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.

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.

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.

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.

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.

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.

Dysregulation of erythropoiesis and altered erythroblastic NMDA receptor-mediated calcium influx in Lrfn2-deficient mice.

LRFN2 encodes a synaptic adhesion-like molecule that physically interacts with N-methyl-D-aspartate (NMDA) receptor 1 and its scaffold proteins. Previous studies in humans and mice have demonstrated its genetic association with neurodevelopmental disorders such as learning deficiency and autism. In this study, we showed that Lrfn2-deficient (KO) mice exhibit abnormalities of erythropoietic systems due to altered NMDA receptor function. In mature Lrfn2 KO male mice, peripheral blood tests showed multilineage abnormalities, including normocytic erythrocythemia, and reduced platelet volume. Colony forming unit assay using bone marrow cells revealed decreases in the counts of erythrocyte progenitors (CFU-E) as well as granulocytes and monocyte progenitors (CFU-GM). Whole bone marrow cell staining showed that serum erythropoietin (EPO) level was decreased and EPO receptor-like immunoreactivity was increased. Flow cytometry analysis of bone marrow cells revealed increased early erythroblast count and increased transferrin receptor expression in late erythroblasts. Further, we found that late erythroblasts in Lrfn2 KO exhibited defective NMDA receptor-mediated calcium influx, which was inhibited by the NMDA receptor antagonist MK801. These results indicate that Lrfn2 has biphasic roles in hematopoiesis and is associated with the functional integrity of NMDA receptors in hematopoietic cells. Furthermore, taken together with previous studies that showed the involvement of NMDA receptors in hematopoiesis, the results of this study indicate that Lrfn2 may regulate erythropoiesis through its regulatory activity on NMDA receptors.

Characterization of the tandem CWCH2 sequence motif: a hallmark of inter-zinc finger interactions.

BACKGROUND: The C2H2 zinc finger (ZF) domain is widely conserved among eukaryotic proteins. In Zic/Gli/Zap1 C2H2 ZF proteins, the two N-terminal ZFs form a single structural unit by sharing a hydrophobic core. This structural unit defines a new motif comprised of two tryptophan side chains at the center of the hydrophobic core. Because each tryptophan residue is located between the two cysteine residues of the C2H2 motif, we have named this structure the tandem CWCH2 (tCWCH2) motif. RESULTS: Here, we characterized 587 tCWCH2-containing genes using data derived from public databases. We categorized genes into 11 classes including Zic/Gli/Glis, Arid2/Rsc9, PacC, Mizf, Aebp2, Zap1/ZafA, Fungl, Zfp106, Twincl, Clr1, and Fungl-4ZF, based on sequence similarity, domain organization, and functional similarities. tCWCH2 motifs are mostly found in organisms belonging to the Opisthokonta (metazoa, fungi, and choanoflagellates) and Amoebozoa (amoeba, Dictyostelium discoideum). By comparison, the C2H2 ZF motif is distributed widely among the eukaryotes. The structure and organization of the tCWCH2 motif, its phylogenetic distribution, and molecular phylogenetic analysis suggest that prototypical tCWCH2 genes existed in the Opisthokonta ancestor. Within-group or between-group comparisons of the tCWCH2 amino acid sequence identified three additional sequence features (site-specific amino acid frequencies, longer linker sequence between two C2H2 ZFs, and frequent extra-sequences within C2H2 ZF motifs). CONCLUSION: These features suggest that the tCWCH2 motif is a specialized motif involved in inter-zinc finger interactions.

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain.

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left-right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1-4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin alpha1/alpha6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS.

Expression of ZIC family genes in meningiomas and other brain tumors.

BACKGROUND: Zic zinc finger proteins are present in the developing rodent meninges and are required for cell proliferation and differentiation of meningeal progenitors. Although human ZIC genes are known to be molecular markers for medulloblastomas, their expression in meningioma has not been addressed to date. METHODS: We examined the mRNA and protein expression of human ZIC1, ZIC2, ZIC3, ZIC4 and ZIC5 genes in meningiomas in comparison to other brain tumors, using RT-PCR, analysis of published microarray data, and immunostaining. RESULTS: ZIC1, ZIC2 and ZIC5 transcript levels in meningiomas were higher than those in whole brain or normal dura mater, whereas all five ZIC genes were abundantly expressed in medulloblastomas. The expression level of ZIC1 in public microarray data was greater in meningiomas classified as World Health Organization Grade II (atypical) than those classified as Grade I (benign). Immunoscreening using anti-ZIC antibodies revealed that 23 out of 23 meningioma cases were ZIC1/2/3/5-immunopositive. By comparison, nuclear staining by the anti-ZIC4 antibody was not observed in any meningioma case, but was strongly detected in all four medulloblastomas. ZIC-positive meningiomas included meningothelial, fibrous, transitional, and psammomatous histological subtypes. In normal meninges, ZIC-like immunoreactivities were detected in vimentin-expressing arachnoid cells both in human and mouse. CONCLUSIONS: ZIC1, ZIC2, and ZIC5 are novel molecular markers for meningiomas whereas ZIC4 expression is highly selective for medulloblastomas. The pattern of ZIC expression in both of these tumor types may reflect the properties of the tissues from which the tumors are derived.

Link between the causative genes of holoprosencephaly: Zic2 directly regulates Tgif1 expression.

One of the causal genes for holoprosencephaly (HPE) is ZIC2 (HPE5). It belongs to the zinc finger protein of the cerebellum (Zic) family of genes that share a C2H2-type zinc finger domain, similar to the GLI family of genes. In order to clarify the role of Zic2 in gene regulation, we searched for its direct target genes using chromatin immunoprecipitation (ChIP). We identified TGIF1 (HPE4), another holoprosencephaly-causative gene in humans. We identified Zic2-binding sites (ZBS) on the 5' flanking region of Tgif1 by in vitro DNA binding assays. ZBS were essential for Zic2-dependent transcriptional activation in reporter gene assays. Zic2 showed a higher affinity to ZBS than GLI-binding sequences. Zic2-binding to the cis-regulatory element near the Tgif1 promoter may be involved in the mechanism underlying forebrain development and incidences of HPE.

Elucidation of penetrance variability of a ZIC3 mutation in a family with complex heart defects and functional analysis of ZIC3 mutations in the first zinc finger domain.

We studied a series of 42 cases of transposition of the great arteries (TGA), a complex heart defect (CHD) that is two times more prevalent in males than in females. A mutation in the X chromosome at the ZIC3 gene was found in two affected siblings (one male, one female) and their unaffected mother. A second factor, skewed X-inactivation pattern explained the discrepancy between the daughter/mother phenotype. In this family, the missense mutation (p.W255G) was found in the first zinc finger of ZIC3, a domain that is relatively specific to each of the five human ZIC genes. It was tested further along with two other mutations of this domain (p.C253S and p.H286R). In transfected 3T3 cells, mutants p.W255G and p.H286R expressed lower protein levels, and an increased protein degradation (p.W255G only). Moreover, mutants p.C253S and p.W255G had a decreased transcription activation of the TK-luciferase reporter gene. Nuclear translocation of the three ZIC3 mutants varied considerably depending on the experimental models. Finally, p.W255G and p.H286R showed diminished activities for both left-right axis disturbance and neural crest induction in Xenopus embryos. These results suggest that mutations in the first zinc finger of ZIC3 mildly affect several functions of the protein.

Autism-like behaviours and enhanced memory formation and synaptic plasticity in Lrfn2/SALM1-deficient mice.

Lrfn2/SALM1 is a PSD-95-interacting synapse adhesion molecule, and human LRFN2 is associated with learning disabilities. However its role in higher brain function and underlying mechanisms remain unknown. Here, we show that Lrfn2 knockout mice exhibit autism-like behavioural abnormalities, including social withdrawal, decreased vocal communications, increased stereotyped activities and prepulse inhibition deficits, together with enhanced learning and memory. In the hippocampus, the levels of synaptic PSD-95 and GluA1 are decreased. The synapses are structurally and functionally immature with spindle shaped spines, smaller postsynaptic densities, reduced AMPA/NMDA ratio, and enhanced LTP. In vitro experiments reveal that synaptic surface expression of AMPAR depends on the direct interaction between Lrfn2 and PSD-95. Furthermore, we detect functionally defective LRFN2 missense mutations in autism and schizophrenia patients. Together, these findings indicate that Lrfn2/LRFN2 serve as core components of excitatory synapse maturation and maintenance, and their dysfunction causes immature/silent synapses with pathophysiological state.

Elfn1 recruits presynaptic mGluR7 in trans and its loss results in seizures.

GABAergic interneurons are highly heterogeneous, and much is unknown about the specification and functional roles of their neural circuits. Here we show that a transinteraction of Elfn1 and mGluR7 controls targeted interneuron synapse development and that loss of Elfn1 results in hyperactivity and sensory-triggered epileptic seizures in mice. Elfn1 protein increases during postnatal development and localizes to postsynaptic sites of somatostatin-containing interneurons (SOM-INs) in the hippocampal CA1 stratum oriens and dentate gyrus (DG) hilus. Elfn1 knockout (KO) mice have deficits in mGluR7 recruitment to synaptic sites on SOM-INs, and presynaptic plasticity is impaired at these synapses. In patients with epilepsy and attention deficit hyperactivity disorder (ADHD), we find damaging missense mutations of ELFN1 that are clustered in the carboxy-terminal region required for mGluR7 recruitment. These results reveal a novel mechanism for interneuron subtype-specific neural circuit establishment and define a common basis bridging neurological disorders.

Gli protein nuclear localization signal.

Drosophila cubitus interruptus (Ci) and its vertebrate homologues, the glioblastoma (Gli) protein family, are the transcription factors belonging to the metazoan Gli/Glis/Zic ZF protein superfamily that shares similar five tandemly repeated C2H2-type zinc finger (ZF) motifs. Nuclear transport of Gli/Ci proteins is regulated by hedgehog (Hh) signaling and is an essential part of the Hh signal transduction pathway. Gli/Ci proteins possess a nuclear localization signal (NLS) and a nuclear export signal (NES), both of which are key signatures for controlling nucleocytoplasmic shuttling. The NLS of the Gli/Ci proteins has been mapped to the fifth ZF domain and its C-terminal side. It contains two clusters of basic residues (classical bipartite-type), which are conserved in metazoan Gli/Ci homologues, but which partially deviate from the intra-ZF domain NLSs in the Glis and Zic proteins. Recently, Importin α3 was identified as a nuclear transport protein for Ci. When we modeled the 3D structure of the Gli NLS-Importin α complex, the two basic clusters were predicted to fit in the two binding interfaces of Importin α. The mechanisms controlling the function of NLSs and NESs involve the elimination of the NES by Hh signaling-dependent protein cleavage in the Ci and the Gli3 proteins, and the phosphorylation of a threonine residue close to the NLS in Gli1. Both processes depend on the activity of protein kinase A, which has a critical role in Hh signaling in fly wing discs. In addition, the Roadkill protein, a substrate recognition component of E3 ubiquitin ligase, competes with the Ci protein to interact with Importin α3 resulting in inhibition of Ci protein nuclear import.

Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients.

ZIC2 is a causal gene for holoprosencephaly and encodes a zinc-finger-type transcriptional regulator. We characterized Zic2(kd/+) mice with a moderate (40%) reduction in Zic2 expression. Zic2(kd/+) mice showed increased locomotor activity in novel environments, cognitive and sensorimotor gating dysfunctions, and social behavioral abnormalities. Zic2(kd/+) brain involved enlargement of the lateral ventricle, thinning of the cerebral cortex and corpus callosum, and decreased number of cholinergic neurons in the basal forebrain. Because these features are reminiscent of schizophrenia, we examined ZIC2 variant-carrying allele frequencies in schizophrenia patients and in controls in the Japanese population. Among three novel missense mutations in ZIC2, R409P was only found in schizophrenia patients, and was located in a strongly conserved position of the zinc finger domain. Mouse Zic2 with the corresponding mutation showed lowered transcription-activating capacity and had impaired target DNA-binding and co-factor-binding capacities. These results warrant further study of ZIC2 in the pathogenesis of schizophrenia.

Mouse Zic5 deficiency results in neural tube defects and hypoplasia of cephalic neural crest derivatives.

Zic family genes encode zinc finger proteins, which are homologues of the Drosophila pair-rule gene odd-paired. In the present study, we characterized the fifth member of the mouse Zic family gene, mouse Zic5. Zic5 is located near Zic2, which is responsible for human brain malformation syndrome (holoprosencephaly, or HPE). In embryonic stages, Zic5 was expressed in dorsal part of neural tissues and limbs. Expression of Zic5 overlapped with those of other Zic genes, most closely with Zic2, but was not identical. Targeted disruption of Zic5 resulted in insufficient neural tube closure at the rostral end, similar to that seen in Zic2 mutant mice. In addition, the Zic5-deficient mice exhibited malformation of neural-crest-derived facial bones, especially the mandible, which had not been observed in other Zic family mutants. During the embryonic stages, there were delays in the development of the first branchial arch and extension of the trigeminal and facial nerves. Neural crest marker staining revealed fewer neural crest cells in the dorsal cephalic region of the mutant embryos without significant changes in their migration. When mouse Zic5 was overexpressed in Xenopus embryos, expression of a neural crest marker was enhanced. These findings suggested that Zic5 is involved in the generation of neural crest tissue in mouse development. ZIC5 is also located close to ZIC2 in humans, and deletions of 13q32, where ZIC2 is located, lead to congenital brain and digit malformations known as the "13q32 deletion syndrome". Based on both their similar expression pattern in mouse embryos and the malformations observed in Zic5-deficient mutant mice, human ZIC5 might be involved in the deletion syndrome.

Myogenic repressor I-mfa interferes with the function of Zic family proteins.

Zinc finger proteins belonging to the Zic family control several developmental processes such as patterning of the axial skeleton. Here we mapped the transcriptional regulatory domains in Zic2 protein and identified a protein which specifically binds to one of them. In the mapping experiments, an amino-terminal region was identified as transcriptional regulatory domains. A search for proteins binding to the amino terminal domain of Zic2 revealed that inhibitor of MyoD family (I-mfa) protein, which has been identified as a repressor of myogenic helix-loop-helix class transcription factors, can physically interact with the amino terminal domain. When Zic1-3 and I-mfa proteins were co-expressed in cultured cells, nuclear import of the Zic proteins was inhibited. Consequently, I-mfa inhibited transcriptional activation by the Zic proteins in cultured cells. These results suggest that the physical and functional interaction between Zic and I-mfa proteins can play a role in the vertebrate development.