ZIC2 Mutations Research Articles

A novel ZIC3 mutation in a Chinese family with heterotaxy and multiple types of congenital heart defect.

A couple was referred for prenatal counseling at gestational age 21weeks for revealed situs inversus with levocardia (HP:0,031,592), atrial situs inversus (HP:0,011,538), congenitally corrected transposition of the great arteries (ccTGA, HP:0,011,540) with ventricular septal defect (HP:0,001,629) and right aortic arch (HP:0,012,020). The couple had multiple prior pregnancies with complex congenital heart defects (CHDs, HP:0,001,627) in male fetuses. Testing was initiated to identify any fetal abnormality. The genetic cause of the observed prenatal defects was investigated. Whole exome sequencing and Sanger sequencing were performed on DNA extracted from parental blood samples and skeletal muscle tissue of the aborted fetuses. A pathogenic hemizygous missense variant in ZIC3 (NM_003413.4: c.895T>C) associated with X-linked heterotaxy-1 (HTX1) and multiple types of congenital heart defect-1 (CHTD1) (OMIM #306955) was identified, which was inherited from the mother. ZIC3 encodes a highly conserved zinc-finger protein that is highly correlated with CHDs. The present study of a Han Chinese family with CHDs expands the mutation spectrum of ZIC3 and provides further evidence that ZIC3 plays important roles in CHDs.

Deregulation of the Epithelial-to-Mesenchymal Transition Process Underlies Zic2-Linked Holoprosencephaly

Mutations in the Zic2 gene cause holoprosencephaly type 5, but the mechanisms that translate Zic2 mutations into this devastating pathology remain unclear. Here, we report that Zic2 is expressed in a few epiblast cells during gastrulation to become transiently upregulated in the primitive streak. Later, during neurulation, Zic2 is expressed in neural crest cells and downregulated as they delaminate from the neural tube. In combination with transcriptomic data from mutant embryos, chromatin occupancy profiles in gastrula and neural crest cells reveal that Zic2 regulates a large number of genes associated with the Wnt, cadherin and TGF-β pathways. Functional experiments in chick and mice demonstrate that Zic2 blocks the activation of the Wnt canonical pathway at the time that induces the TGF-β pathway to trigger cell delamination. All these experiments together demonstrate that Zic2 initiates epithelial-to-mesenchymal transition at early developmental stages.

The Double Bubble Sign: Duodenal Atresia and Associated Genetic Etiologies

Background: The “double bubble” sign is an ultrasonographic finding that commonly represents duodenal atresia and is associated with trisomy 21. Objectives: We sought to evaluate the positive predictive value of a prenatally identified double bubble sign for duodenal atresia and the genetic etiologies associated with it. Methods: We examined a retrospective cohort with prenatal double bubble sign between January 1, 2008, and June 30, 2017. Postnatal diagnoses were determined by review of operative reports and additional postnatal evaluation including cytogenetic analysis, molecular analysis, and/or clinical genetic evaluation. Results: All live births at our institution with a prenatal double bubble sign had confirmed duodenal atresia. Additional anatomic anomalies and/or genetic abnormalities were identified in 62% of cases. Out of 21 cases, 6 had trisomy 21. Of the remaining 15 cases, 8 were nonisolated duodenal atresia, 3 of which had a heterotaxy syndrome. In the 7 isolated cases, 1 likely pathogenic chromosomal microdeletion was identified. Conclusions: Prenatal double bubble sign is a reliable predictor of duodenal atresia. In addition to trisomy 21, heterotaxy may be encountered. ZIC3 mutations as well as microdeletion of 4q22.3 may be underlying genetic etiologies to be considered in the diagnostic evaluation of a prenatal double bubble sign.

Nasal fistula, epidermal cyst and hypernatremia in a girl presenting holoprosencephaly due to a rare ZIC2 point mutation

Holoprosencephaly is the most common brain malformation in humans and it is a complex genetic disorder. We report on a patient with holoprosencephaly caused by a rare ZIC2 mutation presenting a bifid nose associated with a nasal fistula and an epidermal cyst, besides hypernatremia. The patient was a 1 year and 4 months old girl that developed an important neuropsychomotor delay. Currently, she uses a wheelchair to move around and only emits sounds. Computed tomography (CT) scan revealed a semilobar holoprosencephaly and a Dandy-Walker variant. Head magnetic resonance imaging also disclosed corpus callosum agenesis and prefrontal subarachnoid space enlargement. On physical examination at 1 year and 4 months of age, we verified growth retardation, microcephaly, bilateral epicantic fold, upslanting palpebral fissures, bifid nose, and limbs spasticity secondary to hypertonia. Later, she began to present hypernatremia; however, its precise cause was not identified. At 6 years and 10 months of age, a nasal fistula was suspected. Facial CT scan showed an epidermal cyst at cartilaginous portion of the nasal septum. High resolution GTG-Banding karyotype was normal. However, molecular analysis through direct sequencing technique showed a mutation at regulatory region of the ZIC2 gene: c.1599*954T > A, a genetic variation previously described only in a Brazilian patient. Our patient presented findings still not reported in literature among patients with holoprosencephaly, including those with ZIC2 mutations. Thus, the spectrum of abnormalities associated to ZIC2 mutations may be broader and include other defects as those observed in our patient.

A novel ZIC3 gene mutation identified in patients with heterotaxy and congenital heart disease

Heterotaxy syndrome (HTX) is characterized by left-right (LR) asymmetry disturbances associated with severe heart malformations. However, the exact genetic cause of HTX pathogenesis remains unclear. The aim of this study was to investigate the pathogenic mechanism underlying heterotaxy syndrome. Targeted next-generation sequencing (NGS) was performed for twenty-two candidate genes correlated with LR axis development in sixty-six HTX patients from unrelated families. Variants were filtered from databases and predicted in silico using prediction programs. A total of twenty-one potential disease-causing variants were identified in seven genes. Next, we used Sanger sequencing to confirm the identified variants in the family pedigree and found a novel hemizygous mutation (c.890G > T, p.C297F) in the ZIC3 gene in a male patient that was inherited from his mother, who was a carrier. The results of functional indicated that this ZIC3 mutation decreases transcriptional activity, affects the affinity of the GLI-binding site and results in aberrant cellular localization in transfected cells. Moreover, morpholino-knockdown experiments in zebrafish demonstrated that zic3 mutant mRNA failed to rescue the abnormal phenotype, suggesting a role for the novel ZIC3 mutation in heterotaxy syndrome.

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.

A Requirement for Zic2 in the Regulation of Nodal Expression Underlies the Establishment of Left-Sided Identity

ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.

Spina bifida-predisposing heterozygous mutations in Planar Cell Polarity genes and Zic2 reduce bone mass in young mice

Fractures are a common comorbidity in children with the neural tube defect (NTD) spina bifida. Mutations in the Wnt/planar cell polarity (PCP) pathway contribute to NTDs in humans and mice, but whether this pathway independently determines bone mass is poorly understood. Here, we first confirmed that core Wnt/PCP components are expressed in osteoblasts and osteoclasts in vitro. In vivo, we performed detailed µCT comparisons of bone structure in tibiae from young male mice heterozygous for NTD-associated mutations versus WT littermates. PCP signalling disruption caused by Vangl2 (Vangl2Lp/+) or Celsr1 (Celsr1Crsh/+) mutations significantly reduced trabecular bone mass and distal tibial cortical thickness. NTD-associated mutations in non-PCP transcription factors were also investigated. Pax3 mutation (Pax3Sp2H/+) had minimal effects on bone mass. Zic2 mutation (Zic2Ku/+) significantly altered the position of the tibia/fibula junction and diminished cortical bone in the proximal tibia. Beyond these genes, we bioinformatically documented the known extent of shared genetic networks between NTDs and bone properties. 46 genes involved in neural tube closure are annotated with bone-related ontologies. These findings document shared genetic networks between spina bifida risk and bone structure, including PCP components and Zic2. Genetic variants which predispose to spina bifida may therefore independently diminish bone mass.

ZIC2 in Holoprosencephaly.

The ZIC2 transcription factor is one of the most commonly mutated genes in Holoprosencephaly (HPE) probands. HPE is a severe congenital defect of forebrain development which occurs when the cerebral hemispheres fail to separate during the early stages of organogenesis and is typically associated with mispatterning of the embryonic midline. Recent study of genotype-phenotype correlations in HPE cases has defined distinctive features of ZIC2-associated HPE presentation and genetics, revealing that ZIC2 mutation does not produce the craniofacial abnormalities generally thought to characterise HPE but leads to a range of non-forebrain phenotypes. Furthermore, the studies confirm the extent of ZIC2 allelic heterogeneity and that pathogenic variants of ZIC2 are associated with both classic and middle interhemispheric variant (MIHV) HPE which arise from defective ventral and dorsal forebrain patterning, respectively. An allelic series of mouse mutants has helped to delineate the cellular and molecular mechanisms by which one gene leads to defects in these related but distinct embryological processes.

Zic Family Proteins in Emerging Biomedical Studies.

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

Zic2 mutation causes holoprosencephaly via disruption of NODAL signalling.

The ZIC2 transcription factor is one of the genes most commonly mutated in Holoprosencephaly (HPE) probands. Studies in cultured cell lines and mice have shown a loss of ZIC2 function is the pathogenic mechanism but the molecular details of this ZIC2 requirement remain elusive. HPE arises when signals that direct morphological and fate changes in the developing brain and facial primordia are not sent or received. One critical signal is sent from the prechordal plate (PrCP) which develops beneath the ventral forebrain. An intact NODAL signal transduction pathway and functional ZIC2 are both required for PrCP establishment. We now show that ZIC2 acts downstream of the NODAL signal during PrCP development. ZIC2 physically interacts with SMAD2 and SMAD3, the receptor activated proteins that control transcription in a NODAL dependent manner. Together SMAD3 and ZIC2 regulate FOXA2 transcription in cultured cells and Zic2 also controls the foxA2 expression during Xenopus development. Variant forms of the ZIC2 protein, associated with HPE in man or mouse, are deficient in their ability to influence SMAD-dependent transcription. These findings reveal a new mechanism of NODAL signal transduction in the mammalian node and provide the first molecular explanation of how ZIC2 loss-of-function precipitates HPE.

Monozygotic twins with de novo ZIC2 gene mutations discordant for the type of holoprosencephaly.

Middle interhemispheric variant of holoprosencephaly (MIH) is a rare brain malformation; hemispheric fusion does not occur at the rostral forebrain, but rather across the posterior frontal region. Barkovich and Quint1 first described and proposed MIH as part of the holoprosencephaly (HPE) spectrum in 1993. Although classic HPE and MIH share several similarities, they are related to different embryological mechanisms: classic HPE is caused by a defect in the formation of the embryonic floor plate, whereas MIH occurs after a disturbance to the roof plate formation.2 The gene ZIC2 is important for the differentiation of the roof plate in the dorsal midline of the neural tube of the developing embryo. In humans, ZIC2 mutations have been identified in 3%–4% of HPE cases, including individuals with MIH, thus confirming that MIH is a variant of HPE.3

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.

Zic2 Controls the Migration of Specific Neuronal Populations in the Developing Forebrain.

Although the phenotype of Zic2 mutant individuals was reported more than 10 years ago, until now, the main function of this transcription factor during early development has not been precisely defined. Here, we reveal a previously unknown role for Zic2 in the migration of forebrain neurons such as Cajal-Retzius cells, interneurons moving to the ventral lateral geniculate nucleus, and neocortical cells going to the amygdala. We believe that the role of this transcription factor in certain populations of migratory cells contributes to defects in cortical layering and hypocellularity in the ventral LGN and amygdala and will contribute to our understanding of the devastating phenotypes associated with Zic2 mutations in both humans and mice.

Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association

Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor-associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat shock protein 90-related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum-stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle’s loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or CAKUT in VACTERL association.

Rare nasal cleft in a patient with holoprosencephaly due to a mutation in the ZIC2 gene

Holoprosencephaly (HPE) is a spectrum of midline malformations of the prosencephalon generally reflected in a continuum of midline facial anomalies. Patients with mutation in the ZIC2 gene usually present a normal or mildly dysmorphic face associated with a severe brain malformation. Here we present a rare unilateral nasal cleft (Tessier cleft n. 1) with holoprosencephaly in a patient with a ZIC2 mutation. The male newborn presented with alobar HPE, microcephaly, ocular hypertelorism, upslanting palpebral fissures, a bulky nose with a left paramedian alar cleft. Mutational screening for HPE genes revealed the occurrence of a frameshift mutation in the ZIC2 gene. The mutation was inherited from the father who presented only mild ocular hypotelorism but had an affected child with HPE from his first marriage. The occurrence of oral clefts is common in patients with HPE, but unusual in patients with mutation in the ZIC2 gene. To our knowledge, clefts of the nasal alae have been reported only once or twice in patients with ZIC2 mutations. In documented patients from the literature, only 2% of individuals with described pathogenic mutations in the ZIC2 gene (3/171) presented facial clefts, one of them a nasal cleft, while common oral clefts were observed in 27% of individuals (7/26) described with nonpathogenic ZIC2 mutations or presenting a concomitant mutation in another HPE gene. When compared with the general population, nasal clefts are common in ZIC2 mutations and these mutations must be searched for in undiagnosed cases.

Holoprosencephaly: ZIC2 mutation in a case with panhypopituitarism

Holoprosencephaly (HPE), the most common malformation of the brain, results from failed or incomplete separation of the embryonic forebrain (prosencephalon). HPE occurs in approximately 1 in 250 embryos and in about 1 in 10,000 births. It is etiologically heterogeneous, and may be caused by cytogenetic anomalies and teratogenic influences; it occurs as part of a syndrome, or due to heterozygous mutations in 1 of over 10 HPE-associated genes. ZIC2 mutations are the second-most common cause of non-syndromic non-chromosomal HPE (after sonic hedgehog) and occur de novo in 74% of the affected probands. The objective of the study was to describe the first case of ZIC2-related HPE with both anterior and posterior pituitary insufficiencies. We report about a 2-year-8-month-old boy who was born as a second child in a non-consanguineous healthy Turkish family. He has the characteristic ZIC2 phenotype: bitemporal narrowing, upslanting palpebral fissures, large ears, short nose with anteverted nares and broad and deep philtrum. Magnetic resonance imaging revealed alobar HPE. During laboratory investigation, his blood sodium level was 158 mmol/L and the specific gravity of his urine was 1.002. Serum osmolarity was 336 mOsm/L and urine osmolality was 135 mOsm/kg. His FT4 was 0.8 ng/dL and TSH was 0.79 mLU/mL. Response to vasopressin confirmed the diagnosis of central diabetes insipidus and TRH-stimulating test supported the central hypothyroidism. A frameshift mutation (NM_007129.2:c1091_1092 del, p.Gln364Leufs*2) in the ZIC2 gene was detected. Pituitary insufficiency other than isolated diabetes insipidus is a rare finding of HPE, and occurs most frequently in patients with GLI2 mutations (the phenotype of which typically does not include frank neuroanatomic anomalies such as HPE); ours is the only described patient with a ZIC2 mutation and both anterior and posterior pituitary dysfunction.

Molecular analysis of holoprosencephaly in South America.

Holoprosencephaly (HPE) is a spectrum of brain and facial malformations primarily reflecting genetic factors, such as chromosomal abnormalities and gene mutations. Here, we present a clinical and molecular analysis of 195 probands with HPE or microforms; approximately 72% of the patients were derived from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and 82% of the patients were newborns. Alobar HPE was the predominant brain defect in almost all facial defect categories, except for patients without oral cleft and median or lateral oral clefts. Ethmocephaly, cebocephaly, and premaxillary agenesis were primarily observed among female patients. Premaxillary agenesis occurred in six of the nine diabetic mothers. Recurrence of HPE or microform was approximately 19%. The frequency of microdeletions, detected using Multiplex Ligation-dependant Probe Amplification (MLPA) was 17% in patients with a normal karyotype. Cytogenetics or QF-PCR analyses revealed chromosomal anomalies in 27% of the probands. Mutational analyses in genes SHH, ZIC2, SIX3 and TGIF were performed in 119 patients, revealing eight mutations in SHH, two mutations in SIX3 and two mutations in ZIC2. Thus, a detailed clinical description of new HPE cases with identified genetic anomalies might establish genotypic and phenotypic correlations and contribute to the development of additional strategies for the analysis of new cases.

The phenotypic spectrum of ZIC3 mutations includes isolated d‐transposition of the great arteries and double outlet right ventricle

Disease causing mutations for heterotaxy syndrome were first identified in the X-linked laterality gene, ZIC3. Mutations typically result in males with situs ambiguus and complex congenital heart disease; however affected females and one male with isolated d-transposition of the great arteries (d-TGA) have been reported. We hypothesized that a subset of patients with heart defects common to heterotaxy but without laterality defects would have ZIC3 mutations. We also sought to estimate the prevalence of ZIC3 mutations in sporadic heterotaxy. Patients with TGA (n = 169), double outlet right ventricle (DORV; n = 89), common atrioventricular canal (CAVC; n = 41), and heterotaxy (n = 54) underwent sequencing of ZIC3 exons. We tested 90 patients with tetralogy of Fallot (TOF) to correlate genotype with phenotype. Three potentially disease-related missense mutations were detected: c.49G > T (Gly17Cys) in a female with isolated DORV, c.98C > T (Ala33Val) in a male with isolated d-TGA, and c.841C > T (His281Tyr) in a female with sporadic heterotaxy. We also identified a novel insertion (CPFP333ins) in a family with heterotaxy. All were absent in 200 control patients and the 1000 Genomes Project (n = 629). No significant mutations were found in patients with TOF. Functional studies demonstrated reduced transcriptional activity of the ZIC3 His281Tyr mutant protein. ZIC3 mutations were rarely identified in isolated DORV and d-TGA suggesting that a subset of DORV and d-TGA may fall within the spectrum of laterality defects. ZIC3 mutations were found in 3.7% of patients with sporadic heterotaxy; therefore testing should be considered in patients with heterotaxy.

Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning

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.