The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Stephanie M Ware - One of the best experts on this subject based on the ideXlab platform.
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loss of ZIC3 impairs planar cell polarity leading to abnormal left right signaling heart defects and neural tube defects
Human Molecular Genetics, 2021Co-Authors: Helen M Bellchambers, Stephanie M WareAbstract:Loss of function of ZIC3 causes heterotaxy (OMIM #306955), a disorder characterized by organ laterality defects including complex heart defects. Studies using ZIC3 mutant mice have demonstrated that loss of ZIC3 causes heterotaxy due to defects in establishment of left-right (LR) signaling, but the mechanistic basis for these defects remains unknown. Here, we demonstrate ZIC3 null mice undergo cilia positioning defects at the embryonic node consistent with impaired planar cell polarity (PCP). Cell-based assays demonstrate that ZIC3 must enter the nucleus to regulate PCP and identify multiple critical ZIC3 domains required for regulation of PCP signaling. Furthermore, we show that ZIC3 displays a genetic interaction with the PCP membrane protein Vangl2 and the PCP effector genes Rac1 and Daam1 resulting in increased frequency and severity of neural tube and heart defects. Gene and protein expression analyses indicate that ZIC3 null embryos display disrupted expression of PCP components and reduced phosphorylation of the core PCP protein DVL2 at the time of LR axis determination. These results demonstrate that ZIC3 interacts with PCP signaling during early development, identifying a novel role for this transcription factor, and adding additional evidence about the importance of PCP function for normal LR patterning and subsequent heart development.
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ZIC3 in heterotaxy
Advances in Experimental Medicine and Biology, 2018Co-Authors: Helen M Bellchambers, Stephanie M WareAbstract:Mutation of ZIC3 causes X-linked heterotaxy, a syndrome in which the laterality of internal organs is disrupted. Analysis of model organisms and gene expression during early development suggests ZIC3-related heterotaxy occurs due to defects at the earliest stage of left-right axis formation. Although there are data to support abnormalities of the node and cilia as underlying causes, it is unclear at the molecular level why loss of ZIC3 function causes such these defects. ZIC3 has putative roles in a number of developmental signalling pathways that have distinct roles in establishing the left-right axis. This complicates the understanding of the mechanistic basis of ZIC3 in early development and left-right patterning. Here we summarise our current understanding of ZIC3 function and describe the potential role ZIC3 plays in important signalling pathways and their links to heterotaxy.
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genetic and functional analyses of ZIC3 variants in congenital heart disease
Human Mutation, 2014Co-Authors: Jason Cowan, Muhammad Tariq, Stephanie M WareAbstract:Mutations in zinc-finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc-finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N- and C-terminal mutations has, however, prevented similar analyses in these regions. Notably, an N-terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk-based counseling. Eight of 11 were novel, including 5 N-terminal variants. Subsequent functional analyses included four additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc-finger variants, but not for downstream or in-frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains.
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heterotaxy spectrum heart defects in ZIC3 hypomorphic mice
Pediatric Research, 2013Co-Authors: Allison Haaning, Malgorzata E. Quinn, Stephanie M WareAbstract:Mutations in Zinc Finger Protein of the Cerebellum 3 (ZIC3) cause X-linked heterotaxy and isolated cardiovascular malformations. Recent data suggest a potential cell-autonomous role for ZIC3 in myocardium via regulation of Nppa and Tbx5. We sought to develop a hypomorphic ZIC3 mouse to model human heterotaxy and investigate developmental mechanisms underlying variability in cardiac phenotypes. ZIC3 hypomorphic mice were created by targeted insertion of a neomycin cassette and investigated by gross, histologic, and molecular methods. Low-level ZIC3 expression is sufficient for partial rescue of viability as compared with ZIC3 null mice. Concordance of early left–right molecular marker abnormalities and later anatomic abnormalities suggests that the primary effect of ZIC3 in heart development occurs during left–right patterning. Cardiac-specific gene expression of Nppa (atrial natriuretic factor) and Tbx5 marked the proper morphological locations in the heart regardless of looping abnormalities. ZIC3 hypomorphic mice are useful models to investigate the variable cardiac defects resulting from a single genetic defect. Low-level ZIC3 expression rescues the left pulmonary isomerism identified in ZIC3 null embryos. Our data do not support a direct role for ZIC3 in the myocardium via regulation of Nppa and Tbx5 and suggest that the primary effect of ZIC3 on cardiac development occurs during left–right patterning.
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ZIC3 is required in the migrating primitive streak for node morphogenesis and left right patterning
Human Molecular Genetics, 2013Co-Authors: Mardi J Sutherland, Allison M. Haaning, Malgorzata E. Quinn, Shuyun Wang, Stephanie M WareAbstract:In humans, loss-of-function mutations in ZIC3 cause isolated cardiovascular malformations and X-linked heterotaxy, a disorder with abnormal left-right asymmetry of organs. ZIC3 null mice recapitulate the human heterotaxy phenotype but also have early gastrulation defects, axial patterning defects and neural tube defects complicating an assessment of the role of ZIC3 in cardiac development. ZIC3 is expressed ubiquitously during critical stages of left-right patterning but its later expression in the developing heart remains controversial and the molecular mechanism(s) by which it causes heterotaxy are unknown. To define the temporal and spatial requirements, for ZIC3 in left-right patterning, we generated conditional ZIC3 mice and ZIC3-LacZ-BAC reporter mice. The latter provide compelling evidence that ZIC3 is expressed in the mouse node and absent in the heart. Conditional deletion using T-Cre identifies a requirement for ZIC3 in the primitive streak and migrating mesoderm for proper left-right patterning and cardiac development. In contrast, ZIC3 is not required in heart progenitors or the cardiac compartment. In addition, the data demonstrate abnormal node morphogenesis in ZIC3 null mice and identify similar node dysplasia when ZIC3 was specifically deleted from the migrating mesoderm and primitive streak. These results define the temporal and spatial requirements for ZIC3 in node morphogenesis, left-right patterning and cardiac development and suggest the possibility that a requirement for ZIC3 in node ultrastructure underlies its role in heterotaxy and laterality disorders.
Jun Aruga - One of the best experts on this subject based on the ideXlab platform.
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an evolutionarily conserved mesodermal enhancer in vertebrate ZIC3
Scientific Reports, 2018Co-Authors: Yuri S Odaka, Jun Aruga, Atsushi Toyoda, Takahide TohmondaAbstract:ZIC3 encodes a zinc finger protein essential for the development of meso-ectodermal tissues. In mammals, ZIC3 has important roles in the development of neural tube, axial skeletons, left-right body axis, and in maintaining pluripotency of ES cells. Here we characterized cis-regulatory elements required for ZIC3 expression. Enhancer activities of human-chicken-conserved noncoding sequences around Zic1 and ZIC3 were screened using chick whole-embryo electroporation. We identified enhancers for meso-ectodermal tissues. Among them, a mesodermal enhancer (ZIC3-ME) in distant 3' flanking showed robust enhancement of reporter gene expression in the mesodermal tissue of chicken and mouse embryos, and was required for mesodermal ZIC3 expression in mice. ZIC3-ME minimal core region is included in the DNase hypersensitive region of ES cells, mesoderm, and neural progenitors, and was bound by T (Brachyury), Eomes, Lef1, Nanog, Oct4, and Zic2. ZIC3-ME is derived from an ancestral sequence shared with a sequence encoding a mitochondrial enzyme. These results indicate that ZIC3-ME is an integrated cis-regulatory element essential for the proper expression of ZIC3 in vertebrates, serving as a hub for a gene regulatory network including ZIC3.
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zic1 function in normal cerebellar development and human developmental pathology
Advances in Experimental Medicine and Biology, 2018Co-Authors: Jun Aruga, Kathleen J MillenAbstract: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 TGFβ 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.
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Link between the causative genes of holoprosencephaly: Zic2 directly regulates Tgif1 expression
Nature Publishing Group, 2018Co-Authors: Akira Ishiguro, Minoru Hatayama, Maky I. Otsuka, Jun ArugaAbstract:Abstract 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
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RESEARCH ARTICLE Open Access Expression of ZIC family genes in meningiomas and other brain tumors
2013Co-Authors: Jun Aruga, Yayoi Nozaki, Minoru Hatayama, Yuri S Odaka, Naoki YokotaAbstract: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. ZICpositive meningiomas included meningothelial, fibrous, transitional, and psammomatous histological subtypes. In normal meninges, ZIC-like immunoreactivities were detected in vimentin-expressing arachnoid cells both in huma
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xenopus ZIC3 controls notochord and organizer development through suppression of the wnt β catenin signaling pathway
Developmental Biology, 2012Co-Authors: Takahiko J Fujimi, Minoru Hatayama, Jun ArugaAbstract:ZIC3 controls neuroectodermal differentiation and left-right patterning in Xenopus laevis embryos. Here we demonstrate that ZIC3 can suppress Wnt/β-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/β-catenin signaling, we subsequently examined the functional interaction between ZIC3 and Wnt signaling. Co-injection of Xenopus Zic RNAs and β-catenin RNA with a reporter responsive to the Wnt/β-catenin cascade indicated that Zic1, Zic2, ZIC3, Zic4, and Zic5 can all suppress β-catenin-mediated transcriptional activation. In addition, co-injection of ZIC3 RNA inhibited the secondary axis formation caused by ventral-side injection of β-catenin RNA in Xenopus embryos. ZIC3-mediated Wnt/β-catenin signal suppression required the nuclear localization of ZIC3, and involved the reduction of β-catenin nuclear transport and enhancement of β-catenin degradation. Furthermore, ZIC3 co-precipitated with Tcf1 (a β-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/β-catenin signaling.
Katsuhiko Mikoshiba - One of the best experts on this subject based on the ideXlab platform.
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functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain
Human Molecular Genetics, 2008Co-Authors: Minoru Hatayama, Katsuhiko Mikoshiba, T Tomizawa, Kumiko Sakaikato, Patrice Bouvagnet, Shingo Kose, Naoko Imamoto, Shigeyuki Yokoyama, Naoko Utsunomiyatate, T KigawaAbstract: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 α1/α6 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.
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zic deficiency in the cortical marginal zone and meninges results in cortical lamination defects resembling those in type ii lissencephaly
The Journal of Neuroscience, 2008Co-Authors: Takashi Inoue, Katsuhiko Mikoshiba, Masaharu Ogawa, Jun ArugaAbstract:The formation of the highly organized cortical structure depends on the production and correct placement of the appropriate number and types of neurons. The Zic family of zinc-finger transcription factors plays essential roles in regulating the proliferation and differentiation of neuronal progenitors in the medial forebrain and the cerebellum. Examination of the expression of Zic genes demonstrated that Zic1, Zic2, and ZIC3 were expressed by the progenitor cells in the septum and cortical hem, the sites of generation of the Cajal-Retzius (CR) cells. Immunohistochemical studies have revealed that Zic proteins were abundantly expressed in the meningeal cells and that the majority of the CR cells distributed in the medial and dorsal cortex also expressed Zic proteins in the mid-late embryonic and postnatal cortical marginal zones. During embryonic cortical development, Zic1/ZIC3 double-mutant and hypomorphic Zic2 mutant mice showed a reduction in the number of CR cells in the rostral cortex, whereas the cell number remained unaffected in the caudal cortex. These mutants also showed mislocalization of the CR cells and cortical lamination defects, resembling the changes noted in type II (cobblestone) lissencephaly, throughout the brain. In the Zic1/3 mutant, reduced proliferation of the meningeal cells was observed before the thinner and disrupted organization of the pial basement membrane (BM) with reduced expression of the BM components and the meningeal cell-derived secretory factor. These defects correlated with the changes in the end feet morphology of the radial glial cells. These findings indicate that the Zic genes play critical roles in cortical development through regulating the proliferation of meningeal cells and the pial BM assembly.
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zic2 and ZIC3 synergistically control neurulation and segmentation of paraxial mesoderm in mouse embryo
Developmental Biology, 2007Co-Authors: Takashi Inoue, Katsuhiko Mikoshiba, Jun ArugaAbstract:Abstract Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1–5) have been reported. Despite the partly overlapping expression profiles of these genes, mouse mutants for each Zic show distinct phenotypes. To uncover possible redundant roles, we characterized Zic2/ZIC3 compound mutant mice. Zic2 and ZIC3 are both expressed in presomitic mesoderm, forming and newly generated somites with differential spatiotemporal accentuation. Mice heterozygous for the hypomorphic Zic2 allele together with null ZIC3 allele generally showed severe malformations of the axial skeleton, including asymmetric or rostro-caudally bridged vertebrae, and reduction of the number of caudal vertebral bones, that are not obvious in single mutants. These defects were preceded by perturbed somitic marker expression, and reduced paraxial mesoderm progenitors in the primitive streak. These results suggest that Zic2 and ZIC3 cooperatively control the segmentation of paraxial mesoderm at multiple stages. In addition to the segmentation abnormality, the compound mutant also showed neural tube defects that ran the entire rostro-caudal extent (craniorachischisis), suggesting that neurulation is another developmental process where Zic2 and ZIC3 have redundant functions.
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zic1 and ZIC3 regulate medial forebrain development through expansion of neuronal progenitors
The Journal of Neuroscience, 2007Co-Authors: Takashi Inoue, Katsuhiko Mikoshiba, Maya Ota, Miyuki Ogawa, Jun ArugaAbstract:The medial telencephalon is a source of neurons that follow distinct tangential trajectories of migration to various structures such as the cerebral cortex, striatum, and olfactory bulb. In the present study, we characterized the forebrain anomalies in Zic1/ZIC3 compound mutant mice. Zic1 and ZIC3 were strongly expressed in the medial structures, including the septum, medial cerebral cortex, and choroid plexus. Mice homozygous for the Zic1 mutant allele together with the null ZIC3 allele showed medial forebrain defects, which were not obvious in either Zic1 or ZIC3 single mutants. Absence of both Zic1 and ZIC3 caused hypoplasia of the hippocampus, septum, and olfactory bulb. Analysis of the cell cycle revealed that the cell cycle exit rate was increased in the septa of double mutants. Misexpression of ZIC3 in the ventricular layer of the cerebral cortex inhibited neuronal differentiation. These results indicated that both Zic1 and ZIC3 function in maintaining neural precursor cells in an undifferentiated state. The functions of these genes may be essential to increasing neural cell numbers regionally in the medial telencephalon and to proper mediolateral patterning of the telencephalon.
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locomotor and oculomotor impairment associated with cerebellar dysgenesis in ZIC3 deficient bent tail mutant mice
European Journal of Neuroscience, 2004Co-Authors: Jun Aruga, Barbara Franke, Katsuhiko Mikoshiba, Miyuki Ogawa, Hiroo Ogura, Fumihiro Shutoh, Soichi NagaoAbstract:We examined the adult neural phenotypes of the Bent tail mutant mouse. The Bent tail mutant mouse was recently shown to lack a submicroscopic part of the X chromosome containing the ZIC3 gene, which encodes a zinc-finger protein controlling vertebrate neural development. While nearly one-fourth of hemizygous Bent tail (Bn/Y, ZIC3-deficient) mice developed neural tube defects in their midbrain and hindbrain region, the other Bn/Y mice showed apparently normal behaviour in a C57BL/6 genetic background. A battery of behavioural and eye movement tests revealed impaired spontaneous locomotor activity, reduction of muscle tone and impairments of vestibuloocular and optokinetic eye movements in these mice. Morphological examination of the mutant brain showed a significant reduction in the cell numbers in the cerebellar anterior lobe and paraflocculus-flocculus complex. Our results indicate that the cerebellar dysgenesis characterized by subregional hypoplasia affects the locomotor activity, muscle tone and eye movement control of the mice. These findings may have some clinical implications in relation to disorders characterized by cerebellar dysgenesis, such as Joubert syndrome.
John W. Belmont - One of the best experts on this subject based on the ideXlab platform.
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ZIC3 is required in the extra cardiac perinodal region of the lateral plate mesoderm for left right patterning and heart development
Human Molecular Genetics, 2013Co-Authors: Zhengxin Jiang, Lirong Zhu, Timothy C Slesnick, Robia G Pautler, Monica J Justice, John W. BelmontAbstract:Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of ZIC3 demonstrates an early role for ZIC3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in ZIC3(null) mice and the relatively broad expression pattern of ZIC3 suggest its important roles in multiple developmental processes. Here, we report that ZIC3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in ZIC3 deficiency occur not because ZIC3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for ZIC3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of ZIC3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of ZIC3 with signaling pathways involved in the early establishment of laterality.
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differential requirement of ZIC3 function in cardiac development and x linked heterotaxy
Developmental Biology, 2011Co-Authors: Zhengxin Jiang, Lirong Zhu, Robia G Pautler, Monica J Justice, John W. BelmontAbstract:Heterotaxy, contributing to ~5% of congenial heart defects (CHD), arises from abnormal left-right patterning. Mutations of ZIC3 gene (Zinc finger protein of cerebellum 3) are associated with human Xlinked heterotaxy. A mouse model with targeted disruption of ZIC3 exhibited ~75% early lethality, and recapitulated the phenotype seen in human patients. However, it is not knownwhether ZIC3 is required in a single developmental field or whether it has pleiotropic roles in multiple developmental processes, and the detailed mechanism remains elusive. To address these questions, we generated a conditional allele of the ZIC3 gene by flanking its 1st exon with loxP sites. Sox2-cre, Wnt1-cre and T-cre lines were used to delete ZIC3 in epiblast, neural crest and mesoderm, respectively. Deletion of ZIC3 in epiblast and mesoderm, but not in neural crest, led to ~50% early lethality. Examination of epiblast conditional embryos by microscopy revealed multiple CNS and neural tube defects similar to the null embryos. But these defects were not found in mesoderm or neural crest conditional embryos, suggesting that ZIC3's function in CNS development likely remains intact in these mutants. MRI scanning of ZIC3 epiblast and mesoderm conditional embryos also uncovered multiple heterotaxy related visceral abnormalities. Gene expression analysis by microarray in the hearts of embryos at 15.5 dpc revealed a similar expression pattern between ZIC3 epiblast conditional and null males, which was significantly different from control males. Perturbed expression of several cardiac genes and direct targets of ZIC3 suggested that Notch, BMP and TGF-β signaling might be affected, and requires further investigation.
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identification of a novel role of ZIC3 in regulating cardiac development
Human Molecular Genetics, 2007Co-Authors: Lirong Zhu, Karine G. Harutyunyan, Jian Lan Peng, Jun Wang, Robert J Schwartz, John W. BelmontAbstract:Mutations in ZIC3 cause X-linked heterotaxy, a disorder characterized by abnormal lateralization of normally asymmetric thoracic and abdominal organs. Animal models demonstrate an early role for ZIC3 in embryonic left-right (LR) patterning. ZIC3 mutations have also been described in patients with isolated cardiovascular malformations. We wished to address the hypothesis that ZIC3 has plieotropic effects in development and may regulate cardiac development independent of its role in LR patterning. We observed significantly reduced expression of several markers of cardiac lineage commitment in ZIC3(null/y) embryonic stem cells including atrial natriuretic factor (ANF), Nkx2.5 and Tbx5. Likewise, ANF expression-a molecular marker of trabecular myocardium and a direct target of multiple cardiac-specific transcription factors-was severely reduced in E9.5 ZIC3 null hearts. Trabecular myocardium was reduced in these embryos. This finding was similar to that observed in embryos with cardiac-specific ablation of serum response factor (SRF), a direct transcriptional regulator of ANF expression. While ZIC3 by itself had no effect on the ANF promoter, it could bind to and inhibit a cardiac alpha-actin promoter through its zinc finger domains. We observed that ZIC3 could function as a coactivator of SRF on both cardiac alpha-actin and ANF promoters. The zinc fingers of ZIC3 and the mcm1, agamous deficiens SRF (MADS) box motif of SRF were found to mediate their physical and functional interactions. These findings reveal a novel role of ZIC3 in regulating cardiac gene expression and may explain, in part, the association of ZIC3 mutation with cardiovascular malformations.
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craniofacial skeletal and cardiac defects associated with altered embryonic murine ZIC3 expression following targeted insertion of a pgk neo cassette
Frontiers in Bioscience, 2007Co-Authors: Lirong Zhu, Karine G. Harutyunyan, Monica J Justice, Jian Lan Peng, Monica D Garcia, John W. BelmontAbstract:Mutation in ZIC3 (OMIM #306955), a zinc finger transcription factor, causes heterotaxy (situs ambiguus) or isolated congenital heart defects in humans. Mice bearing a null mutation in ZIC3 have left-right patterning defects with associated cardiovascular, vertebra/rib, and central nervous system malformations. Although XZIC3 is thought to play a critical role in Xenopus neural crest development, no defects in tissues derived from neural crest are apparent in adult ZIC3(null) mice. In this study we have characterized the effect of a PGK-neo cassette insertion 5' of the ZIC3 locus. The ZIC3 transcript in this new allele is up-regulated in ES cells and in E9.0 embryos, but no ectopic expression was detected. Unlike the ZIC3(null) mutation in which only 20% of mutant animals survive to adulthood, there was no evidence of excess fetal death caused by the ZIC3(neo) allele. ZIC3(neo) mutant mice exhibited hemifacial microsomia, asymmetric low set ears, axial skeletal defects, kyphosis and scoliosis; a combination of defects which mimics Goldenhar Syndrome. Some ZIC3(neo) mice had evidence of left-right axis patterning defects, but cardiac malformation was much less common than in the ZIC3(null) mutants. A six-week old hemizygous mouse was found to have thoraco-cervical ectopia cordis, an extremely rare congenital malformation in humans and for which there is no precedent in a mouse model.
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Characterization of the interactions of human ZIC3 mutants with GLI3.
Human mutation, 2007Co-Authors: Lirong Zhu, Guisheng Zhou, Suzanne Poole, John W. BelmontAbstract:ZIC3, a GLI superfamily transcription factor, is involved in establishing normal embryonic left–right patterning. Multiple abnormalities in the central nervous system (CNS) and axial skeleton have also been observed in mice bearing a ZIC3 null allele, mice with a ZIC3 overexpression allele, and the majority of patients carrying ZIC3 mutations. Previous studies indicate that ZIC3 protein can bind to the GLI consensus binding site (GLIBS) and physically interact with GLI3, a transcription factor involved in multiple aspects of neural and skeletal development. We investigated in vitro interactions of ZIC3 with GLI3 and the effect of ZIC3 mutations identified in patients with either heterotaxy or isolated cardiovascular malformations. Electrophoresis mobility shift assay (EMSA) revealed that all five intact zinc finger (ZF) domains were necessary for binding of ZIC3 to GLIBS. Inclusion of GLIBS upstream of a basal TK promoter had no effect on the activation of the promoter by ZIC3 alone, but it enhanced the synergistic activation of ZIC3 and GLI3. Wild-type (WT) ZIC3 showed specific binding to GLI3 in GST-pull-down assays. Nonsense and frameshift ZIC3 mutants lacking one or more of the zinc finger domains did not physically interact with GST-GLI3; however, two missense mutants c.1213A>G (p.K405E, fifth ZF domain), and c.649C>G (p.P217A, conserved N-terminal domain) retained binding. Luciferase reporter assays indicated that both p.P217A and p.K405E mutants also retained coactivation with GLI3 of reporter gene expression activity, while all the GLI3-nonbinding ZIC3 mutants lacked this activity. Interestingly, no CNS or skeletal abnormalities were observed in patients bearing the p.P217A or p.K405E mutations. Hum Mutat 29(1), 99–105, 2008. Published 2007 Wiley-Liss, Inc.
Lirong Zhu - One of the best experts on this subject based on the ideXlab platform.
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ZIC3 is required in the extra cardiac perinodal region of the lateral plate mesoderm for left right patterning and heart development
Human Molecular Genetics, 2013Co-Authors: Zhengxin Jiang, Lirong Zhu, Timothy C Slesnick, Robia G Pautler, Monica J Justice, John W. BelmontAbstract:Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of ZIC3 demonstrates an early role for ZIC3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in ZIC3(null) mice and the relatively broad expression pattern of ZIC3 suggest its important roles in multiple developmental processes. Here, we report that ZIC3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in ZIC3 deficiency occur not because ZIC3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for ZIC3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of ZIC3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of ZIC3 with signaling pathways involved in the early establishment of laterality.
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differential requirement of ZIC3 function in cardiac development and x linked heterotaxy
Developmental Biology, 2011Co-Authors: Zhengxin Jiang, Lirong Zhu, Robia G Pautler, Monica J Justice, John W. BelmontAbstract:Heterotaxy, contributing to ~5% of congenial heart defects (CHD), arises from abnormal left-right patterning. Mutations of ZIC3 gene (Zinc finger protein of cerebellum 3) are associated with human Xlinked heterotaxy. A mouse model with targeted disruption of ZIC3 exhibited ~75% early lethality, and recapitulated the phenotype seen in human patients. However, it is not knownwhether ZIC3 is required in a single developmental field or whether it has pleiotropic roles in multiple developmental processes, and the detailed mechanism remains elusive. To address these questions, we generated a conditional allele of the ZIC3 gene by flanking its 1st exon with loxP sites. Sox2-cre, Wnt1-cre and T-cre lines were used to delete ZIC3 in epiblast, neural crest and mesoderm, respectively. Deletion of ZIC3 in epiblast and mesoderm, but not in neural crest, led to ~50% early lethality. Examination of epiblast conditional embryos by microscopy revealed multiple CNS and neural tube defects similar to the null embryos. But these defects were not found in mesoderm or neural crest conditional embryos, suggesting that ZIC3's function in CNS development likely remains intact in these mutants. MRI scanning of ZIC3 epiblast and mesoderm conditional embryos also uncovered multiple heterotaxy related visceral abnormalities. Gene expression analysis by microarray in the hearts of embryos at 15.5 dpc revealed a similar expression pattern between ZIC3 epiblast conditional and null males, which was significantly different from control males. Perturbed expression of several cardiac genes and direct targets of ZIC3 suggested that Notch, BMP and TGF-β signaling might be affected, and requires further investigation.
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identification of a novel role of ZIC3 in regulating cardiac development
Human Molecular Genetics, 2007Co-Authors: Lirong Zhu, Karine G. Harutyunyan, Jian Lan Peng, Jun Wang, Robert J Schwartz, John W. BelmontAbstract:Mutations in ZIC3 cause X-linked heterotaxy, a disorder characterized by abnormal lateralization of normally asymmetric thoracic and abdominal organs. Animal models demonstrate an early role for ZIC3 in embryonic left-right (LR) patterning. ZIC3 mutations have also been described in patients with isolated cardiovascular malformations. We wished to address the hypothesis that ZIC3 has plieotropic effects in development and may regulate cardiac development independent of its role in LR patterning. We observed significantly reduced expression of several markers of cardiac lineage commitment in ZIC3(null/y) embryonic stem cells including atrial natriuretic factor (ANF), Nkx2.5 and Tbx5. Likewise, ANF expression-a molecular marker of trabecular myocardium and a direct target of multiple cardiac-specific transcription factors-was severely reduced in E9.5 ZIC3 null hearts. Trabecular myocardium was reduced in these embryos. This finding was similar to that observed in embryos with cardiac-specific ablation of serum response factor (SRF), a direct transcriptional regulator of ANF expression. While ZIC3 by itself had no effect on the ANF promoter, it could bind to and inhibit a cardiac alpha-actin promoter through its zinc finger domains. We observed that ZIC3 could function as a coactivator of SRF on both cardiac alpha-actin and ANF promoters. The zinc fingers of ZIC3 and the mcm1, agamous deficiens SRF (MADS) box motif of SRF were found to mediate their physical and functional interactions. These findings reveal a novel role of ZIC3 in regulating cardiac gene expression and may explain, in part, the association of ZIC3 mutation with cardiovascular malformations.
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craniofacial skeletal and cardiac defects associated with altered embryonic murine ZIC3 expression following targeted insertion of a pgk neo cassette
Frontiers in Bioscience, 2007Co-Authors: Lirong Zhu, Karine G. Harutyunyan, Monica J Justice, Jian Lan Peng, Monica D Garcia, John W. BelmontAbstract:Mutation in ZIC3 (OMIM #306955), a zinc finger transcription factor, causes heterotaxy (situs ambiguus) or isolated congenital heart defects in humans. Mice bearing a null mutation in ZIC3 have left-right patterning defects with associated cardiovascular, vertebra/rib, and central nervous system malformations. Although XZIC3 is thought to play a critical role in Xenopus neural crest development, no defects in tissues derived from neural crest are apparent in adult ZIC3(null) mice. In this study we have characterized the effect of a PGK-neo cassette insertion 5' of the ZIC3 locus. The ZIC3 transcript in this new allele is up-regulated in ES cells and in E9.0 embryos, but no ectopic expression was detected. Unlike the ZIC3(null) mutation in which only 20% of mutant animals survive to adulthood, there was no evidence of excess fetal death caused by the ZIC3(neo) allele. ZIC3(neo) mutant mice exhibited hemifacial microsomia, asymmetric low set ears, axial skeletal defects, kyphosis and scoliosis; a combination of defects which mimics Goldenhar Syndrome. Some ZIC3(neo) mice had evidence of left-right axis patterning defects, but cardiac malformation was much less common than in the ZIC3(null) mutants. A six-week old hemizygous mouse was found to have thoraco-cervical ectopia cordis, an extremely rare congenital malformation in humans and for which there is no precedent in a mouse model.
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Characterization of the interactions of human ZIC3 mutants with GLI3.
Human mutation, 2007Co-Authors: Lirong Zhu, Guisheng Zhou, Suzanne Poole, John W. BelmontAbstract:ZIC3, a GLI superfamily transcription factor, is involved in establishing normal embryonic left–right patterning. Multiple abnormalities in the central nervous system (CNS) and axial skeleton have also been observed in mice bearing a ZIC3 null allele, mice with a ZIC3 overexpression allele, and the majority of patients carrying ZIC3 mutations. Previous studies indicate that ZIC3 protein can bind to the GLI consensus binding site (GLIBS) and physically interact with GLI3, a transcription factor involved in multiple aspects of neural and skeletal development. We investigated in vitro interactions of ZIC3 with GLI3 and the effect of ZIC3 mutations identified in patients with either heterotaxy or isolated cardiovascular malformations. Electrophoresis mobility shift assay (EMSA) revealed that all five intact zinc finger (ZF) domains were necessary for binding of ZIC3 to GLIBS. Inclusion of GLIBS upstream of a basal TK promoter had no effect on the activation of the promoter by ZIC3 alone, but it enhanced the synergistic activation of ZIC3 and GLI3. Wild-type (WT) ZIC3 showed specific binding to GLI3 in GST-pull-down assays. Nonsense and frameshift ZIC3 mutants lacking one or more of the zinc finger domains did not physically interact with GST-GLI3; however, two missense mutants c.1213A>G (p.K405E, fifth ZF domain), and c.649C>G (p.P217A, conserved N-terminal domain) retained binding. Luciferase reporter assays indicated that both p.P217A and p.K405E mutants also retained coactivation with GLI3 of reporter gene expression activity, while all the GLI3-nonbinding ZIC3 mutants lacked this activity. Interestingly, no CNS or skeletal abnormalities were observed in patients bearing the p.P217A or p.K405E mutations. Hum Mutat 29(1), 99–105, 2008. Published 2007 Wiley-Liss, Inc.
