Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities.

Mutations in SLITRK1 are found in patients with Tourette's syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an alpha2-adrenergic agonist that is frequently used to treat patients with Tourette's syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourette's syndrome and trichotillomania.

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene.

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

Predominant expression of human zic in cerebellar granule cell lineage and medulloblastoma.

Zic is a novel zinc finger protein which displays a highly restricted expression pattern in the adult and developing mouse cerebellum and is highly homologous to the recently cloned Drosophila pair-rule gene Opa. To clarify the mechanism for the development of the human cerebellum and its involvement in human nervous system diseases, we have isolated human Zic cDNA and examined its expression by using monoclonal antibody against recombinant Zic protein. The nucleotide sequence of human Zic cDNA is 85% homologous to that of mouse Zic cDNA. Its putative amino acid sequence is highly conserved (> 99%) except for substitution of only two amino acid residues. In situ chromosome hybridization localized the human Zic gene to chromosome band 3q24. Human Zic protein was immunohistochemically detected in the nuclei of the cerebellar granule cell lineage from the progenitor cells of the external germinal layer to the postmigrated cells of the internal granular layer. Furthermore, Zic protein was detected in medulloblastoma (26/29 cases), whereas no other tumors examined (over 70 cases including primitive neuroectodermal tumors) expressed this protein. These findings suggest that Zic is a potential biomarker for medulloblastoma as well as the human cerebellar granule cell lineage.

Xmeis1, a protooncogene involved in specifying neural crest cell fate in Xenopus embryos.

Meis1 (Myeloid Ecotropic viral Integration Site 1) is a homeobox gene that was originally isolated as a common site of viral integration in myeloid tumors of the BXH-2 recombinant inbred mice strain. We previously isolated a Xenopus homolog of Meis1 (Xmeis1). Here we show that Xmeis1 may play a significant role in neural crest development. In developing Xenopus embryos, Xmeis1 displays a broad expression pattern, but strong expression is observed in tissue of neural cell fate, such as midbrain, hindbrain, the dorsal portion of the neural tube, and neural crest derived branchial arches. In animal cap explants, overexpression of Xmeis1b, an alternatively spliced form of Xmeis1, induces expression of neural crest marker genes in the absence of mesoderm. Moreover, Xmeis1b induces XGli-3 and XZic3, pre-pattern genes involved at the earliest stages of neural crest development, and like these two genes, can induce ectopic pigmented cell masses when overexpressed in developing embryos. Misexpression of Xmeis1b also induces ectopic expression of neural crest markers along the antero-posterior axis of the neural tube in developing Xenopus embryos. In contrast, Xmeis1a, another splice variant, is much less effective at inducing these effects. These data suggest that Xmeis1b is involved in neural crest cell fate specification during embryogenesis, and can functionally intersect with the Gli/Zic signal transduction pathway.

A novel member of the Xenopus Zic family, Zic5, mediates neural crest development.

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.

Xenopus Zic family and its role in neural and neural crest development.

We characterized two new members of the Zic family, Xenopus Zic1 and Zic2. They are very similar to mouse Zic1 and Zic2 in the protein coding region including the zinc finger domain. In early gastrula, Zic1 expression was restricted to the prospective neural plate region whereas Zic2 was expressed widely in the ectoderm. We observed enhanced neural and neural crest-derived tissue formation in the Zic1 or Zic2 overexpressed embryos and neural and neural crest marker induction in the Zic1 or Zic2 overexpressed animal cap explants. Our findings suggest that Zic1 and Zic2 have essentially the same properties as Zic3 and that the Xenopus Zic family may act cooperatively in the initial phase of neural and neural crest development.

Zic1 regulates the patterning of vertebral arches in cooperation with Gli3.

Skeletal abnormalities are described that appeared in Zic1-deficient mice. These mice show multiple abnormalities in the axial skeleton. The deformities are severe in the dorsal parts of the vertebrae, vertebral arches, but less so in the vertebral bodies (spina bifida occulta). The proximal ribs are deformed having ectopic processes. The abnormalities found in the vertebral arches can be traced back to disturbed segmental patterns of dorsal sclerotome. The Zic1/Gli3 double mutants showed severe abnormalities of vertebral arches not found in single mutants. The abnormalities in the vertebral arches were less severe in Zic1/Pax1 mutants than Zic1/Gli3 mutants, but significantly more pronounced than in Zic1 single mutants. The three genes may act synergistically in the development of the vertebral arches.

A neural cell-type-specific expression system using recombinant adenovirus vectors.

We demonstrated neural cell-type-specific gene expression using an adenovirus vector, which is useful for delivering foreign genes into quiescent neural cells. We produced eight recombinant replication-defective adenoviruses carrying the lacZ reporter gene driven by various promoters, including those of the L7/PCP2 gene (highly restricted expression in cerebellar Purkinje cells), and the myelin basic protein (in oligodendrocytes) gene. We demonstrated in vitro and in vivo promoter-driven, neural cell-type-specific gene expression by recombinant adenoviruses. The genes were transferred into these recombinant adenoviruses and expressed with high levels of efficiency in vitro and in vivo. In primary culture, the recombinant adenoviruses AdexL7-NL-LacZ and AdexMBP-NL-LacZ appeared to be expressed in a Purkinje cell and oligodendrocyte-specific manner. Introduction of 10(8) pfu of these viruses into the adult rat cerebellum by stereotactic injection yielded neural cell-type-specific expression without apparent toxicity to the animals. Thus, adenovirus vectors are useful for cell-type-specific therapeutic uses and in studies requiring neural cell-type-specific gene expression.

Promoter structure and gene expression of the mouse inositol 1,4,5-trisphosphate receptor type 3 gene.

Inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) is a ubiquitously expressed IP(3)R gene in the IP(3)R gene family. We identified an upstream region of the mouse IP(3)R3 genomic DNA. Transcription start points for the IP(3)R3 gene were found to be located mainly at four sites between nucleotide position -325 and -285 relative to the first ATG codon. The major start point was mapped around -325. Transcription promotion ability was detected between -325 and -285 in an IP(3)R3 proximal promoter sequence. The promoter had no TATA-box but was highly GC-rich and contained two putative Sp1-binding sites. There was no sequence similarity between promoter regions of IP(3)R3 and IP(3)R2, another ubiquitous gene, except for GC-boxes. By using a series of 5'-truncation versions and a transient luciferase assay, we detected multiple common and cell-type-dependent regulatory regions within the distal promoter sequence downstream from -4.0 kb that function positively or negatively. The IP(3)R3 gene was highly transcribed in the kidney, spleen, heart, and skeletal muscle, and this tissue distribution pattern was nearly complementary to that of IP(3)R2. We found that IP(3)R3 gene expression was repressed in retinoic acid-treated and neural differentiated P19 mouse embryonic carcinoma cells.

Identification and characterization of Zic4, a new member of the mouse Zic gene family.

The mouse Zic genes encode zinc-finger (Zf) proteins expressed only in the cerebellum of the adult brain. The genes are the vertebrate homologues of the Drosophila pair-rule gene, odd-paired (opa). We identified a novel gene, Zic4, which belongs to the Zic gene family, through a genomic and cDNA cloning study. Zic4 is highly similar to Zic1, Zic2 and Zic3, especially in its Zf motif. An analysis of the genomic organization of Zic4 showed that the gene shares a common exon-intron boundary with Zic1, Zic2, Zic3 and opa. The chromosomal location of Zic4 was determined to be mouse chromosome 9 in the vicinity of Zic1, using an interspecific backcross panel. An RNase protection study showed that Zic4 is expressed only in the cerebellum during the adult stage, as are the other Zic genes. The temporal profile of mRNA expression in the developing cerebellum is similar to that of Zic3 which has a peak on postnatal day 5. These findings suggest that Zic4 is a gene which works cooperatively with other Zic genes during cerebellar development.

Distribution of a reeler gene-related antigen in the developing cerebellum: an immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice.

We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.

A 5' segment of the mouse Zic1 gene contains a region specific enhancer for dorsal hindbrain and spinal cord.

Zic1 encodes a zinc finger protein, which is required for the development of the dorsal neural tissue. The gene is a mammalian homologue of the Drosophila odd-paired. We examined the regulatory elements in the 5' flanking region of the Zic1 gene as an initial step to understanding how the Zic1 expression is restricted to the dorsal neural tissue. When a 2.9-kb fragment of the 5' flanking segment of the mouse Zic1 gene was linked to the E. coli beta-galactosidase gene, the enzyme was consistently expressed in the dorsal half of the embryonic spinal cord and in the vestibulocochlear nucleus in all four transgenic mouse lines. The transgene expression mimics the Zic1 expression with respect to the region where it occurs. But this is not so for the neuronal cell types. This suggests that the segment contains a region-specific enhancer. In vivo and in vitro deletion analyses indicated that there are essential regions between -2.0 and -0.9 kb and within the proximal 0.9 kb. The distal element is necessary for the transgene expression in the embryonic dorsal spinal cord whereas the adult vestibulocochlear nucleus expression is regulated by both elements. In these regions, there are sequences similar to the binding sequences for potential regulatory proteins.

Calcium-dependent phospholipid binding to the C2A domain of a ubiquitous form of double C2 protein (Doc2 beta).

Rabphilin 3A and Doc2 alpha are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2 alpha plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2 beta) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2 alpha (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (M(r)) of 45,837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2 beta is expressed in all cell lines and tissues tested. Ca(2+)-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2 beta binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca(2+)-dependent, with an EC50 value of approximately 1 microM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2 alpha. Our results suggest that Doc2 beta is involved in constitutive membrane trafficking.

Chimeric and molecular genetic analysis of myelin-deficient (shiverer and mld) mutant mice.

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing the MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

Molecular biology of myelin basic protein: gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants.

1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer (shi) and myelin-deficient (mld. shimid), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3-7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9. The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice.

Identification of the new isoforms of mouse myelin basic protein: the existence of exon 5a.

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.

Behavioral abnormalities of Zic1 and Zic2 mutant mice: implications as models for human neurological disorders.

Zic1 and Zic2 encode closely related zinc finger proteins expressed in dorsal neural tube and its derivatives. In previous studies, we showed that the homozygous Zic1 null mutation (Zic1-/-) results in cerebellar malformation with severe ataxia and that holoprosencephaly and spina bifida occur in homozygotes for Zic2 knockdown mutation (Zic2kd/kd). Since human ZIC2 haploinsufficiency is a cause of holoprosencephaly, the Zic2kd/kd mice are regarded as an animal model for holoprosencephaly in humans. In this study, the behavioral characteristics of the Zic1 and Zic2 mutant mice were investigated in heterozygotes (Zic1-/+ or Zic2kd/+), and significant abnormalities were found in the hanging, spontaneous locomotor activity, stationary rod (Zic1-/+), acoustic startle response, and prepulse inhibition tests (Zic2kd/+). The abnormalities in the Zic1-/+ mice may be explained in part by the hypotonia caused by hypoplasia of the cerebellar anterior vermis, and these mice are regarded as a model of Joubert syndrome. In contrast, the sensorimotor gating abnormality in the Zic2kd/+ mice may be attributable to the presumed abnormality in the dorsomedial forebrain, which was strongly affected in the Zic2kd/kd mice. Zic2kd/+ mice can serve as a model for diseases involving sensorimotor gating abnormalities, such as schizophrenia.

Molecular properties of Zic proteins as transcriptional regulators and their relationship to GLI proteins.

Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into cultured cells, Zic proteins enhanced or suppressed sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.

Xenopus Zic3, a primary regulator both in neural and neural crest development.

Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.