Identification and Characterization of Novel Conserved Domains in Metazoan Zic Proteins.

Zic family genes encode C2H2-type zinc finger proteins that act as critical toolkit proteins in the metazoan body plan establishment. In this study, we searched evolutionarily conserved domains (CDs) among 121 Zic protein sequences from 22 animal phyla and 40 classes, and addressed their evolutionary significance. The collected sequences included those from poriferans and orthonectids. We discovered seven new CDs, CD0-CD6, (in order from the N- to C-terminus) using the most conserved Zic protein sequences from Deuterostomia (Hemichordata and Cephalochordata), Lophotrochozoa (Cephalopoda and Brachiopoda), and Ecdysozoa (Chelicerata and Priapulida). Subsequently, we analyzed the evolutionary history of Zic CDs including the known CDs (ZOC, ZFD, ZFNC, and ZFCC). All Zic CDs are predicted to have existed in a bilaterian ancestor. During evolution, they have degenerated in a taxa-selective manner with significant correlations among CDs. The N terminal CD (CD0) was largely lost, but was observed in Brachiopoda, Priapulida, Hemichordata, Echinodermata, and Cephalochordata, and the C terminal CD (CD6) was highly conserved in conserved-type-Zic possessing taxa, but was truncated in vertebrate Zic gene paralogues (Zic1/2/3), generating a vertebrate-specific C-terminus critical for transcriptional regulation. ZOC was preferentially conserved in insects and in an anthozoan paralogue, and it was bound to the homeodomain transcription factor Msx in a phylogenetically conserved manner. Accordingly, the extent of divergence of Msx and Zic CDs from their respective bilaterian ancestors is strongly correlated. These results suggest that coordinated divergence among the toolkit CDs and among toolkit proteins is involved in the divergence of metazoan body plans.

Lophotrochozoan Zic Genes.

Lophotrochozoa is a sister taxon of Ecdysozoa in the Protostomia that includes mollusks, annelids, brachiopods, and platyhelminths. Recent studies have clarified the structure, expression, and roles of lophotrochozoan Zic family genes. Zic genes in oligochaete annelid Tubifex tubifex (freshwater sludge worm) and polychaete annelid Capitella teleta (bristle worm) are commonly expressed in a subset of developing brain and mesoderm derivatives. The latter includes the naïve mesoderm and the associated chaetal sacs in each body segment, although the segmentation processes differ between the two species. Furthermore, in brachiopod Terebratalia transversa (lamp shell), Zic is expressed in the anterior ectodermal domains and mesodermal derivatives, including those associated with the chaetal sacs. This result suggests the common involvement of Zic genes in the development of chaetae, a lophotrochozoan novelty acquired in the course of evolution. In addition, the highly simplified lophotrochozoan Dicyema acuticephalum (dicyemid mesozoan, a cephalopod endoparasite), which lost its gut, nervous system, and muscles during evolution, expresses its Zic genes in hermaphroditic gonads, highlighting the role of Zic genes in germ cell development. The role of Zic in head regeneration was revealed in studies on platyhelminth Schmidtea mediterranea (freshwater planarian). Planarian Zic expression was induced in a subpopulation of neoblasts that includes adult pluripotent stem cells. It is needed for head regeneration and production of an anterior signaling center. Suppression of Wnt-ß-catenin signaling underlies Zic-mediated head regeneration, reminiscent of Wnt-ß-catenin suppression by vertebrate Zic genes. Taken together, studies on the lophotrochozoan Zic genes are essential to understanding not only the roles of these genes in body plan evolution but also the molecular mechanism underlying adult stem cell regulation.

Zic Family Proteins in Emerging Biomedical Studies.

Zic family proteins have been investigated in various biomedical studies. Here we summarize the contact points between Zic proteins and recent medical research. The topics cover a wide range, reflecting the pleiotropic roles of these proteins in early embryogenesis and organogenesis. Zic1, Zic2, and Zic3 proteins play important roles in the development of axial and limb bones, and of muscles, among the derivatives of the notochord and somites. Zic1 is involved in bone's response to mechanical stress, and it also serves as a marker specific for brown adipocytes. Zic1, Zic2, Zic3, and Zic5 proteins are required for the development of neural crest derivatives, including the meningeal membrane and facial bones, and deficiency of these proteins causes cortical lamination defects resembling those in type II lissencephaly. In vascular systems, Zic3 is associated not only with normal cardiovascular development, failure of which causes congenital heart anomalies, but also controls maturation of the blood-brain barrier. Zic1 is also expressed in the brain pericytes possessing stem cell properties that control the blood-brain barrier activity and capillary hemodynamic responses. The possible involvement of Zic proteins in neuropsychiatric disorders has been indicated by the analyses of mutant mice behaviors. Zic1 and Zic3 mutant mice show hypotonia and decreased locomotor activities. Zic2 hypomorphic mutant mice exhibit schizophrenia-related behavioral abnormalities such as cognitive dysfunction and impaired sensorimotor gating and social behaviors, and ZIC2 mutations found in schizophrenia patients included a severely functionally defective one. Based on these facts, the application of Zic protein activities in translational medicine might be considered.

Comparative Genomics of the Zic Family Genes.

Zic family genes encode five C2H2-type zinc finger domain-containing proteins that have many roles in animal development and maintenance. Recent phylogenetic analyses showed that Zic family genes are distributed in metazoans (multicellular animals), except Porifera (sponges) and Ctenophora (comb jellies). The sequence comparisons revealed that the zinc finger domains were absolutely conserved among the Zic family genes. Zic zinc finger domains are similar to, but distinct from those of the Gli, Glis, and Nkl gene family, and these zinc finger protein families are proposed to have been derived from a common ancestor gene. The Gli-Glis-Nkl-Zic superfamily and some other eukaryotic zinc finger proteins share a tandem CWCH2 (tCWCH2) motif, a hallmark for inter-zinc finger interaction between two adjacent C2H2 zinc fingers. In Zic family proteins, there exist additional evolutionally conserved domains known as ZOC and ZFNC, both of which may have appeared before cnidarian-bilaterian divergence. Comparison of the exon-intron boundaries in the Zic zinc finger domains revealed an intron (A-intron) that was absolutely conserved in bilaterians (metazoans with bilateral symmetry) and a placozoan (a simple nonparasitic metazoan). In vertebrates, there are five to seven Zic paralogs among which Zic1, Zic2, and Zic3 are generated through a tandem gene duplication and carboxy-terminal truncation in a vertebrate common ancestor, sharing a conserved carboxy-terminal sequence. Several hypotheses have been proposed to explain the Zic family phylogeny, including their origin, unique features in the first and second zinc finger motif, evolution of the nuclear localization signal, significance of the animal taxa-selective degeneration, gene multiplication in the vertebrate lineage, and involvement in the evolutionary alteration of the animal body plan.

ZIC1 Function in Normal Cerebellar Development and Human Developmental Pathology.

Zic genes are strongly expressed in the cerebellum. This feature leads to their initial identification and their name "zic," as the abbreviation of "zinc finger protein of the cerebellum." Zic gene function in cerebellar development has been investigated mainly in mice. However, association of heterozygous loss of ZIC1 and ZIC4 with Dandy-Walker malformation, a structural birth defect of the human cerebellum, highlights the clinical relevance of these studies. Two proposed mechanisms for Zic-mediated cerebellar developmental control have been documented: regulation of neuronal progenitor proliferation-differentiation and the patterning of the cerebellar primordium. Clinical studies have also revealed that ZIC1 gain of function mutations contribute to coronal craniosynostosis, a rare skull malformation. The molecular pathways contributing to these phenotypes are not fully explored; however, embryonic interactions with sonic hedgehog signaling, retinoic acid signaling, and TGFß signaling have been described during mouse cerebellar development. Further, Zic1/2 target a multitude of genes associated with cerebellar granule cell maturation during postnatal mouse cerebellar development.

Role of Zic Family Proteins in Transcriptional Regulation and Chromatin Remodeling.

Proper functions of Zic proteins are essential for animals in health and disease. Here, we summarize our current understanding of the molecular properties and functions of the Zic family across animal species and paralog subtypes. Zics are basic proteins with some posttranslational modifications and can move to the cell nucleus via importin- and CRM1-based nucleocytoplasmic shuttling mechanisms. Degradation is mediated by the ubiquitin proteasome system. Many Zic proteins are capable of binding to two types of target DNA sequences (CTGCTG-core-type and GC-stretch-type). Recent chromatin immunoprecipitation assays showed that CTGCTG-core-type target sequences are enriched in enhancers. Nonetheless, the DNA binding is not always required for transcriptional regulation by Zic proteins. On the other hand, Zic proteins bind many proteins including transcription factors (Gli1-3, Tcf1 or Tcf4, Smad2 or Smad3, Oct4, Pax3, Cdx, and SRF), chromatin-remodeling factors (NuRD and NURF), and other nuclear enzymes (DNA-PK, PARP1, and RNA helicase A). Zic family-mediated gene expression control involves both their actions near the transcription start site and those affecting the global gene expression via binding to enhancers. Although Zic proteins perform essential functions in transcriptional regulation of Oct4 and Nanog expression via their promoters, recent genome-wide analyses of the Zic-binding sites and their downstream targets indicate that Zic proteins are associated with distant regulatory elements and are the critical enhancer-priming nuclear regulators in organismal development. Chromatin-remodeling complexes such as NuRD and NURF that interact with Zic proteins have been shown to participate in Zic-mediated enhancer regulation.

Transcriptional regulatory networks in epiblast cells and during anterior neural plate development as modeled in epiblast stem cells.

Somatic development initiates from the epiblast in post-implantation mammalian embryos. Recent establishment of epiblast stem cell (EpiSC) lines has opened up new avenues of investigation of the mechanisms that regulate the epiblast state and initiate lineage-specific somatic development. Here, we investigated the role of cell-intrinsic core transcriptional regulation in the epiblast and during derivation of the anterior neural plate (ANP) using a mouse EpiSC model. Cells that developed from EpiSCs in one day in the absence of extrinsic signals were found to represent the ANP of ~E7.5 embryos. We focused on transcription factors that are uniformly expressed in the E6.5 epiblast but in a localized fashion within or external to the ANP at E7.5, as these are likely to regulate the epiblast state and ANP development depending on their balance. Analyses of the effects of knockdown and overexpression of these factors in EpiSCs on the levels of downstream transcription factors identified the following regulatory functions: cross-regulation among Zic, Otx2, Sox2 and Pou factors stabilizes the epiblastic state; Zic, Otx2 and Pou factors in combination repress mesodermal development; Zic and Sox2 factors repress endodermal development; and Otx2 represses posterior neural plate development. All of these factors variably activate genes responsible for neural plate development. The direct interaction of these factors with enhancers of Otx2, Hesx1 and Sox2 genes was demonstrated. Thus, a combination of regulatory processes that suppresses non-ANP lineages and promotes neural plate development determines the ANP.

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Slitrk4 is required for the development of inhibitory neurons in the fear memory circuit of the lateral amygdala.

The Slitrk family consists of six synaptic adhesion molecules, some of which are associated with neuropsychiatric disorders. In this study, we aimed to investigate the physiological role of Slitrk4 by analyzing Slitrk4 knockout (KO) mice. The Slitrk4 protein was widely detected in the brain and was abundant in the olfactory bulb and amygdala. In a systematic behavioral analysis, male Slitrk4 KO mice exhibited an enhanced fear memory acquisition in a cued test for classical fear conditioning, and social behavior deficits in reciprocal social interaction tests. In an electrophysiological analysis using amygdala slices, Slitrk4 KO mice showed enhanced long-term potentiation in the thalamo-amygdala afferents and reduced feedback inhibition. In the molecular marker analysis of Slitrk4 KO brains, the number of calretinin (CR)-positive interneurons was decreased in the anterior part of the lateral amygdala nuclei at the adult stage. In in vitro experiments for neuronal differentiation, Slitrk4-deficient embryonic stem cells were defective in inducing GABAergic interneurons with an altered response to sonic hedgehog signaling activation that was involved in the generation of GABAergic interneuron subsets. These results indicate that Slitrk4 function is related to the development of inhibitory neurons in the fear memory circuit and would contribute to a better understanding of osttraumatic stress disorder, in which an altered expression of Slitrk4 has been reported.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

Comparative anatomy of marmoset and mouse cortex from genomic expression.

Advances in mouse neural circuit genetics, brain atlases, and behavioral assays provide a powerful system for modeling the genetic basis of cognition and psychiatric disease. However, a critical limitation of this approach is how to achieve concordance of mouse neurobiology with the ultimate goal of understanding the human brain. Previously, the common marmoset has shown promise as a genetic model system toward the linking of mouse and human studies. However, the advent of marmoset transgenic approaches will require an understanding of developmental principles in marmoset compared to mouse. In this study, we used gene expression analysis in marmoset brain to pose a series of fundamental questions on cortical development and evolution for direct comparison to existing mouse brain atlas expression data. Most genes showed reliable conservation of expression between marmoset and mouse. However, certain markers had strikingly divergent expression patterns. The lateral geniculate nucleus and pulvinar in the thalamus showed diversification of genetic organization between marmoset and mouse, suggesting they share some similarity. In contrast, gene expression patterns in early visual cortical areas showed marmoset-specific expression. In prefrontal cortex, some markers labeled architectonic areas and layers distinct between mouse and marmoset. Core hippocampus was conserved, while afferent areas showed divergence. Together, these results indicate that existing cortical areas are genetically conserved between marmoset and mouse, while differences in areal parcellation, afferent diversification, and layer complexity are associated with specific genes. Collectively, we propose that gene expression patterns in marmoset brain reveal important clues to the principles underlying the molecular evolution of cortical and cognitive expansion.

Xenopus Zic3 controls notochord and organizer development through suppression of the Wnt/ß-catenin signaling pathway.

Zic3 controls neuroectodermal differentiation and left-right patterning in Xenopus laevis embryos. Here we demonstrate that Zic3 can suppress Wnt/ß-catenin signaling and control development of the notochord and Spemann's organizer. When we overexpressed Zic3 by injecting its RNA into the dorsal marginal zone of 2-cell-stage embryos, the embryos lost mesodermal dorsal midline structures and showed reduced expression of organizer markers (Siamois and Goosecoid) and a notochord marker (Xnot). Co-injection of Siamois RNA partially rescued the reduction of Xnot expression caused by Zic3 overexpression. Because the expression of Siamois in the organizer region is controlled by Wnt/ß-catenin signaling, we subsequently examined the functional interaction between Zic3 and Wnt signaling. Co-injection of Xenopus Zic RNAs and ß-catenin RNA with a reporter responsive to the Wnt/ß-catenin cascade indicated that Zic1, Zic2, Zic3, Zic4, and Zic5 can all suppress ß-catenin-mediated transcriptional activation. In addition, co-injection of Zic3 RNA inhibited the secondary axis formation caused by ventral-side injection of ß-catenin RNA in Xenopus embryos. Zic3-mediated Wnt/ß-catenin signal suppression required the nuclear localization of Zic3, and involved the reduction of ß-catenin nuclear transport and enhancement of ß-catenin degradation. Furthermore, Zic3 co-precipitated with Tcf1 (a ß-catenin co-factor) and XIC (I-mfa domain containing factor required for dorsoanterior development). The findings in this report produce a novel system for fine-tuning of Wnt/ß-catenin signaling.

A role for Zic1 and Zic2 in Myf5 regulation and somite myogenesis.

Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. Here, we report that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog(-/-) mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog(-/-) and in Zic2(kd/kd) mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation.

Developmental control of noradrenergic system by SLITRK1 and its implications in the pathophysiology of neuropsychiatric disorders.

SLITRK1 is a neuronal transmembrane protein with neurite development-and synaptic formation-controlling abilities. Several rare variants of SLITRK1 have been identified and implicated in the pathogenesis of Tourette's syndrome, trichotillomania, and obsessive-compulsive disorder, which can be collectively referred to as obsessive-compulsive-spectrum disorders. Recent studies have reported a possible association between bipolar disorder and schizophrenia, including a revertant of modern human-specific amino acid residues. Although the mechanisms underlying SLITRK1-associated neuropsychiatric disorders are yet to be fully clarified, rodent studies may provide some noteworthy clues. Slitrk1-deficient mice show neonatal dysregulation of the noradrenergic system, and later, anxiety-like behaviors that can be attenuated by an alpha 2 noradrenergic receptor agonist. The noradrenergic abnormality is characterized by the excessive growth of noradrenergic fibers and increased noradrenaline content in the medial prefrontal cortex, concomitant with enlarged serotonergic varicosities. Slitrk1 has both cell-autonomous and cell-non-autonomous functions in controlling noradrenergic fiber development, and partly alters Sema3a-mediated neurite control. These findings suggest that transiently enhanced noradrenergic signaling during the neonatal stage could cause neuroplasticity associated with neuropsychiatric disorders. Studies adopting noradrenergic signal perturbation via pharmacological or genetic means support this hypothesis. Thus, Slitrk1 is a potential candidate genetic linkage between the neonatal noradrenergic signaling and the pathophysiology of neuropsychiatric disorders involving anxiety-like or depression-like behaviors.

Leucine-Rich Repeats and Transmembrane Domain 2 Controls Protein Sorting in the Striatal Projection System and Its Deficiency Causes Disturbances in Motor Responses and Monoamine Dynamics.

The striatum is involved in action selection, and its disturbance can cause movement disorders. Here, we show that leucine-rich repeats and transmembrane domain 2 (Lrtm2) controls protein sorting in striatal projection systems, and its deficiency causes disturbances in monoamine dynamics and behavior. The Lrtm2 protein was broadly detected in the brain, but it was enhanced in the olfactory bulb and dorsal striatum. Immunostaining revealed a strong signal in striatal projection output, including GABAergic presynaptic boutons of the SNr. In subcellular fractionation, Lrtm2 was abundantly recovered in the synaptic plasma membrane fraction, synaptic vesicle fraction, and microsome fraction. Lrtm2 KO mice exhibited altered motor responses in both voluntary explorations and forced exercise. Dopamine metabolite content was decreased in the dorsal striatum and hypothalamus, and serotonin turnover increased in the dorsal striatum. The prefrontal cortex showed age-dependent changes in dopamine metabolites. The distribution of glutamate decarboxylase 67 (GAD67) protein and gamma-aminobutyric acid receptor type B receptor 1 (GABA B R1) protein was altered in the dorsal striatum. In cultured neurons, wild-type Lrtm2 protein enhanced axon trafficking of GAD67-GFP and GABA B R1-GFP whereas such activity was defective in sorting signal-abolished Lrtm2 mutant proteins. The topical expression of hemagglutinin-epitope-tag (HA)-Lrtm2 and a protein sorting signal abolished HA-Lrtm2 mutant differentially affected GABA B R1 protein distribution in the dorsal striatum. These results suggest that Lrtm2 is an essential component of striatal projection neurons, contributing to a better understanding of striatal pathophysiology.

SLITRK1-mediated noradrenergic projection suppression in the neonatal prefrontal cortex.

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-associated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiens-specific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit.

Slitrk2 deficiency causes hyperactivity with altered vestibular function and serotonergic dysregulation.

SLITRK2 encodes a transmembrane protein that modulates neurite outgrowth and synaptic activities and is implicated in bipolar disorder. Here, we addressed its physiological roles in mice. In the brain, the Slitrk2 protein was strongly detected in the hippocampus, vestibulocerebellum, and precerebellar nuclei-the vestibular-cerebellar-brainstem neural network including pontine gray and tegmental reticular nucleus. Slitrk2 knockout (KO) mice exhibited increased locomotor activity in novel environments, antidepressant-like behaviors, enhanced vestibular function, and increased plasticity at mossy fiber-CA3 synapses with reduced sensitivity to serotonin. A serotonin metabolite was increased in the hippocampus and amygdala, and serotonergic neurons in the raphe nuclei were decreased in Slitrk2 KO mice. When KO mice were treated with methylphenidate, lithium, or fluoxetine, the mood stabilizer lithium showed a genotype-dependent effect. Taken together, Slitrk2 deficiency causes aberrant neural network activity, synaptic integrity, vestibular function, and serotonergic function, providing molecular-neurophysiological insight into the brain dysregulation in bipolar disorders.

IP3 signaling is required for cilia formation and left-right body axis determination in Xenopus embryos.

Vertebrate left-right (LR) body axis is manifested as an asymmetrical alignment of the internal organs such as the heart and the gut. It has been proposed that the process of LR determination commonly involves a cilia-driven leftward flow in the mammalian node and its equivalents (Kupffer's vesicle in zebrafish and the gastrocoel roof plate in Xenopus). Recently, it was reported that Ca(2+) flux regulates Kupffer's vesicle development and is required for LR determination. As a basis of Ca(2+) flux in many cell types, inositol 1,4,5-trisphosphate (IP(3)) receptor-mediated calcium release from the endoplasmic reticulum (ER) plays important roles. However, its involvement in LR determination is poorly understood. We investigated the role of IP(3) signaling in LR determination in Xenopus embryos. Microinjection of an IP(3) receptor-function blocking antibody that can inhibit IP(3) calcium channel activity randomized the LR axis in terms of left-sided Pitx2 expression and organ laterality. In addition, an IP(3) sponge that could inhibit IP(3) signaling by binding IP(3) more strongly than the IP(3) receptor impaired LR determination. Examination of the gastrocoel roof plate revealed that the number of cilia was significantly reduced by IP(3) signal blocking. These results provide evidence that IP(3) signaling is involved in LR asymmetry formation in vertebrates.

Role of BMP, FGF, calcium signaling, and Zic proteins in vertebrate neuroectodermal differentiation.

More than a decade has passed since Zic family zinc finger proteins were discovered to be transcription factors controlling neuroectodermal differentiation (neural induction) in Xenopus laevis embryos. Although BMP-signal blocking has been shown to be a major upregulator of Zic genes in neuroectodermal differentiation, recent studies have revealed that FGF signaling and intracellular calcium elevation are also involved in regulating the expression of Zic genes. Different regulatory mechanisms have been found for the Zic1 and Zic3 genes, raising the possibility that functional synergism between them partly accounts for the integration of BMP-signal blocking and FGF signaling in neuroectodermal differentiation. Furthermore, mammalian Zic1 and Zic3 have been found to be neural-cell-fate-inducing and pluripotency-maintaining factors, respectively, leading us to the intriguing question of whether the mechanism underlying amphibian neuroectodermal differentiation is applicable to mammals. Comprehensive understanding of the Zic family genes is therefore essential for the study of the neuroectodermal differentiation and stem cell biology.

Trans-Synaptic Regulation of Metabotropic Glutamate Receptors by Elfn Proteins in Health and Disease.

Trans-regulation of G protein-coupled receptors (GPCRs) by leucine-rich repeat (LRR) transmembrane proteins has emerged as a novel type of synaptic molecular interaction in the last decade. Several studies on LRR-GPCR interactions have revealed their critical role in synapse formation and in establishing synaptic properties. Among them, LRR-GPCR interactions between extracellular LRR fibronectin domain-containing family proteins (Elfn1 and Elfn2) and metabotropic glutamate receptors (mGluRs) are particularly interesting as they can affect a broad range of synapses through the modulation of signaling by glutamate, the principal excitatory transmitter in the mammalian central nervous system (CNS). Elfn-mGluR interactions have been investigated in hippocampal, cortical, and retinal synapses. Postsynaptic Elfn1 in the hippocampus and cerebral cortex mediates the tonic regulation of excitatory input onto somatostatin-positive interneurons (INs) through recruitment of presynaptic mGluR7. In the retina, presynaptic Elfn1 binds to mGluR6 and is necessary for synapse formation between rod photoreceptor cells and rod-bipolar cells. The repertoire of binding partners for Elfn1 and Elfn2 includes all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), and both Elfn1 and Elfn2 can alter mGluR-mediated signaling through trans-interaction. Importantly, both preclinical and clinical studies have provided support for the involvement of the Elfn1-mGluR7 interaction in attention-deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and epilepsy. In fact, Elfn1-mGluR7-associated disorders may reflect the altered function of somatostatin-positive interneuron inhibitory neural circuits, the mesolimbic and nigrostriatal dopaminergic pathway, and habenular circuits, highlighting the need for further investigation into this interaction.