Polyunsaturated fatty acid deficiency during neurodevelopment in mice models the prodromal state of schizophrenia through epigenetic changes in nuclear receptor genes.

The risk of schizophrenia is increased in offspring whose mothers experience malnutrition during pregnancy. Polyunsaturated fatty acids (PUFAs) are dietary components that are crucial for the structural and functional integrity of neural cells, and PUFA deficiency has been shown to be a risk factor for schizophrenia. Here, we show that gestational and early postnatal dietary deprivation of two PUFAs-arachidonic acid (AA) and docosahexaenoic acid (DHA)-elicited schizophrenia-like phenotypes in mouse offspring at adulthood. In the PUFA-deprived mouse group, we observed lower motivation and higher sensitivity to a hallucinogenic drug resembling the prodromal symptoms in schizophrenia. Furthermore, a working-memory task-evoked hyper-neuronal activity in the medial prefrontal cortex was also observed, along with the downregulation of genes in the prefrontal cortex involved in oligodendrocyte integrity and the gamma-aminobutyric acid (GABA)-ergic system. Regulation of these genes was mediated by the nuclear receptor genes Rxr and Ppar, whose promoters were hyper-methylated by the deprivation of dietary AA and DHA. In addition, the RXR agonist bexarotene upregulated oligodendrocyte- and GABA-related gene expression and suppressed the sensitivity of mice to the hallucinogenic drug. Notably, the expression of these nuclear receptor genes were also downregulated in hair-follicle cells from schizophrenia patients. These results suggest that PUFA deficiency during the early neurodevelopmental period in mice could model the prodromal state of schizophrenia through changes in the epigenetic regulation of nuclear receptor genes.

Neurogenesis and sensorimotor gating: bridging a microphenotype and an endophenotype.

Human genetic data on psychiatric disorders repeatedly demonstrate the involvement of various genes that are associated with neural development and neurogenesis. Neurogenesis is a biological process that is critical in brain development and continues throughout life. Neurogenesis is a multi-step process starting from the division of neural stem cells/progenitor cells, leading to self-renewal and simultaneously to the production of lineage-committed cells, including neurons and glial cells. Minor defects in the neurogenesis process, such as production of fewer new neurons and malformation of neural circuits, could represent phenotypes of psychiatric disorders at molecular and cellular levels in animal models (here termed as "microphenotypes"). However, microphenotypes are not easily used as biomarkers. We have focused on a physiological condition, sensorimotor gating deficits, that can be scored by a prepulse inhibition (PPI) test. Impaired PPI is considered to be one of the compelling endophenotypes (biological markers) of mental disorders such as schizophrenia, autism, and other neurodevelopmental disorders. Because the neural circuit for PPI involves the hippocampus, a unique brain region where neurogenesis occurs postnatally, we hypothesize that an impairment of preadolescent neurogenesis is critical for the onset of sensorimotor gating defects. To test this hypothesis, we investigated a critical period of neurogenesis that can affect PPI. In this paradigm, we introduced an enriched environment to restore neurogenesis, thereby recovering PPI deficits in mice. We noted impairments in the maturation of newborn neurons in the hippocampal dentate gyrus (DG) and GABAergic neurons in the hippocampus, which could be considered as microphenotypes associated with PPI defects. More precise genetically controlled neurogenesis models (with precise time points or periods) are needed to be studied in further investigation to support our hypothesis.

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart.

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

Ectopically localized HNK-1 epitope perturbs migration of the midbrain neural crest cells in Pax6 mutant rat.

Small eye rats, which have a mutation in a gene encoding transcription factor Pax6, exhibit impaired migration of the midbrain neural crest cells, thereby showing severe craniofacial defects. Orthotopic grafting of the midbrain neural crest cells taken from the wild-type into Pax6 mutant embryos has suggested environmental defects along the migratory pathway of the midbrain crest cells. In the present study we found that the HNK-1 carbohydrate epitope was ectopically localized in the frontonasal epithelium of Pax6 mutant embryos. The GlcAT-P gene, encoding an enzyme for the synthesis of the HNK-1 epitope, was also expressed ectopically in the frontonasal epithelium of the mutant. In explant cultures, the migration rate of neural crest cells from the midbrain, but not from the forebrain, was significantly less in HNK-1-coated dishes than in non-coated dishes. These results suggest that the arrested migration of the midbrain crest cells in Pax6 mutant embryos may, at least in part, be due to the inhibitory effect of the HNK-1 epitope ectopically localized in the frontonasal epithelium.

Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.

Body patterning.

Early patterning of the body during animal development is a fundamental process to subsequent events including cell differentiation, tissue and organ formation, and correct function of the adult body. We focused on two major topics: body segmentation and brain patterning, both of which are essential for conferring a functional complexity to the body, repetition of skeletal elements and complex neural network, respectively.

Gene transfer into cultured mammalian embryos by electroporation.

To gain a better understanding of mammalian development at the molecular level, technology is needed that allows the transfer of exogenous genes into desired embryonic regions at defined stages of development. Our strategy has been to use electroporation (EP) of plasmid DNA following whole-embryo culture (WEC). In our gene transfer system, postimplantation rodent embryos are taken out of the uterus and a purified DNA solution of mammalian expression plasmid constructs is injected into the neural tube. A square-pulse current is delivered using an electroporator with an optimizer. Electroporated embryos are allowed to develop in the WEC system for 24--48 h. Within the targeted area, the proportion of transfected cells varied from 10% to approximately 100% depending on the test conditions (e.g., DNA concentration, voltage, duration of EP, and pulse number). The EP--WEC system has several advantages including rapid gene expression, minimal laboratory work, precisely targeted regions, and no risk for human beings. Application of the method is useful in improving our understanding of early neural development (E7--E12 in mice), e.g., alteration of gene function via ectopic expression, interference with dominant negative proteins, and fate mapping with marker genes. In addition, EP can complement genetic approaches such as the generation of knockout and transgenic mice.

The role of Pax6 in brain patterning.

Pax6 g ene encodes a transcription factor that plays a pivotal role in various aspects of brain development. Here I review the molecular and cellular mechanisms of how the early brain is patterned, and introduce recent studies on the role of Pax6 in brain patterning, neuronal specification, neuronal migration and axonal extension.

Current topics in comparative developmental biology of vertebrate brains.

Little is known about how unique features in a species can emerge along the conserved body plan beyond species. For example, mammals, including human beings, have acquired the neocortex with distinct function and morphology. Here we review current topics in comparative developmental biology of vertebrate brains, especially focusing on the cerebral neocortex as a suitable model for considering species-specific aspects.

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.

Identification of RALDH-3, a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina.

In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system.

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors.

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.

Detection of spatial localization of Hst-1/Fgf-4 gene expression in brain and testis from adult mice.

HST-1, a member of the fibroblast growth factor (FGF) family (FGF-4), has been shown to be a signaling molecule whose expression is essential for embryonic development. However, HST-1/FGF-4 expression has not been detected or reported in adult tissues so far analysed. To investigate whether there is a possible role of HST-1/FGF-4 in adult stage, we have carried out a highly sensitive RT-PCR analysis of Hst-1/Fgf-4 gene expression in adult mice tissues. Results show Hst-1/Fgf-4 gene expression in the nervous system, intestines, and testis of normal adult mice. In situ hybridization technique was used to localize Hst-1/Fgf-4 gene expression in the cerebellum and testis from 10-week-old mice. Cell type-specific gene expression was detected: Purkinje cells in the cerebellum and Sertoli cells in testis. These findings suggest that the Hst-1/Fgf-4 gene also plays an important role in adult tissues, and may offer insights into the biological significance of HST-1/FGF-4 in cerebellar and testicular functions.

Neocortical origin and tangential migration of guidepost neurons in the lateral olfactory tract.

The early-generated neurons designated as lot cells specifically mark the future site of the lateral olfactory tract (LOT) and guide LOT axons. We investigated the mechanism of how lot cells develop and get localized in the LOT position. Lot cells differentiated from neuroepithelial cells in all regions of the neocortex but not from those in the ganglionic eminence in culture. Cell tracing analyses demonstrated that lot cells generated from the neocortex subsequently followed a tangential migration stream ventrally toward the LOT position. Mutant mouse embryos lacking the function of transcription factor Gli3 showed disturbances of the migration stream and translocation of lot cells in the dorsal telencephalon. These results reveal a new type of neuronal migration in the telencephalon and introduce an unexpected dramatic feature of the earliest regionalization of the telencephalon.

Fate mapping of the mouse prosencephalic neural plate.

Little is known about the behavior of cells within the anterior neural plate or tube in developing mammalian embryos in utero due to technical limitations. Here we labeled neuroepithelial cells with vital dye and traced their siblings for 1 or 2 days using the whole-embryo culture system. The results demonstrated that rostral cell movement from the midbrain to the forebrain in the mouse neural plate was restricted at the boundary by the five-somite stage. Coincident with restriction of cell intermingling, expression of a transcription factor, Pax6, and a cell adhesion molecule, cadherin-6, commmenced to demarcate the forebrain compartment. Within this compartment, we also mapped several prospective regions of the telencephalon and diencephalon to the eyes. The fate map of the mouse prosencephalic neural plate was very similar to those of other vertebrates, providing evidence that mammalian-specific brain structures, represented in the cerebral neocortex, could evenly develop along the conserved framework of neuromeres.

Histogenesis of the cerebral cortex in rat fetuses with a mutation in the Pax-6 gene.

The embryonic development of the cerebral cortex was histologically examined in rat homozygotes with a mutation of the Paired box (Pax)-6 gene, rat Small eye (rSey(2)/rSey(2)). Although the cerebral wall was thinner in rSey(2)/rSey(2) than in the wild type at embryonic day 16 (E16), cortical cells of mutants labeled with 5'-bromodeoxyuridine (BrdU) at E13 migrated as normal, settling in superficial layer at E16. Mitotic activity in the ventricular zone, estimated by immunoreactivity for proliferating cell nuclear antigen (PCNA), was also retained. On the other hand, after E20 cells were clustered in abnormally expanded ventricular and intermediate zones of the rSey(2)/rSey(2) cortex. Birthdating studies using BrdU revealed that most of these clustered cells were generated between E18 and E20. Most of clustered cells were immunoreactive for PCNA and highly polysialylated NCAM, while immunoreaction for neurofilament and microtubule-associated protein-2 (MAP-2) was hardly detected in the clusters. Furthermore, apoptosis detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) was rarely seen, suggesting that the clustered cells remain in an undifferentiating state, but not degenerated by the end of the gestational period. Considering that Pax-6 immunoreactivity was exclusively localized in the ventricular zone of the normal rat cortex throughout the fetal period, the present results suggest that Pax-6 is crucial for differentiation and migration of late-generated cortical neurons.

Demarcation of early mammalian cortical development by differential expression of fringe genes.

Fringe has originally been found in Drosophila as a gene encoding a putative secreted protein which regulates the sensitivity of Notch signaling pathway to different ligands. We show that three members of murine fringe gene family, Lunatic fringe (L-fng), Manic fringe (M-fng) and Radical fringe (R-fng), show related patterns of expression in the developing cerebral wall. L-fng is expressed in immature cells in the ventricular zone. M-fng is upregulated transiently in maturing neurons when they leave the ventricular zone (VZ). R-fng is upregulated in more mature neurons when they enter the preplate and cortical plate. These patterns suggest that the transition from immature to mature neurons involves sequential changes in the member of fringe family genes expressed. More detailed expression analyses of fringe genes and immunohistochemistry for neuron-specific class III beta-tubulin suggest a mode of neurogenesis which might underlie the histogenesis of the cerebral cortex. A proliferative population situated outside of the VZ is defined as M-fng-positive/BrdU-positive cells, which constitutes about 10-20% of the total S-phase cells in the cerebral wall of embryonic day 10.5-12.5. We found that M-fng is expressed in mitotic figures outside the VZ and some of them react with the antibody against class III beta-tubulin. These observations suggest that a significant number of proliferative cells exist outside the VZ, which supply neurons during early cortical development.

Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems.

Hu proteins are mammalian embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding proteins that contain three RNA recognition motifs. Although Drosophila ELAV is required for the correct differentiation and survival of neurons, the roles played by the Hu genes in the mammalian nervous system remain largely unknown. To explore the in vivo functions of mouse Hu proteins, we overexpressed them in rat pheochromocytoma PC12 cells, where they induced neuronal phenotype in the absence of nerve growth factor. We have characterized the functions of various forms of mHuB and mHuC bearing point mutations or deletions. Mutants of mHuC that had amino acid exchanges in the RNP1 domain of the first or second RNA recognition motifs (RRMs) lost biologic activity as well as RNA-binding activity. In addition, the mutants containing only the third RRM failed to induce the neuronal phenotype in PC12 cells and inhibited the biologic activity of cotransfected wild-type mHuB and mHuC, thus acting as a dominant-negative form. However, these mutants could not suppress the nerve growth factor-induced differentiation of PC12 cells. Further, we misexpressed wild-type and dominant-negative Hu in E9.5 mouse embryos, by using electroporation into the neural tube at the level of the rhombencephalon. mHuB and mHuC induced the ectopic expression of neuronal markers, whereas the dominant-negative forms of mHuB and mHuC suppressed the differentiation of central nervous system motor neurons. From these results, we suggest that Hu proteins are required for neuronal differentiation in the mammalian nervous system.

Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.

We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.

Pax-6 is required for thalamocortical pathway formation in fetal rats.

Pax-6, a transcription regulatory factor, has been demonstrated to play important roles in eye, nose, and brain development by analyzing mice, rats, and humans with a Pax-6 gene mutation. We examined the role of Pax-6 with special attention to the formation of efferent and afferent pathways of the cerebral cortex by using the rat Small eye (rSey2), which has a mutation in the Pax-6 gene. In rSey2/rSey2 fetuses, cortical efferent axons develop with normal trajectory, at least within the cortical anlage, when examined with immunohistochemistry of the neuronal cell adhesion molecule TAG-1 and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling from the cortical surface. A remarkable disorder was found in the trajectory of dorsal thalamic axons by immunostaining of the neurofilament and the neural cell adhesion molecule L1 and DiI labeling from the dorsal thalamus. In normal rat fetuses, dorsal thalamic axons curved laterally in the ventral thalamus without invading a Pax-6-immunoreactive cell cluster in the ventral part of the ventral thalamus. These axons then coursed up to the cortical anlage, passing just dorsal to another Pax-6-immunoreactive cell cluster in the amygdaloid region. In contrast, in rSey2/rSey2 fetuses, dorsal thalamic axons extended downward to converge in the ventrolateral corner of the ventral thalamus and fanned out in the amygdaloid region without reaching the cortical anlage. These results suggest that Pax-6-expressing cell clusters along the thalamocortical pathway (ventral part of the ventral thalamus and amygdala) are responsible for the determination of the axonal pathfinding of the thalamocortical pathway.