non-conserved sequences having certain functionally important nucleotides. Not only non-conserved sequences and non-conserved nucleotides in conserved sequences, but also the spacings between the three conserved regions, play a crucial role in species specificity.

Novel repetitive sequence families showing size and frequency polymorphism in the genomes of mice.

A middle repetitive sequence, PR1, originally found in mouse rDNA appeared as satellite-like bands when EcoRI and BglII digests of genomic DNA were subjected to Southern blot hybridization using PR1 as probe. The copy number and sizes of PR1-related satellite-like bands, designated as PR1 families, differed remarkably among the subspecies and laboratory strains of mice when the EcoRI digests of genomic DNAs were compared. These bands were not detected in rat and human DNAs. A unit of PR1 sequence was determined by examining cloned EcoRI 3.5 kb (kb, 10(3) bases) fragment and 6.6 kb rDNA by cross-hybridization and sequence analysis: 3.5 kb and 6.6 kb DNAs are composed of homologous PR1 regions and the flanking non-homologous sequences. The results indicate that amplification of different sequences containing PR1 has occurred in different subspecies and strains of mice, and that the segments of satellite-like bands are likely to have been created by recombination of the PR1 sequence with other DNA segments before amplification. The chromosomal distribution of the 3.5 kb PR1 family was studied by back-crossing the female F1 between BALB/c and DDD/1 to male DDD/1. The segregation data strongly suggest that most, if not all, of this family are located on a single chromosome. The stability of these PR1 families in the genomes of cultured cells of a given strain was also examined. An extra band homologous to PR1 appeared in their genomes, but was not detected in other tissues, indicating that some PR1 families may change even during cell propagation.

A polymorphic repetitive-sequence PR1 family. Evidence for meiotic instability.

When EcoRI digests of mouse genomic DNA were subjected to Southern blot analysis with the polymorphic repetitive sequence PR1 as a probe, one satellite-like band of 3.5 X 10(3) base-pairs, designated as PR1 family B, was detected in BALB/c-strain mice, but not in the DDD/1- or MOA-strain mice. Analysis of recombinant phage clones revealed that the repeating unit of the PR1 family B was 13.5 X 10(3) base-pairs long. This family consisted of a tandem array of repeating units and occupied as much as 2% of one BALB/c chromosome. Since the BALB/c-specific PR1 family B is not present in DDD/1 or MOA mice, the unpaired portion of the BALB/c chromosome may be looped out in a synaptonemal complex during meiosis in F1 hybrids of the BALB/c strain with DDD/1 or MOA. To determine the fate of this extra DNA, we examined the genotypes of the F1 hybrid mice and of the segregating populations. Although the PR1 patterns of F1 and most N2 mice are consistent with typical Mendelian inheritance, some N2 progeny showed an abnormal 3.5 X 10(3) base-pair band of unexpectedly reduced intensity. This indicated that the extra DNA of PR1 family B occasionally underwent recombination during meiosis in F1 mice, resulting in its apparent excision. Examination of PstI digests supported this interpretation.

Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.

Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse.

Here we report the identification of a novel homeobox gene family Dbx in mouse, which consists of Dbx and Dbx2. The two genes share similar structural organization and are encoded by different chromosomes. The predicted Dbx and Dbx2 proteins share 85% identity in their homeodomain amino acid sequences, but otherwise showed no significant similarity. Characterization of the expression of these two genes in the embryos suggested their role in the development of the CNS. In the forebrain, Dbx is expressed in various regions, while Dbx2 showed a more restricted pattern of expression. In the midbrain, the expression domains of Dbx and Dbx2 overlap along the dorso-lateral wall of the ventricle. In the hindbrain and spinal cord, both genes are expressed in the boundary separating the basal and alar plates, which seems to correspond to the sulcus limitans. Expression of the Dbx/Dbx2 genes is restricted to the ventricular region of the embryonic CNS except for that of Dbx in the septum of the telencephalon. Together these observations indicate possible participation of the members of the Dbx family in regionalization of the CNS. While the expression of Dbx was restricted to the CNS, Dbx2 was also expressed in some of the mesenchymal cells, such as limb buds and tooth germs.