[Protocadherins: classification, structures and functions].
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Biomedical subjects
Shun Hamada
Publications and source records attributed to Shun Hamada.
Developmental pluripotency of the nuclei of neurons in the cerebral cortex of juvenile mice.
Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14-21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.
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Novel function of neuronal PAS domain protein 1 in erythropoietin expression in neuronal cells.
The basic helix loop helix-PAS (bHLH-PAS) transcription factors have diverse roles in physiologic responses to the environment and in early development. One bHLH-PAS protein, neuronal PAS domain protein 1 (NPAS1), is reported to be expressed only in the central nervous system beginning at the late embryonic stage, but its function is unknown. Using an anti-NPAS1 antibody, we have shown that NPAS1 expression in cerebral cortex was observed first around embryonic Day 16.5 (E16.5) and was then dispersed throughout the region as cortical development progressed. From the similarity with hypoxia-inducible factor-1alpha (HIF-1alpha), we investigated whether NPAS1 regulates transcription of erythropoietin (EPO), which is the target of HIF-1alpha and is expressed in the brain during the early developmental stage. In the present study, we have shown that NPAS1 binds to the enhancer region of Epo in vivo. A luciferase reporter assay revealed a repressive effect of NPAS1 on hypoxia-responsive element-regulated gene expression. These results raise the possibility that NPAS1 plays a role in late central nervous system development by modulating EPO expression in response to cellular oxygen level.
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Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.
Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.
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Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex.
Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.
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CNR/Pcdhalpha family in subplate neurons, and developing cortical connectivity.
The cadherin-related neuronal receptor (CNR)/protocadherin (Pcdh) alpha family is one of the diverse protocadherin families identified as a candidate diversified membrane-associated component regulating the formation of neuronal connectivity. However, its expression during neural circuit formation has not been examined in detail. Here, we used a conserved sequence to study the expression of this protein family during the development of neocortical connectivity, by immunohistochemistry and in situ hybridization. The proteins were detected in developing thalamocortical and corticofugal axons, and in subplate neurons, which pioneer these axon tracts. The expression in subplate neurons was confirmed by birth-date labeling with BrdU, and by examination in homozygous reeler mice. This pattern of CNR/Pcdhalpha expression suggests its involvement in the development of neocortical connectivity.
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Exposure to hexanal odor induces extraordinary Fos expression in the medial preoptic area and amygdala of Fyn tyrosine kinase-deficient mice.
Our previous study revealed that the odor of hexanal, which is derived from the wood chips used as bedding, influenced mouse maternal behavior and induced the neonatal death of Fyn-deficient (fyn(-/-)) pups born of fyn(-/-) parents. To clarify what regions of the brain are involved in this effect, we examined which brain regions of fyn(-/-) and Fyn-heterozygous (fyn(+/-)) females were activated by hexanal odor using Fos immunohistochemistry. Hexanal induced Fos immunoreactivity in the primary olfactory cortex and in the medial and central amygdala of both fyn(+/-) and fyn(-/-) mice. In fyn(-/-) females, hexanal also induced the activation of the medial preoptic area and the basolateral and posteriomedial cortical amygdala, which are known to be involved in the control of maternal and emotional behavior.
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Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha.
The protocadherin-alpha (CNR/Pcdhalpha) and protocadherin-gamma (Pcdhgamma) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdhalpha proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdhalpha and Pcdhgamma in HEK293T cells and found that they formed a protein complex and that Pcdhgamma enhanced the surface expression of CNR/Pcdhalpha. This enhanced surface expression was confirmed by flow cytometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdhalpha and Pcdhgamma proteins. The surface expression activity was enhanced by the extracellular domains of the proteins, which could bind each other. Their cytoplasmic domains also had binding activity and influenced their localization. Their protein-protein interaction was also detected in extracts of mouse brain and two neuroblastoma cell lines. Thus, interactions between CNR/Pcdhalpha and Pcdhgamma regulate their surface expression and contribute to the combinatorial diversity of cell recognition proteins in the brain.
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Distinct genomic sequence of the CNR/Pcdhalpha genes in chicken.
CNR/Pcdhalpha family proteins have been first identified as a receptor family that corporate with Fyn, a family of the Src family of tyrosine kinase, and known as synaptic cadherins. Here we report the complete genomic sequence and organization of the chicken (Gallus gallus) CNR/Pcdhalpha The total length of chicken CNR/Pcdhalpha is 177kb. The chicken CNR/Pcdhalpha cluster encodes 12 variable and 3 constant exons. The genomic organizations of the chicken, rat, mouse, and human CNR/Pcdhalpha are basically orthologous. The constant-region exons (CP1, CP2, and CP3) are highly conserved between chicken and mammals, with percent identities of 90.9%, 90.7%, and 91.8% at the amino-acid level for chicken versus rat, mouse, and human, respectively. In contrast, the percent identities of the variable-region exons between chicken and mammals were lower: 51.8%, 51.3%, and 52.7%, on average, for chicken versus rat, mouse, and human, respectively, at the amino-acid level. Moreover, the chicken variable-region exons (from v1 to v12) are highly conserved paralogously (91.4%: nucleic acid, 92.4%: amino acid) in comparison with those of mammals. The CG content of each variable exon in the chicken (v1 to v12) is 74% on average and the CpG dinucleotide frequency in each variable-region exon is twice that of mammals. Due to the high CG content, chicken variable exons (from v1 to v12) encode 3 to 4 frame-shifted open reading frames, which span 1.5-3.0kb, in both the sense and anti-sense orientations.
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Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1.
Cadherin-related neuronal receptor (CNR) proteins are a diverse set of synaptic protocadherins, but little is known about its adhesive properties. We found that overexpressed CNR1 protein localized on the cell surface of HEK293T cells and increased the calcium-dependent cell aggregation potential. However, we could not detect the strong homophilic binding activity of CNR1 EC-Fc fusion protein in vitro. Parental HEK293T cells adhered to Arg-Gly-Asp (RGD) motif of EC1 domain of CNR1-Fc fusion protein. The fusion protein that the Asp73 of EC1 point-mutated to Glu (RGE-Fc) lost the adhesive activity. The adhesion activity of HEK293T cells to CNR1 EC-Fc fusion protein was completely blocked by inhibitors of integrins, including RGDS peptide and anti-beta1 integrin antibodies. The increased cell-aggregative property of CNR1 transfectants was also blocked by RGDS peptides. At cell-cell junctions of the CNR1 transfectants, co-localization between CNR1 and HEK293T endogenous beta1 integrin was observed. Furthermore, the spatiotemporal expression patterns of CNR and beta1 integrin nearly overlapped in the molecular layer of the developing mouse cerebellum in the main stage of synaptogenesis. These results indicate that CNR1 has a heterophilic, calcium-dependent cell adhesion activity with the beta1 integrin subfamily, and raise the possibility of CNR-beta1 integrin association in synaptogenesis.
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Exposure to hexanal odor influences maternal behavior and induces neonatal death in Fyn tyrosine kinase-deficient mice.
Fyn-deficient pups born of Fyn-deficient parents die because they fail to suckle within 1-2 days after birth. Here we demonstrate that the neonatal death phenotype was influenced by the genetic background and an environmental odor. The odor of hexanal (C6-aldehyde) partially impaired mouse maternal behavior and induced the neonatal death of Fyn-deficient pups born of Fyn-deficient parents. This death phenotype was first observed in the breeding environment using autoclaved chips of Douglas fir. An analysis of the volatile chemicals in the autoclaved chips revealed an approximately 10-fold greater amount of hexanal than in non-autoclaved chips. Hexanal influenced the length of time virgin female mice engage in the maternal crouching behavior. In addition, Fyn-deficient females exhibited defects in the maternal behavior of nest building and pup retrieval, regardless of exposure to hexanal. These observations provide new insights into the regulation of maternal behavior by environmental and genetic factors.
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Myelination triggers local loss of axonal CNR/protocadherin alpha family protein expression.
The cadherin-related neuronal receptor (CNR)/protocadherin (Pcdh) alpha family is one of the diverse protocadherin families expressed in developing axons. We observed a strong axonal expression of these proteins at late embryonic and early postnatal stages corresponding to regions where fibers had not yet been myelinated. We therefore followed the postnatal localization of CNR/Pcdh alpha protein in major axonal tracts, such as the internal capsule, lateral olfactory tract, and optic nerve, and found that its axonal localization was dramatically lost in parallel with the increased expression of myelin markers. Moreover, the hypomyelinated optic nerve tracts of the myelin-deficient Shiverer mouse exhibited elevated levels of CNR/Pcdh alpha expression. These axonal expression patterns of CNR/Pcdh alpha in wild-type and Shiverer mice were similar to those of growth associated protein 43 (GAP-43) and L1, both of which are associated with axonal maturation. Thus, myelination may be a trigger for the local loss of axonal CNR/Pcdh alpha protein, and this process may be important in the maturation of neural circuits.
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