Zic3 Expression Research Articles

Transcriptional expression of ZICs as an independent indicator of survival in gliomas

The functional significance of the zinc-finger of the cerebellum (ZIC) gene family in gliomas remains to be elucidated. Clinical data from patients with gliomas, containing expression levels of ZIC genes, were extracted from CCLE, GEPIA2 and The Human Protein Atlas (HPA). Univariate survival analysis adjusted by Cox regression via OncoLnc was used to determine the prognostic significance of ZIC expression. We used cBioPortal to explore the correlation between gene mutations and overall survival (OS). ZIC expression was found to be related to immune cell infiltration in gliomas via TIMER analysis. GO term and KEGG pathway enrichment analyzes were performed with Metascape. PPI networks were constructed using STRING. The expression levels of ZIC1/3/4/5 in gliomas were significantly different from those in normal samples. High expression levels of ZIC1/5 were associated with poor OS in brain low-grade glioma (LGG) patients, while low ZIC3 expression combined was related to favorable OS in glioblastoma multiforme (GBM). ZIC alterations were associated with poor prognosis in LGG patients and related to favorable prognosis in GBM patients. We observed that the expression of ZICs was related to immune cell infiltration in glioma patients. ZICs were enriched in several pathways and biological processes involving Neuroactive ligand-receptor interaction (hsa04080). The PPI network revealed that some proteins coexpressed with ZICs played a role in the pathogenesis of gliomas. Differences in the expression levels of ZIC genes could provide a significant marker for predicting prognosis in gliomas.

An Evolutionarily Conserved Mesodermal Enhancer in Vertebrate Zic3

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.

Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells.

Vascular endothelial cell (EC) function depends on appropriate organ-specific molecular and cellular specializations. To explore genomic mechanisms that control this specialization, we have analyzed and compared the transcriptome, accessible chromatin, and DNA methylome landscapes from mouse brain, liver, lung, and kidney ECs. Analysis of transcription factor (TF) gene expression and TF motifs at candidate cis-regulatory elements reveals both shared and organ-specific EC regulatory networks. In the embryo, only those ECs that are adjacent to or within the central nervous system (CNS) exhibit canonical Wnt signaling, which correlates precisely with blood-brain barrier (BBB) differentiation and Zic3 expression. In the early postnatal brain, single-cell RNA-seq of purified ECs reveals (1) close relationships between veins and mitotic cells and between arteries and tip cells, (2) a division of capillary ECs into vein-like and artery-like classes, and (3) new endothelial subtype markers, including new validated tip cell markers.

Abstract 506: Modulation of ZIC3 Expression in Pluripotent Stem Cells Using CRISPR-interference to Model Heterotaxy

Failure of proper left-right patterning during embryogenesis is an underlying mechanism of congenital heart disease (CHD). Heterotaxy (HTX) is a complex laterality syndrome in which multiple organs, including the heart, are abnormally arranged and mal-rotated. Loss-of-function mutations in ZIC3 , a zinc finger transcription factor within the GLI superfamily, have been associated with X-linked HTX. The mechanism behind how ZIC3 causes laterality defects remains unknown. To investigate this relationship, we developed a method to directly explore ZIC3 using induced pluripotent stem cells (iPSCs) at a molecular, cellular, and functional level through utilization of CRISPR interference (CRISPRi). A stable CRISPRi transgenic line was generated through lentiviral transduced vectors encoding inducible endonuclease-dead Cas9 and an sgRNA targeting the ZIC3 promoter. As ZIC3 is necessary for iPSC pluripotency, this inducible system allowed for a controllable knockdown while maintaining parent cell line pluripotency. RT-PCR confirmed that doxycycline induced a robust knockdown of ZIC3 expression over 72 hours with a fold change of 0.12 compared to non-induced. RNA-Seq was used to generate a gene expression profile of ZIC3 loss. This showed that 446 genes were significantly differentially expressed (adjusted p < 0.01), and of those, 163 genes had an absolute fold change of greater than 1.5. Of the top 15 most significantly differentially expressed genes, 47% (7 of 15) are associated with the cytoskeleton, including gamma actin 1 and myosin light chain 9. These results were confirmed by RT-PCR. Confocal imaging revealed significant changes to the actin cytoskeleton in response to ZIC3 knockdown, with a notable reduction in filamentous actin. Current ongoing studies are focusing on the impact of ZIC3 knockdown on the structural and mechanical differences in differentiated cardiomyocytes. In summary, we have developed a method by which to explore CHD using iPSCs in order to better understand the genetic mechanisms of heterotaxy, and in future studies, other forms of CHD.

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association.

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.

Heterotaxy-spectrum heart defects in Zic3 hypomorphic mice.

BackgroundMutations in 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.MethodsZic3 hypomorphic mice were created by targeted insertion of a neomycin cassette and investigated by gross, histologic, and molecular methodsResultsLow level Zic3 expression is sufficient for partial rescue of viability as compared to Zic3 null mice. Concordance of early left-right molecular marker abnormalities and later anatomic abnormalities suggests the primary effect of Zic3 in heart development occurs during left-right patterning. Cardiac specific gene expression of Nppa (ANF) and Tbx5 marked the proper morphological locations in the heart regardless of looping abnormalities.ConclusionsZic3 hypomorphic mice are a useful model 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 the primary effect of Zic3 on cardiac development occurs during left-right patterning.

Calcium signalling during neural induction in Xenopus laevis embryos.

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.

Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference-induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

Craniofacial, skeletal, and cardiac defects associated with altered embryonic murine Zic3 expression following targeted insertion of a PGK-NEO cassette

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.

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.

Transcriptional regulation of Zic3 by heterodimeric AP-1(c-Jun/c-Fos) during Xenopus development

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.

Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation

The Zic genes are the vertebrate homologues of the Drosophila Odd-paired gene. Mutations in two of these genes are associated with human congenital genetic disorders. Mutation of human and mouse Zic2 is associated with holoprosencephaly which is caused by a defect of ventral forebrain development and mutation of human and mouse Zic3 is associated with a X-linked heterotaxy syndrome that results from a failure of left–right axis formation. The embryological role of the Zic genes in these disorders is not well understood. Here we show that both of these genes are expressed prior to and throughout gastrulation. The genes show some broad similarities in their expression domains. Both genes however are also uniquely expressed in some tissues and these unique domains correlate with regions that potentially play a role in the aetiology of the respective genetic disorders. During primitive streak stages Zic2 is expressed transiently and uniquely in the node and the head process mesendoderm. The head process is known to be required for the establishment or maintenance of the ventral forebrain, which is the region disrupted in holoprosencephaly. Zic3 is not expressed in the node during primitive streak stages but is expressed in and around the node beginning from the head fold stages of development. This expression of Zic3 correlates well with the first steps in the establishment of the left–right axis. We also examined the expression of the closely related gene, Zic1, and did not detect any transcripts in gastrulation stage embryos.

Disruption of gradient expression of Zic3 resulted in abnormal intra-retinal axon projection.

The targeting of retinal ganglion axons toward the optic disc is the first step in axon pathfinding in the visual system. The molecular mechanisms involved in guiding the retinal axons to project towards the optic disc are not well understood. We report that a gene encoding a zinc-finger transcription factor, Zic3, is expressed in a periphery-high and center-low gradient in the retina at the stages of active axon extension inside the retina. The gradient expression of Zic3 recedes towards the periphery over the course of development, correlating with the progression of retinal cell differentiation and axonogenesis. Disruption of gradient expression of Zic3 by retroviral overexpression resulted in mis-targeting of retinal axons and some axons misrouted to the sub-retinal space at the photoreceptor side of the retina. Misexpression of Zic3 did not affect neurogenesis or differentiation inside the retina, or grossly alter retinal lamination. By stripe assay, we show that misexpression of Zic3 may induce the expression of an inhibitory factor to the retinal axons. Zic3 appears to play a role in intra-retinal axon targeting, possibly through regulation of the expression of specific downstream genes involved in axon guidance.

Calcium transients triggered by planar signals induce the expression of ZIC3 gene during neural induction in Xenopus

In intact Xenopus embryos, an increase in intracellular Ca 2+ in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca 2+ increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca 2+ signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca 2+ transients, and neural induction to be explored. We have imaged Ca 2+ dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca 2+ transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type Ca 2+ channels. The accumulated Ca 2+ pattern correlated with the expression of the early neural precursor gene, Zic3. When the transients were blocked with pharmacological agents, the level of Zic3 expression was dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca 2+ -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca 2+ transients over time may play a role in controlling the expression pattern of Zic3.

Expression of ZIC genes in the development of the chick inner ear and nervous system.

ZIC genes, vertebrate homologues of the Drosophila pair-rule gene odd-paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up-regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear.

Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left-right asymmetry.

The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left-right (L-R) axis in mouse and zebrafish. However, the role of Brachyury in L-R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L-R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L-R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant-negative type Xbra (Xbra-EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal-related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra-GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L-R specification may be the direct regulation of Zic3 expression.

Zic Gene expression marks anteroposterior pattern in the presumptive neurectoderm of the zebrafish gastrula.

Anteroposterior (A/P) patterning of the vertebrate neurectoderm begins early during development. In vitro explant assays have been used to show that an A/P subdivision of the zebrafish neurectoderm is in place by early gastrulation. However, no direct markers of this subdivision had been described. We report isolation of two members of the zic gene family, zic2 and zic3, which mark distinct regions in the developing nervous system. zic3 is expressed in early gastrula embryos in a posterior domain, directly demonstrating that A/P pattern is present in the future neurectoderm by this stage. Analysis of mutants in genes encoding bone morphogenetic proteins (BMPs) shows that dorsal restriction of zic3 expression requires intact BMP function, and that posterior restriction of zic3 expression is regulated independently of BMP signaling. These data show an early subdivision of the A/P axis in the presumptive neurectoderm.

Zic3 is involved in the left-right specification of the Xenopus embryo.

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.

Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.