ZIC2 Research Articles

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 Zic2kd/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.

The robe of transcription factors in genetics mechanism of congenital atrioventricular septal defect

Atrioventricular septal defect (AVSD)is a frequently diagnosed congenital heart malformation affecting the atrioventricular valves and septa. Heart transcription factors play fundamental roles in atrioventricular valves development and atrioventricular septa formation. Now cardiac transcription factors ( GATA4, NKX2. 5,TBX5 ,ZIC3)have been identified to involve in the pathogenesis of AVSD in humans. Their abnormal expression or function change may cause atrioventricular valve and septa developmental anomalies. Here we review advances in the understanding of the relationship between these transcription factors and AVSD. Key words: Transcription factors; Atrioventricular septal defect ; Genetics

Disruption of GLI3–ZIC3 interaction in the cadmium-induced omphalocele chick model

Administration of heavy metal cadmium (Cd) after 60-h incubation induces omphalocele spectrum in the chick embryo. Although previous studies have shown that the earliest detectable histological changes in the chick Cd model occurs commencing at 4-h post-treatment (4H). However, the molecular mechanism by which Cd acts in the critical period of early embryogenesis still remains unclear. Zic3, a Gli superfamily transcription factor, is expressed in somites and plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Gli3 is also expressed in somites and interacts with Zic3 physically and functionally. It has been reported that Gli3 homozygous double mutants display omphalocele. Zic3 mutant mice have also been known to result in omphalocele phenotype. We designed this study to test the hypothesis that Gli3 and Zic3 gene expression is downregulated during the critical period of very early embryogenesis in the Cd-induced omphalocele in the chick model. After 60-h incubation, chick embryos were exposed to either saline or 50μM cadmium and divided into two groups: control and Cd (n=24 for each group). Real-time reverse transcription polymerase chain reaction was performed to evaluate the relative mRNA expression levels of Gli3 and Zic3 in the Cd-induced omphalocele chick model. Differences between the two groups at each time point were analyzed statistically and the significance was accepted at p<0.05. Immunohistochemistry was also performed to evaluate the expression/distribution of those proteins in chick embryo. The relative mRNA expression level of Gli3 and Zic3 was significantly decreased in the Cd group at 4H when compared with controls (p<0.05). However, there were no significant differences at the other time points. At 4H, immunoreactivity of GLI3 and ZIC3 was also markedly decreased in Cd-treated embryos, whereas strong expression of them was seen in the somite in controls. We provide evidence, for the first time, that Gli3 and Zic3 gene expression is downregulated during the narrow window of very early embryogenesis in Cd chick model. Disruption of Gli3-Zic3 interaction in the critical period for ventral body wall formation may contribute to omphalocele phenotype in Cd chick model.

Expression and phylogenetic analysis of the zic gene family in the evolution and development of metazoans

Backgroundzic genes are members of the gli/glis/nkl/zic super-family of C2H2 zinc finger (ZF) transcription factors. Homologs of the zic family have been implicated in patterning neural and mesodermal tissues in bilaterians. Prior to this study, the origin of the metazoan zic gene family was unknown and expression of zic gene homologs during the development of early branching metazoans had not been investigated.ResultsPhylogenetic analyses of novel zic candidate genes identified a definitive zic homolog in the placozoan Trichoplax adhaerens, two gli/glis/nkl-like genes in the ctenophore Mnemiopsis leidyi, confirmed the presence of three gli/glis/nkl-like genes in Porifera, and confirmed the five previously identified zic genes in the cnidarian Nematostella vectensis. In the cnidarian N. vectensis, zic homologs are expressed in ectoderm and the gastrodermis (a bifunctional endomesoderm), in presumptive and developing tentacles, and in oral and sensory apical tuft ectoderm. The Capitella teleta zic homolog (Ct-zic) is detectable in a subset of the developing nervous system, the foregut, and the mesoderm associated with the segmentally repeated chaetae. Lastly, expression of gli and glis homologs in Mnemiopsis. leidyi is detected exclusively in neural cells in floor of the apical organ.ConclusionsBased on our analyses, we propose that the zic gene family arose in the common ancestor of the Placozoa, Cnidaria and Bilateria from a gli/glis/nkl-like gene and that both ZOC and ZF-NC domains evolved prior to cnidarian-bilaterian divergence. We also conclude that zic expression in neural ectoderm and developing neurons is pervasive throughout the Metazoa and likely evolved from neural expression of an ancestral gli/glis/nkl/zic gene. zic expression in bilaterian mesoderm may be related to the expression in the gastrodermis of a cnidarian-bilaterian common ancestor.

Expression of the zic1, zic2, zic3, and zic4 genes in early chick embryos

BackgroundThe zic genes encode a family of transcription factors with important roles during early development. Since little is known about zic gene expression in chick embryos, we have characterized the expression patterns of the zic1, zic2, zic3, and zic4 (zic1-4) genes during neurulation and somitogenesis.FindingsWe used in situ hybridization to analyze the expression patterns of the zic1-4 genes during early chick development (HH stages 7-19). The zic1-3 genes showed both overlapping and gene-specific expression patterns along the length of the dorsal neural tube and in the dorsal parts of the somites. In addition, unique expression domains of zic genes included: zic2 in the neural plate, periotic mesoderm and limb buds; zic3 in the paraxial mesoderm surrounding the neural plate, in presomitic mesoderm and in the most recently formed epithelial somites; zic2 and zic3 in developing eyes. zic4 expression was limited to dorsal fore- and midbrain regions and, unlike the expression of the zic1-3 genes, zic4 expression was not detected in the hindbrain and trunk. This was in contrast to more extensive zic4 expression in other vertebrates.ConclusionsThe zic1-3 genes were expressed in both overlapping and unique domains within the neural tube, somites and other ectoderm and mesoderm-derived structures in the future head and trunk. zic4 expression, however, was limited to dorso-anterior regions of the future brain. This is the first comprehensive study of zic1-4 gene expression in chick embryos during neurulation and somitogenesis.

The unfolding clinical spectrum of holoprosencephaly due to mutations in SHH, ZIC2, SIX3 and TGIF genes.

Holoprosencephaly is a severe malformation of the brain characterized by abnormal formation and separation of the developing central nervous system. The prevalence is 1:250 during early embryogenesis, the live-born prevalence is 1:16 000. The etiology of HPE is extremely heterogeneous and can be teratogenic or genetic. We screened four known HPE genes in a Dutch cohort of 86 non-syndromic HPE index cases, including 53 family members. We detected 21 mutations (24.4%), 3 in SHH, 9 in ZIC2 and 9 in SIX3. Eight mutations involved amino-acid substitutions, 7 ins/del mutations, 1 frame-shift, 3 identical poly-alanine tract expansions and 2 gene deletions. Pathogenicity of mutations was presumed based on de novo character, predicted non-functionality of mutated proteins, segregation of mutations with affected family-members or combinations of these features. Two mutations were reported previously. SNP array confirmed detected deletions; one spanning the ZIC2/ZIC5 genes (approx. 100 kb) the other a 1.45 Mb deletion including SIX2/SIX3 genes. The mutation percentage (24%) is comparable with previous reports, but we detected significantly less mutations in SHH: 3.5 vs 10.7% (P=0.043) and significantly more in SIX3: 10.5 vs 4.3% (P=0.018). For TGIF1 and ZIC2 mutation the rate was in conformity with earlier reports. About half of the mutations were de novo, one was a germ line mosaic. The familial mutations displayed extensive heterogeneity in clinical manifestation. Of seven familial index patients only two parental carriers showed minor HPE signs, five were completely asymptomatic. Therefore, each novel mutation should be considered as a risk factor for clinically manifest HPE, with the caveat of reduced clinical penetrance.

Zic1 transcription factor in bone: neural developmental protein regulates mechanotransduction in osteocytes

A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.

Expression of ZIC family genes in meningiomas and other brain tumors

BackgroundZic 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.MethodsWe 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.ResultsZIC1, 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.ConclusionsZIC1, 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.

Familial transposition of the great arteries caused by multiple mutations in laterality genes

BackgroundThe pathogenesis of transposition of the great arteries (TGA) is still largely unknown. In general, TGA is not associated with the more common genetic disorders nor with extracardiac anomalies, whereas...

BMP inhibition initiates neural induction via FGF signaling and Zic genes

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun-unlike the other BMP inhibitors used previously-can trigger conversion of Xenopus epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3 and foxD5a. Conversely, we found that zic1 can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.

The role of zebrafish zic genes in neural crest development

The role of zebrafish zic genes in neural crest development

A novel genetic mechanism regulates dorsolateral hinge-point formation during zebrafish cranial neurulation

During neurulation, vertebrate embryos form a neural tube (NT), the rudiment of the central nervous system. In mammals and birds, a key step in cranial NT morphogenesis is dorsolateral hinge-point (DLHP) bending, which requires an apical actomyosin network. The mechanism of DLHP formation is poorly understood, although several essential genes have been identified, among them Zic2, which encodes a zinc-finger transcription factor. We found that DLHP formation in the zebrafish midbrain also requires actomyosin and Zic function. Given this conservation, we used the zebrafish to study how genes encoding Zic proteins regulate DLHP formation. We demonstrate that the ventral zic2a expression border predicts DLHP position. Using morpholino (MO) knockdown, we show zic2a and zic5 are required for apical F-actin and active myosin II localization and junction integrity. Furthermore, myosin II activity can function upstream of junction integrity during DLHP formation, and canonical Wnt signaling, an activator of zic gene transcription, is necessary for apical active myosin II localization, junction integrity and DLHP formation. We conclude that zic genes act downstream of Wnt signaling to control cytoskeletal organization, and possibly adhesion, during neurulation. This study identifies zic2a and zic5 as crucial players in the genetic network linking patterned gene expression to morphogenetic changes during neurulation, and strengthens the utility of the zebrafish midbrain as a NT morphogenesis model.

Transcription factor-binding sites responsible for NO-dependent repression of the histone H2B promoter

New comparative genome hybridization technology on NotI-microarrays is presented (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-probes from tumor and normal genomic DNA with the principle of new DNA NotI-microarrays. Using this method 181 NotI linking loci from human chromosome 3 were analyzed in 200 malignant tumor samples from different organs: kidney, lung, breast, ovary, cervical, prostate. Most frequently (more than in 30%) aberrations--deletions, methylation,--were identified in NotI-sites located in MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4 genes, that suggests they probably are involved in cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results demonstrate perspective of using this method to solve some oncogenomic problems.

Use of NotI microarrays in analysis of epigenetic and structural changes in epithelial tumor genomes by the example of human chromosome 3

A new comparative genome hybridization technology using NotI microarrays is described (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-enriched probes from tumor and normal genomic DNA with radically new NotI microarrays. A total of 181 NotI-binding loci of human chromosome 3 were assayed in 200 human malignant tissue samples from various organs: kidney, lung, breast, ovary, cervix, and prostate. The most significant portion (above 30%) of aberrations (deletions and methylation) were detected in NotI sites located in the MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, and ZIC4 genes. This indicates that they may be associated with cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results confirm that the proposed method can contribute to cancer genomics.

Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: An updated map of candidate loci

Holoprosencephaly (HPE) is the most frequent malformation of the brain. To date, 12 different HPE loci and 8 HPE genes have been identified from recurrent chromosomal rearrangements or from the sequencing of genes from Nodal and SHH pathways. Our cohort of HPE patients presents a high genetic heterogeneity. Point mutations were found in SHH, ZIC2, SIX3, and TGIF genes in about 20% of cases (with 10% in SHH). Deletions in these same genes were found in 7.5% of the patients and 4.4% presented with other subtelomeric gain or losses. Consequently, the molecular basis of HPE remains unknown in 70% of our cohorts. To detect new HPE candidate genes, we used array-CGH to refine the previous karyotype based HPE loci map. We analyzed 111 HPE patients with high-performance Agilent oligonucleotidic arrays and found that 28 presented anomalies involving known or new potential HPE loci located on different chromosomes but with poor redundancy. This study showed an impressive rate of 19 patients among 111 with de novo chromosomal anomalies giving evidence that microrearrangements could be a major molecular mechanism in HPE. Additionally, this study opens new insights on HPE candidate genes identification giving an updated HPE candidate loci map.

Brpf1, a subunit of the MOZ histone acetyl transferase complex, maintains expression of anterior and posterior Hox genes for proper patterning of craniofacial and caudal skeletons

The epigenetic mechanism involving chromatin modification plays a critical role in the maintenance of the expression of Hox genes. Here, we characterize a mutant of the medaka fish, named biaxial symmetries ( bis), in which brpf1, a subunit of the MOZ histone acetyl transferase (HAT) complex, is mutated. The bis mutant displayed patterning defects both in the anterior–posterior axis of the craniofacial skeleton and the dorsal–ventral axis of the caudal one. In the anterior region, the bis mutant exhibited craniofacial cartilage homeosis. The expression of Hox genes was decreased in the pharyngeal arches, suggesting that the pharyngeal segmental identities were altered in the bis mutant. In the posterior region, the bis mutant exhibited abnormal patterning of the caudal skeleton, which ectopically formed at the dorsal side of the caudal fin. The expression of Zic genes was decreased at the posterior region, suggesting that the dorsal–ventral axis formation of the posterior trunk was disrupted in the bis mutant. We also found that the MOZ-deficient mice exhibited an abnormal patterning of their craniofacial and cervical skeletons and a decrease of Hox transcripts. We propose a common role of the MOZ HAT complex in vertebrates, a complex which is required for the proper patterning for skeletal development.

The full spectrum of holoprosencephaly-associated mutations within theZIC2gene in humans predicts loss-of-function as the predominant disease mechanism

Mutations of the ZIC2 transcription factor gene are among the most common heterozygous variations detected in holoprosencephaly (HPE) patients, a patient group who lack critical midline forebrain specification due to defective embryonic signaling during development. Recent studies indicate that complete deficiency of the related murine Zic2 transcription factor can also be a contributing factor to variable midline deficiencies, presenting during mid-gastrulation, that could explain similar forebrain anomalies in this model system. Here we collect and summarize all available mutations in the human ZIC2 gene detected in HPE patients (21 published and 62 novel). Our analysis corroborates this mechanism proposed in mice by predicting loss-of-function as the likely pathogenetic mechanism common to most, if not all, of these mutations in HPE.

Twelve new patients with 13q deletion syndrome: Genotype–phenotype analyses in progress

13q deletion is characterized by a wide phenotypic spectrum resulting from a partial deletion of the long arm of chromosome 13. The main clinical features are mental retardation, growth retardation, craniofacial dysmorphy and various congenital defects. Only one recent Italian study was aimed at determining genotype–phenotype correlations among 13q deletions from a group of mainly live born children, using array-CGH and FISH. In order to improve the molecular characterization of 13q monosomy, 12 new patients (9 foetuses and 3 children) were collected based on a cohort of holoprosencephaly (HPE) linked to ZIC2 gene deletion and/or patients with 13q deletion diagnosed by standard karyotype. First, quantitative gene screening using MLPA (Multiplex Ligation dependent Probe Amplification) was performed to look for ZIC2 gene deletion and then, CGH array analysis was carried out using the Agilent Human Genome CGH microarray 4 × 44K (Agilent Technologies, Santa Clara, USA). All the foetuses had severe cerebral midline malformations associated with a deletion including the ZIC2 gene. We report one patient with Steinfeld phenotype linked to this chromosomal anomaly, and suggest that some of the associations between cerebral midline malformation and limb defects might be related to 13q deletion. Further candidate genes are suspected to explain the malformations associated with cerebral anomalies in the hypothesis of a contiguous gene syndrome: SPRY2 in 13q31.1 is implicated in lens cell proliferation and differentiation for congenital cataract; GPC5 in 13q32 is mainly expressed in the mesenchyme of the developing limb bud for upper limb anomalies.

Zic2 -associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation

The putative transcription factor ZIC2 is associated with a defect of forebrain development, known as Holoprosencephaly (HPE), in humans and mouse, yet the mechanism by which aberrant ZIC2 function causes classical HPE is unexplained. The zinc finger domain of all mammalian Zic genes is highly homologous with that of the Gli genes, which are transcriptional mediators of Shh signalling. Mutations in Shh and many other Hh pathway members cause HPE and it has been proposed that Zic2 acts within the Shh pathway to cause HPE. We have investigated the embryological cause of Zic2-associated HPE and the relationship between Zic2 and the Shh pathway using mouse genetics. We show that Zic2 does not interact with Shh to produce HPE. Moreover, molecular defects that are able to account for the HPE phenotype are present in Zic2 mutants before the onset of Shh signalling. Mutation of Zic2 causes HPE via a transient defect in the function of the organizer region at mid-gastrulation which causes an arrest in the development of the prechordal plate (PCP), a structure required for forebrain midline morphogenesis. The analysis provides genetic evidence that Zic2 functions during organizer formation and that the PCP develops via a multi-step process.

Zic genes act directly downstream of FGF signalling and BMP inhibition to promote a pre-neural state in Xenopus

zic genes act directly downstream of FGF signalling and BMP inhibition to promote a pre-neural state in Xenopus