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Hum Mol Genet. 2013 May 15; 22(10): 1913–1923.
Published online 2013 January 8. doi:  10.1093/hmg/ddt001
PMCID: PMC3633368

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

Abstract

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.

INTRODUCTION

Congenital cardiac malformations occur in ~1% of all live births (1,2). The molecular and genetic mechanisms responsible for these malformations are largely unknown. Zinc finger protein of the cerebellum 3 (ZIC3), a zinc-finger transcription factor, is one of the few genes known to cause cardiovascular malformations in humans (3). Mutations in ZIC3 are the known cause of X-linked heterotaxy (4,5). Additionally, mutations in ZIC3 account for ~1% of sporadic heterotaxy cases and can also cause isolated cardiovascular malformations (46). Heterotaxy (HTX1, MIM 306955), also known as situs ambiguus, is a disorder characterized by an abnormal arrangement of the thoracic and/or abdominal organs relative to the left–right axis. Cardiovascular malformations typical of abnormal heart looping such as double outlet right ventricle, transposition of the great arteries and atrial isomerism, among others, are frequently coupled with multiple congenital anomalies.

The Zic3 null mouse recapitulates the human heterotaxy phenotype ranging in severity and complexity similar to humans (7). In addition to left–right defects, Zic3 null mice display other anomalies such as gastrulation defects, disturbances in axial patterning and neural tube defects (79). During early development, Zic3 is highly expressed in neuroectoderm and mesoderm during gastrulation of mouse, chick, Xenopus and zebrafish embryos, and has recently been shown to play a conserved role in convergent extension morphogenesis (10). The phenotypic spectrum identified in Zic3 null mice suggests that there are multiple roles for the transcription factor during development. Because of its known role in human cardiovascular malformations, it is important to understand the mechanistic basis of Zic3 function during cardiac development and left–right patterning.

In mouse, a group of ‘organizer’ cells is located at the anterior tip of the primitive streak as it elongates. There are three organizers within the primitive streak during the process of gastrulation, the early-gastrula organizer, mid-gastrula organizer and the node (also called the posterior notochordal plate) which forms at late streak stage of the embryo (1115). The Tgf-β ligand, Nodal, is initially expressed symmetrically in the crown cells of the node, the left–right organizer. Subsequently, the first molecular evidence of asymmetry is observed at the node with the asymmetric expression of Nodal at the 2 to 3 somite stage (16,17). Once Nodal is expressed asymmetrically at the node, a cascade of left-sided gene expression occurs in the left lateral plate mesoderm (LPM) (18,19). Nodal induces its own expression in the left LPM as well as expression of feedback inhibitors Lefty1 and Lefty2 (20,21). Nodal also induces left-sided expression of a homeobox transcription factor Pitx2, important for the downstream development of organ laterality (22). Importantly, the activation and left-sided expression of Nodal at the node are necessary for the proper expression of Nodal itself and downstream genes in the LPM necessary to properly transduce left–right signals to organ primordia (23).

There are two cell populations at the node thought to be critical for the establishment and/or maintenance of left–right asymmetry. Approximately 250 columnar epithelial ‘pit’ cells which have a single monocilium on their surface are required to produce a leftward flow of extracellular fluid known as nodal flow (24). There are ~20–30 cells surrounding the pit cells of the node, known as crown cells. The asymmetric expression of Nodal in these crown cells is dependent on nodal flow (24,25).

During left–right development, Zic3 has been shown to function upstream of the highly conserved Nodal signal transduction pathway (7,9). In Zic3 null mice, Nodal expression is initiated in perinodal crown cells but lost around the 2-somite stage. A loss of Nodal expression at the node prior to its asymmetric expression results in randomization of Nodal and Pitx2 in the LPM.

The mechanisms by which mutations in Zic3 result in abnormal heart looping are unknown. The linear heart tube is formed when two fields of precardiac mesoderm merge at the ventral midline of the embryo. Rightward bending of the linear heart tube is the first visible evidence of morphological asymmetry in the embryo. Following rightward bending, the heart tube undergoes a series of rotational and torsional movements which ultimately properly orient segments of the developing heart and great vessels. This process of heart looping morphogenesis uses positional information from all axes, including the left–right axis (26,27). Although the importance of left–right patterning for subsequent cardiac development is well established, it has recently been proposed that Zic3 may have a primary role in cardiac development independent of its role in left–right patterning (28).

In this study, we delineate the tissue-specific requirement for Zic3 in the establishment of the left–right axis using a conditional loss-of-function approach. We examined the requirement for Zic3 in the cardiac compartment, node and migrating primitive streak using seven different Cre lines. Using a ZIC3-LacZ-BAC reporter mouse line, we examined the temporal activity of Zic3 during the establishment of left–right asymmetry and heart looping. We found Zic3 to be present in the node during the establishment of left–right asymmetry but absent in the heart during looping morphogenesis. Deleting Zic3 from heart progenitors and the heart compartment did not affect the viability of Zic3 deficient embryos, corroborating the importance of Zic3 expression in extra-cardiac tissues for looping morphogenesis.

This study identifies a novel role for Zic3 in node formation. The spatial requirement for Zic3 in this process was defined in the migrating mesoderm of the primitive streak. Further, we defined the temporal requirement to occur between E7.0 and E8.0 at the end of mouse gastrulation. Taken together, these results identify the tissue-specific functions of Zic3 in left–right asymmetry and cardiac development, and highlight the importance of Zic3 in proper node formation, affecting node function and later left–right patterning.

RESULTS

Epiblast deletion of Zic3 recapitulates the Zic3 null phenotype

To determine the tissue-specific requirement of Zic3 in left–right patterning, a conditional allele of Zic3 was constructed in which loxP sites flank exon 1 (Fig. 1A). The translational start site is located in exon 1, predicting a loss of function of Zic3 upon tissue-specific deletion. To confirm functional deletion, we mated Zic3flox/flox females with Sox2-Cre males (27). Sox2-Cre is expressed in all cells of the epiblast at E6.25 prior to gastrulation. As expected, Zic3flox/flox;Sox2-Cre+ embryos recapitulated the Zic3 null phenotype exhibiting gastrulation (data not shown), heart (Fig. 1E) and neural tube (Fig. 1F) defects compared with Cre-negative male littermates. Additionally, Zic3 expression was absent in Cre-positive males compared with their Cre-negative male littermates (Fig. 1B and C). These results indicate that the Zic3 floxed allele can be effectively removed in a tissue-expressing Cre recombinase and left–right patterning defects and cardiovascular malformations result from epiblast-specific deletion of Zic3.

Figure 1.
Epiblast deletion of Zic3 recapitulates the Zic3 null phenotype. (A) A schematic of the Zic3 conditional allele. Targeted insertion of a neomycin cassette in intron 1 of mouse Zic3 (top) allowed for selection for recombination. Removal of the neomycin ...

Specific deletion of Zic3 from heart and heart progenitors does not result in increased lethality

ZIC3 mutations have been identified in individuals with isolated cardiovascular malformations, indicating that laterality defects are not an obligate feature of loss of function of this transcription factor (46). In addition, the cardiovascular malformations identified in Zic3 null mice include some defects not previously associated with cardiac looping abnormalities such as isolated septal defects and failure of right ventricular development (7). These facts, along with the identification of low levels of Zic3 transcripts in the developing murine heart, the ability of Zic3 to physically interact with the serum response factor in vitro, and a requirement of Zic3 for expression of heart-specific transcripts have led to speculation about a primary role of Zic3 in the developing heart, distinct from its role in left–right patterning (28). Arguing against such a role, we find that expression is absent in the heart when analyzed with whole-mount in situ (WISH) hybridization (Fig. 1C, data not shown) or a Zic3-LacZ-BAC reporter mouse during heart looping morphogenesis (Fig. 2C). In the Zic3-LacZ-BAC mouse, exon 1 of Zic3 is replaced with LacZ. β-Galactosidase (β-gal) staining recapitulates endogenous expression of Zic3 in the reporter mouse (Fig. 2 and (29)). To further investigate whether cardiovascular malformations are a result of a cell autonomous role for Zic3 in the developing heart, conditional deletion was used to evaluate the requirement of Zic3 in the cardiac compartment and heart progenitors. Four cardiac-specific Cre lines were used to assess the role of Zic3 in cardiac development. To analyze an early requirement in cardiac progenitors in the cardiac crescent and cardiomyocytes of the looping heart tube, we mated Zic3flox/flox females with Nkx2.5-Cre (30) males; Mef2c-Cre (31) was used to analyze a requirement in the anterior heart field and its derivatives; Wnt1-Cre (32) was used to analyze a requirement in cardiac neural crest cells; and a later requirement in embryonic and fetal cardiomyocytes was evaluated using βMyHC-Cre (33). Despite the wide range of heart defects observed in Zic3 null mice, heart looping defects were not observed (data not shown) and all conditional mutants were viable (Table 1). These results indicate that Zic3 is not temporally required in the heart from the early cardiac crescent stage through heart looping morphogenesis stages. Therefore, despite a previously reported requirement of Zic3 for expression of key transcriptional regulators of cardiac development, Zic3’s function in cardiac development is non-cell autonomous.

Table 1.
Specific deletion of Zic3 from heart and heart progenitors does not result in increased lethality
Figure 2.
Zic3 is present in the node during the establishment of left–right asymmetry. A transgenic Zic3-LacZ-BAC reporter mouse was generated to monitor Zic3 expression throughout mouse development. (A) and (B) show E7.75 embryos with strong β-gal ...

Zic3 is present in the node during the establishment of left–right asymmetry

The data from cardiac-specific deletion indicate that extra-cardiac expression of Zic3, possibly during left–right patterning, is responsible for the development of cardiac defects. The first evidence of molecular asymmetry is seen in the mouse node at approximately the 2 to 3 somite stage. At this stage, left-biased Nodal expression is first observed in the crown cells of the node following leftward nodal flow created by cilia on pit cells. Previous evaluation during these stages has shown that Zic3 is expressed in embryonic mesoderm, definitive endoderm and ectoderm but is reportedly absent from extraembryonic tissues (34). However, expression in the node is questionable when analyzed with WISH. To more closely examine Zic3 activity in the node, we analyzed Zic3-LacZ-BAC reporter mice. Node stage embryos were embedded in plastic resin to achieve better cellular resolution. Whole embryo staining revealed expression in the node (Fig. 2A and B). Sections throughout node stage embryos revealed staining in all three germ layers (data not shown). At E8.5 (Fig. 2C), LacZ staining is widespread but not in the heart. Sections in the region of the notochord and node (Fig. 2D) identify strong expression. These results demonstrate that Zic3 is present in node cells at the time that left–right patterning is initiated.

Zic3 is required in the migrating primitive streak for left–right patterning and normal cardiac development

The node and axial midline arise from the anterior primitive streak and are important organizers of the left–right body axis. In order to examine the temporal and spatial requirements of Zic3 prior to and during left–right patterning, we used a mouse with Cre driven by the T (Brachyury) promoter (3537). The T-Cre promoter is driven by primitive streak-specific regulatory elements and is active in the migrating streak and mesodermal lineage derivatives (36). We intercrossed Zic3flox/flox females with male mice expressing T-Cre. Analysis of embryos at E10.5 and E13.5 demonstrates that T-Cre conditional mutants have heart looping defects similar to Zic3 null embryos (Fig. 3B and D). To determine whether these cardiac defects are a result of abnormal left–right patterning, we performed WISH analysis for asymmetric molecular markers Pitx2 and Lefty2 (Fig. 4). As expected, T-Cre mutants (Fig. 4B and D) express left-sided markers abnormally when compared with control embryos (Fig. 4A and C) indicating abnormal left–right patterning. These results indicate that the function of Zic3 in left–right patterning and cardiac development occurs between ~E7.0 and E8.0 in the tissue-specific domains defined by T-Cre activity.

Figure 3.
Zic3 is required in the migrating mesoderm and primitive streak for normal heart looping. B) and (D) Representative images of heart defects observed at E10.5 (B) and E13.5 (D) in Zic3del/y;T-Cre+ males. (A) and (C) Stage-matched control embryos. Scale ...
Figure 4.
Zic3 is required in the migrating mesoderm and primitive streak for normal molecular left–right patterning. (A–D) Images of E8.5 embryos following WISH with Pitx2 (A and B) and Lefty2 (C and D) riboprobes. (A) demonstrates normal left-sided ...

Deletion of Zic3 in node cells after gastrulation does not alter left–right patterning

Node precursors emerge from the primitive streak to form the node. Following the observation that Zic3 is present in the node during the initiation of nodal flow and asymmetric Nodal expression, we hypothesized that Zic3 is required within the node to initiate left–right asymmetry. To further test the cell autonomous requirement for Zic3 in the node, we performed conditional deletion using two node-specific Cre lines which should delete Zic3 specifically in node cells with the timing similar to T-Cre. The Nodal-dependent enhancer Cre mouse line (hereafter NDE-Cre) (19) drives Cre expression in lateral crown cells of the node and experiments with the Rosa R26R reporter mouse demonstrate efficient recombination in the peripheral crown cells of the node by the 2 somite stage. The same temporal results are observed in ciliated pit cells of the node when the FOXJ1-Cre mouse (38) is crossed with the reporter mouse (39). Foxj1 is a transcription factor expressed in many tissues containing ciliated cells, including the node, and has been shown to regulate genes important for ciliogenesis (3841). The cell-type specificity of these two Cre lines led us to hypothesize that they would be useful in deleting Zic3 from the two types of node cells prior to the breaking of symmetry at the node. Surprisingly, mating Zic3flox/flox females with males expressing Cre in each cell type of the node led to viable progeny (Table 2) which displayed normal left–right patterning (Fig. 5A–H and Table 3).

Table 2.
Specific deletion of Zic3 from node cells does not result in increased lethality
Table 3.
Embryos exhibit normal morphological and molecular left–right patterning when Zic3 is deleted from node cells
Figure 5.
Zic3 RNA deficiency in node cells following gastrulation does not alter left–right patterning. (A)–(D) show WISH of E8.5 embryos using an antisense riboprobe for Pitx2 expression. Zic3 mutants (B) and (D) and their control littermates ...

To determine the timing of Zic3 recombination within the node, fluorescent-activated cell sorting (FACS) was used to isolate node cells for RNA analysis. These experiments made use of a transgenic FOXJ1-EGFP reporter mouse line bred into the relevant conditional matings. Embryos were pooled according to the genotype and node cells were isolated from the surrounding embryonic tissue at 0–4 somite stages. Zic3 RNA was analyzed in Zic3del/y;FOXJ1-Cre+;FOXJ1-EGFP node cells, Zic3flox/y; FOXJ1-Cre−; FOXJ1-EGFP node cells and wildtype (WT) FOXJ1-EGFP node cells (Fig. 3I and J). In addition, GFP-negative cells surrounding the node (indicated in blue in Fig. 5I) were collected. Zic3 RNA is absent in the node cells of conditionally deleted embryos at these stages. Therefore, deficiency of Zic3 RNA in node cells does not cause left–right patterning defects or abnormal cardiac development.

Zic3 is required for normal node morphogenesis

Events essential for left–right patterning occur over the span of a few hours and protein perdurance makes it challenging to unequivocally address a cell-autonomous role in node cells via conditional deletion. Nevertheless, the discrepant results of left–right patterning identified with T-Cre-mediated deletion versus NDE-Cre or FOXJ1-Cre deletion is surprising and led us to investigate whether there is an earlier requirement for Zic3 in node formation. To examine this hypothesis, Zic3 null nodes (n = 6) were compared with WT (n = 5) using scanning electron microscopy (SEM) (Fig. 6A and B). In WT nodes, a cohesive teardrop-shaped depression at the distal tip of the embryo forms (Fig. 6A; n = 5/5 embryos). In Zic3 null embryos, the teardrop structure is irregular and forms non-cohesive groups of node cells, characterized by small apical surfaces (Fig. 6B; n = 4/6 embryos). Similar results were demonstrated using immunohistochemistry (IHC) (Fig. 6CF). Cilia are present but shorter than stage-matched WT embryos (Fig. 6G–I). Additionally, endoderm cells appear larger (Fig. 6F and I) while the cell number was not affected (Fig. 6I). Overall, Zic3 null nodes are abnormal in structure with shorter cilia. It is unclear whether the shorter cilia are a primary defect or secondary to abnormal node formation and/or developmental delay.

Figure 6.
Zic3 is required for normal node morphogenesis. (A) and (B) show SEM of node ultrastructure in WT (A) and Zic3 null embryos (B). The node of WT embryos is a distinct triangular-shaped depression (A). The node of Zic3 mutant embryos is disorganized and ...

To determine whether abnormal node morphogenesis underlies the defects identified in T-Cre conditional mutants, SEM was used for analysis of Zic3del/y;T-Cre+ nodes (Fig. 7C and D). The results demonstrate dysmorphic node morphology (n = 5/6) similar to Zic3 null embryos (Fig. 6B and H) while the Cre-negative controls (Fig. 7A and B) displayed normal node morphology (n = 4/4) similar to WT embryos (Fig. 6A and G). These results demonstrate a requirement for Zic3 in the late migrating primitive streak for proper node formation.

Figure 7.
Zic3 is required in the migrating mesoderm and primitive streak for normal node morphogenesis. (A) and (B) SEM images of a Cre-negative control embryo exhibiting normal node morphology. (C) and (D) SEM images demonstrating abnormal node morphology when ...

DISCUSSION

Zic3 null mice exhibit a wide spectrum of phenotypic abnormalities including left–right defects recapitulating the human heterotaxy phenotype (7). However, there are also early gastrulation and later neural tube defects which complicate an understanding of the temporal and spatial, tissue-specific role of Zic3 in left–right patterning. Here, we use a conditional Zic3 mouse model to delineate the tissue-specific requirement for Zic3 in left–right patterning and cardiac development.

Zic3 does not function cell autonomously in the development of cardiac defects

There is significant phenotypic variability in individuals with ZIC3 mutations as well as in Zic3 null mice. Cardiovascular malformations are highly, but incompletely, penetrant (6,7) and may occur independently or in conjunction with other signs and symptoms of a left–right patterning defect. Given the low-level expression of Zic3 in the cardiac compartment, detectable only by RT–PCR, a cell autonomous role for Zic3 in cardiac development has been controversial. However, recent evidence that Zic3 can bind to serum response factor and affect cardiac transcription has suggested a potential primary role (28). Herein, we demonstrate that Zic3 is not detectable in the cardiac compartment using a sensitive reporter transgenic line. In addition, four cardiac-specific Cre lines demonstrate normal cardiac development and viability, thereby indicating that the mechanistic basis for cardiovascular malformations is extra-cardiac in origin.

Zic3 is required in the migrating mesoderm and primitive streak for normal left–right patterning and cardiac development

The data indicate that deletion of Zic3 expression in tissues in which the T-Cre transgene is active underlies the development of both left–right defects and subsequent cardiac malformations. In contrast, gastrulation defects and neural tube defects were not identified upon conditional deletion with the T-Cre transgene. We further demonstrate that deletion of Zic3 using T-Cre leads to abnormal node morphogenesis, an ultrastructural abnormality that is known to cause left–right patterning defects.

Previously, we have demonstrated that Zic3 functions upstream of Nodal in the development of left–right patterning and that Zic3 null mutants fail to establish properly lateralized Nodal expression within the left-sided crown cells at the 2 to 3 somite stage (7,9). Here, Zic3 is shown to be expressed in node cells using Zic3-LacZ-BAC reporter mice and FACS sorted node cells from FOXJ1-EGFP transgenic mice. In addition, Zic3 is expressed in the LPM, another tissue critical for the propagation of left–right asymmetry. Is it possible that Zic3 has dual functions in left–right patterning, acting early in node formation and slightly later within the cells of the node or LPM? Node expression is addressed using T-Cre, NDE-Cre and FOXJ1-Cre transgenic lines, whereas Zic3 expression in the LPM is deleted only using T-Cre.

The discrepant findings with the three Cre lines were surprising. It is possible that the timing of Zic3 deletion from the crown cells and pit cells does not occur rapidly enough to address its function in the node in left–right patterning. If this were the explanation for the discrepant findings, one would expect the temporal relationship for node deletion to differ between these lines. In fact, published data demonstrate that both NDE-Cre and Foxj1-Cre drive activity in the node, as assessed using R26R reporter mice, at 0–2 somite stages, whereas the T-Cre transgenic line does not show evidence of node deletion until 3 to 4 somites or later (19,35,36,39). Although we cannot exclude Zic3 protein perdurance as an explanation for the lack of phenotype identified with Foxj1-Cre and NDE-Cre conditional deletion, this caveat would apply for the T-Cre conditional deletion as well and therefore, cannot explain the presence of left–right defects in T-Cre mutants.

Kumar et al. (35) demonstrate that T-Cre activity occurs early enough in the mesodermal lineage to cause complete deletion of gene expression in the LPM, although a significant level of expression still occurs in the node before eventual elimination. Thus, the LPM represents a second tissue which may underlie the phenotypic effects mediated by deletion with the T-Cre transgene. However, the mechanisms by which Zic3 deficiency in the LPM would lead to failure to properly lateralize Nodal expression within the left-sided crown cells at the 2 to 3 somite stage are not clear. Future studies using LPM or node-specific rescue of Zic3 activity may further elucidate the function of Zic3 in these tissues. Taken together, the results are consistent with abnormal node morphogenesis due to Zic3 deficiency as a primary mechanism for left–right patterning defects and subsequent cardiovascular malformations, but it remains possible that Zic3 has a secondary role slightly later within mesodermal derivatives of the T-Cre transgene expression domain.

Zic3 is essential for normal node morphogenesis

Little is known about the mechanism(s) underlying normal node morphogenesis. Studies have examined late gastrula mouse embryos using SEM and cell lineage tracing studies (15,42,43). The location of the future node is covered with endoderm at E7.25. Node precursors undergo epithelial to mesenchymal transition at the anterior primitive streak (44). Shortly after, ciliated clusters of what appear to be node pit cells are identifiable and ultimately coalesce to form the tear drop-shaped node following mesenchymal to epithelial transition and reorganization of apical–basal polarity. We observed a range of defects in the Zic3 mutant nodes including some resembling this middle stage of node development (Fig. 6B, D, F, H), in which the cells are unable to form the final node structure and complete morphogenesis. Studies using SEM and confocal microscopy have shown that the ciliated cells are located underneath the larger endoderm cells (15,43). Experiments using GFP knocked into the Noto locus (43) reveal that the pit cells may form in an epithelial sheet underneath the endoderm layer prior to the emergence of the disconnected clusters of pit cells.

Cell migration, cytoskeletal dynamics including reorganization of the actin cytoskeleton, and extracellular matrix modification play important roles in the movement of node precursors from the anterior primitive streak, arrangement into ciliated cell clusters, assembly into a contiguous epithelia and interaction with surrounding endoderm (15,4446). Mouse mutants with defects in these processes, including fibronectin mutants (46), Rac1 epiblast-deleted mutants (45) and Lulu/Epb4.l1.5 mutants (44), share features of abnormal node morphogenesis, defective midline patterning and aberrant left–right asymmetry and bear a striking phenotypic similarity to Zic3 mutants. Like Lulu or Rac1 mutants, Zic3 null and Zic3 T-Cre mutants fail to form a single, coalesced node and tightly packed midline. Instead, several clusters of node cells are present, no pit was formed, and the midline is broad, irregular and kinked (this study and (8)). We have previously shown defects of primitive streak mesoderm, midline patterning and convergent extension of the axial mesoderm as a result of Zic3 deficiency or knockdown in mouse, Xenopus and zebrafish (7,8,10). Given the defects in convergent extension of the axial mesoderm in Zic3 mutants, it is interesting to note that Rac1 has been implicated as a downstream effector of planar cell polarity-mediated convergent extension in vertebrates and future studies are necessary to better define potential interactions. Abnormal node morphology has also been observed in mouse mutants in signaling pathways including Notch (4749), Wnt (50) and BMP (51) and the precise role of these pathways in node development remains to be identified.

In addition to the overall abnormal node structure observed in Zic3 mutants, node cilia were shorter. It is well established that cilia play an integral role in left–right patterning (24,25,52,53). In normal node development, cilia are shorter at the earlier stages of morphogenesis and elongate as a normal progression of node development (15,42). It is unclear whether Zic3 plays a primary role in ciliogenesis or whether the abnormalities detected are secondary to abnormal node formation. Developmental delay does not appear to be the culprit, given that the endoderm cells are larger in Zic3 null mice and this is not the characteristic of an earlier stage of node development. Mutants lacking Noto have an abnormal node and cilia phenotype similar to Zic3 mutants (54). In a recent study, the role of Noto in cilia and node formation was dissected (55). Mice expressing Foxj1 instead of Noto were examined for node and ciliary defects. While the ciliary defects were rescued, the node defects were not. Therefore, the authors were able to dissect the Foxj1-dependent role of Noto in cilia formation and the independent role of Noto in node morphogenesis. Similar studies will be necessary to define specific aspects of Zic3 in node and cilia formation.

Overall, this study has identified the tissue-specific requirement for Zic3 in left–right patterning and cardiac development. We have discovered a novel tissue-specific role for Zic3 in murine node morphogenesis which results in abnormal heart looping. The temporal requirement for Zic3 in this process occurs between E7.0 and E8.0. Evidence in recent studies (10,4446,56,57) including this one indicate that proper node ultrastructure is a necessary prerequisite to the development of normal left–right asymmetry. These data have clinical relevance since ZIC3 mutations underlie human cardiovascular malformations and heterotaxy spectrum laterality disorders and understanding the molecular requirements for normal heart looping is a necessary prerequisite for the development of genetic diagnostics and therapeutic treatments. Determining the mechanism by which Zic3 loss of function results in abnormal node morphology will be useful to uncover novel downstream targets and molecular pathway(s) important for left–right and cardiac development.

MATERIALS AND METHODS

Animals

Mouse embryos were collected from E7.5 to E13.5 for gene expression or phenotype analyses. The Zic3 null mice (7) and Zic3-LacZ-BAC transgenic mice (29) have been described previously. In order to generate the Zic3 conditional mice, Zic3flox/flox, an ~12.0 kb Zic3 genomic region was subcloned from a positively identified BAC clone (BAC PAC Resources). The targeting vector has a short homology arm that extends 2.3 kb 5′ to a loxP/FRT flanked Neo cassette. The long homology arm contains 9.7 kb 3′ to loxP/FRT flanked Neo cassette. The single loxP site was inserted upstream of exon 1 and the loxP/FRT-flanked Neo cassette was inserted downstream of exon 1. The targeting vector was confirmed by restriction analysis after each modification step and by sequencing across all junctions. The targeting construct was electroporated into embryonic stem cells. Following selection, clones were picked for screening. Five correctly targeted clones were injected into mouse blastocysts to create male chimeras. Germline transmission of the conditional allele was identified by coat color of offspring and confirmed by polymerase chain reaction (PCR)-based genotyping followed by Southern blot analysis of tail clip genomic DNA. Subsequent genotyping was performed by PCR of ear clip DNA using the following primers: forward-5′-CGT TCT CAA GGT GGT GAG GCA GCA G-3′; reverse-5′-GAA AGG GAT CCG CCG GGT TTG CG-3′. Cre mice were genotyped with the following primers: F-5′-CGA TGC AAC GAG TGA TGA GG-3′; R-5′GCA TTG CTG TCA CTT GGT CGT-3′.

The Sox2-Cre (58), Nkx2.5-Cre (30), Mef2c-Cre (31), βMyHC-Cre (33), Wnt1-Cre (32), FOXJ1-Cre (38,39), NDE-Cre (19), FOXJ1-EGFP (59) and T-Cre (36) have all been described previously. Because Zic3 is on the X chromosome, all experiments compared Cre-positive male embryos with stage-matched Cre-negative male embryos.

All mice were housed in the AALAC accredited Cincinnati Children's Hospital Research Foundation Animal Facility and experiments were approved by the Institutional Animal Care and Use Committee.

Whole-mount in situ hybridization and β-galactosidase staining

Embryos were collected in ice-cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (PFA) in PBS and dehydrated through a methanol series. WISH was performed as described previously (8). Probes were labeled using a DIG RNA labeling kit (Roche Applied Science). Staining for β-gal was performed on Zic3-LacZ-BAC embryos according to the standard protocols following a 20 min fixation in 4%PFA in PBS. For sections, β-gal-stained embryos were embedded in plastic resin (JB-4 Embedding Kit, Polysciences, Inc.) and sectioned at 5 μm with a glass knife as described previously (8).

Viability analysis

Embryos were genotyped with primers described above at postnatal day 0 (P0) and weaning age. Male embryos with genotype Zic3flox/y;Cre− were compared with Zic3del/y;Cre+ males for expected Mendelian ratios. Statistical significance was evaluated using Chi-square analysis.

Node cell isolation and quantification of RNA

FOXJ1-Cre males were intercrossed with FOXJ1-EGFP female mice to obtain double transgenic FOXJ1-Cre+;FOXJ1-EGFP males. Double transgenic males were intercrossed with Zic3flox/flox females. Embryos ranging from 0 to 4 somites were dissected in ice-cold PBS at E7.75 and analyzed with a Nikon SMZ1500 Zoom stereomicroscope using an EXFO X-Cite 120 fluorescence illuminator. The GFP-positive node and surrounding GFP-negative tissue was dissected away from the remaining embryo and stored in 5% fetal bovine serum (FBS) at 4°C during rapid genotyping. The remaining tissue was used for genotyping with primers described above. Zic3del/y;FOXJ1-Cre+;FOXJ1-EGFP and Zic3flox/y;FOXJ1-Cre-;FOXJ1-EGFP nodes were pooled according to the genotype. Single-cell suspensions were accomplished by incubating pooled nodes in 1% collagenase A (Roche) at 37°C for 5 min. Collagenase A was quenched using 10% FBS/PBS. Following centrifugation at 3000 rpm in 4°C for 5 min, the supernatant was removed from the pelleted cells. The cells were resuspended in 2% FBS/PBS with 1 mm EDTA and transferred to a 5 ml polypropylene tube (BD Falcon) on ice. The cells were immediately sorted using FACS (BD FACSAria II; CCHMC Research Flow Cytometry Core) into RNA lysis buffer (Qiagen). RNA purification was performed according to the RNeasy Micro kit protocol (Qiagen). Total purified RNA samples were converted to Complementary DNA (cDNA) using the Ovation RNA-seq System v2 assay (NuGEN Technologies). RT–PCR was performed on cDNA using the following primers: Zic3 F-5′ CGG GCT GCG GGA AGA T-3′; Zic3 R-5′ CTC ACC TGT ATG GGT CCT CTT GT-3′; Gapdh F-5′ TGC GAC TTC AAC AGC AAC TC -3′ and Gapdh R-5′ GCC TCT CTT GCT CAG TGT CC-3′.

Antibodies

Primary antibodies include mouse monoclonal anti-acetylated tubulin clone 6-11B-1 (Sigma) used at 1:200 dilution and rabbit anti-ZO-1 (N-terminal) polyclonal antibody (Zymed) used at 1:100 for whole-mount embryos. Secondary antibodies include goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 568 (Molecular Probes) all used at 1:150 dilution.

Whole-mount immunohistochemistry

Embryos were dissected at E7.75 in ice-cold PBS and fixed with 4% PFA in PBS overnight. After fixation, embryos were dehydrated with graded methanol series for storage at −20°C. Embryos with 0–2 somites were selected for further analysis. After stepwise rehydration, the embryos were permeabilized with 0.2% Triton X-100 in PBS (PBT), blocked in 3% bovine serum albumin in PBT for 1 h and subsequently incubated with primary antibodies for 24–48 h at 4°C with gentle rocking. After thorough washing with PBT, the embryos were stained with secondary antibodies. After washing, embryos were mounted in Pro-long antifade (Molecular Probes) and imaged using a confocal laser-scanning microscope (Nikon PCM 2000) at ×60 magnification.

Measurement of node cilia length, cell number and endoderm cell area

NLS-Elements D2.30 software was used to trace and measure the length of cilia in SEM captured images. Endoderm cells were defined as a surrounding horseshoe ring around node pit cells. To determine the endoderm cell area, ZO-1 staining was traced using NLS-Elements D2.30 software. To determine the node cell number, embryos were stained with ZO-1 and DAPI then manually counted (WT embryos, n = 10; Zic3 null embryos, n = 8).

Scanning electron microscopy

Embryos were dissected in ice-cold PBS, immediately fixed in an SEM fixative (0.1 m sodium cacodylate buffer, pH 7.3, with 2% glutaraldehyde) overnight at 4°C. Following a 24 h fixation, embryos were genotyped and transferred to individual tubes containing the SEM fixative.

FUNDING

This work is supported by the National Institutes of Health (RO1 HL088639, HL088639-03S1 to S.M.W.). M.J.S. is supported by a National Institutes of Health T32 Pulmonary and Cardiovascular Developmental Training Grant (HL007752-16 to J.A.W.).

ACKNOWLEDGEMENTS

We thank Brian L. Black for the Mef2c-Cre line, Mark Lewandoski for the T-Cre line, Michael J. Holtzman for the FOXJ1-Cre line, Yukio Saijoh for the NDE-Cre line and Jeffery D. Molkentin for the βMyHC-Cre line.

Conflict of Interest statement. None declared.

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