Morpholinos for splice modificatio

Morpholinos for splice modification

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Summary

Inactivation of the left-right asymmetry gene Pitx2 has been shown, in mice, to result in right isomerism with associated defects that are similar to that found in humans. We show that the Pitx2c isoform is expressed asymmetrically in a presumptive secondary heart field within the branchial arch and splanchnic mesoderm that contributes to the aortic sac and conotruncal myocardium. Pitx2c was expressed in left aortic sac mesothelium and in left splanchnic and branchial arch mesoderm near the junction of the aortic sac and branchial arch arteries. Mice with an isoform-specific deletion of Pitx2c had defects in asymmetric remodeling of the aortic arch vessels. Fatemapping studies using a Pitx2 cre recombinase knock-in allele showed that daughters of Pitx2-expressing cells populated the right and left ventricles, atrioventricular cushions and valves and pulmonary veins. In Pitx2 mutant embryos, descendents of Pitx2-expressing cells failed to contribute to the atrioventricular cushions and valves and the pulmonary vein, resulting in abnormal morphogenesis of these structures. Our data provide functional evidence that the presumptive secondary heart field, derived from branchial arch and splanchnic mesoderm, patterns the forming outflow tract and reveal a role for Pitx2c in aortic arch remodeling. Moreover, our findings suggest that a major function of the Pitx2-mediated left right asymmetry pathway is to pattern the aortic arches, outflow tract and atrioventricular valves and cushions.

INTRODUCTION

Pitx2 is a paired-related homeobox gene that has been shown to play a central role in the late aspects of left-right asymmetric morphogenesis (Capdevila et al., 2000; Harvey, 1998). Identified as the gene mutated in Rieger syndrome 1, Pitx2 also functions in eye, tooth and abdominal wall development (Alward, 2000; Semina et al., 1996). Importantly, Pitx2 has been shown to be a direct target of the left-right signaling pathways that originate early in development through the function of nodal (Shiratori et al., 2001). Loss-of-function experiments performed in mice have revealed a role for Pitx2 in left-right asymmetry of many organs but its role in heart and vascular development is less clear (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999b).

Although Pitx2-null mice have severe cardiac phenotypes that are similar to those observed in humans with laterality defects, the Pitx2-null phenotype suggests that Pitx2 function is important after looping morphogenesis, as Pitx2 mutant hearts loop correctly to the right (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Liu et al., 2001; Lu et al., 1999b). Analysis of individuals with laterality defects has revealed a spectrum of associated cardiac septation and valve anomalies, including abnormalities in conotruncal and right ventricular development, atrial lateralization and atrioventricular (AV) septation (Brown and Anderson, 1999; Icardo and Sanchez de Vega, 1991). These observations suggest that the genetic pathways regulating left-right asymmetry may also directly regulate valve and cushion morphogenesis and that subtle defects in left-right asymmetry may be a common etiologic factor for congenital heart disease.

In common with human patients, Pitx2-null mice display atrial septal defects (ASD), abnormal AV septation (resulting in complete AV canal) and abnormal arterioventricular connections (Kitamura et al., 1999; Liu et al., 2001). Pitx2 mutants also have a hypoplastic right ventricle. Although Pitx2 mutant mice have severe cardiac anomalies, the primary function of Pitx2 in heart development remains unclear as the Pitx2 mutant heart phenotypes could be secondary to delayed looping morphogenesis or embryonic rotation.

In this work, we have used a combination of gene expression analysis and gene targeting approaches to investigate Pitx2 function in cardiovascular development in more detail. Our data demonstrate that the Pitx2c isoform is expressed in a presumptive secondary heart field that invades the heart after looping morphogenesis. Pitx2c was expressed in a subpopulation of left branchial arch and splanchnic mesoderm apposed to forming branchial arch arteries (BAAs) and in left aortic sac mesothelium. An isoform-specific deletion of Pitx2c, generated by gene targeting in embryonic stem cells, revealed that Pitx2c functions to regulate asymmetric BAA remodeling and to pattern the outflow tract (OFT). Fate-mapping studies with a Pitx2 cre knock-in allele revealed that Pitx2 daughter cells invade the AV cushions and valves in a Pitx2-dependent fashion, suggesting a role for Pitx2 in local cell movement or survival within the heart. Our results provide insight into Pitx2 function in post-looping cardiac morphogenesis and in BAA remodeling.

MATERIALS AND METHODS

Gene targeting in ES cells

To generate the Pitx2 δc neo targeting vector, we replaced the Pitx2 exon 4 that encodes the Pitx2c-specific exon with a LoxP flanked PGKneomycin cassette. The Pitx2 δc neo targeting vector introduced a novel EcoRV site into the mutant Pitx2 locus that we used to screen for homologous recombination events by Southern blot using flanking probes.

After homologous recombination, the Pitx2 δc neo allele resulted in deletion of the majority of exon 4, including all coding sequences within this exon. The Pitx2 δc neo targeting vector was electroporated into AK7 ES cells, targeted clones identified by Southern blot, and injected into 3.5 dpc C57BL/6J mouse embryos to generate chimeras. To induce recombination between the two loxP sites and remove PGKneomycin cassette, we crossed Pitx2 δc neo chimeras to CMVCre recombinase deleter strain. Pitx2 δc neo and δc alleles were maintained on a mixed 129/Sv×C57BL/6J genetic background.

The Pitx2 δabccreneo will be described elsewhere. Briefly, to generate this allele, an IRES cre PGKneomycin cassette was introduced into the PvuII and NruI sites of Pitx2 exon 5, that encodes part of the homeodomain, generating a null allele of Pitx2. Wholemount in situ with a cre recombinase (cre) probe confirmed that expression of cre recapitulated endogenous Pitx2 expression pattern.

Whole-mount in situ hybridization

Whole-mount in situ hybridization was performed as described (Lu et al., 1999b). The Pitx2c probe was a 1 kb genomic fragment containing exon 4 that was linearized with XhoI and transcribed with T7 polymerase. The semaphorin 3c probe has been described previously (Brown et al., 2001) and the cre probe was a cDNA fragment that was linearized with EcoRI and transcribed with T7 polymerase.

lacZ staining and histology

For histology, embryos were fixed overnight in buffered formalin, dehydrated through graded ethanol and paraffin embedded. Sections were cut at 7-10 μm and H&E stained. lacZ staining was described (Lu et al., 1999a).

Corrosion cast and casting dye injections

Injection of casting dye: 18.5 dpc embryos were harvested and sternum removed. Yellow casting dyes (Connecticut Valley Biological Supply) were injected into right ventricles using a capillary pipette, followed by blue dye into left ventricle. Corrosion casts: 18.5 dpc embryos were isolated and the heart exposed by a thoracic incision. Batson number 17 acrylic (Polysciences) was injected into right and left ventricles until great arteries were filled. After hardening overnight in distilled water at 4°C, tissues were removed with Maceration Solution at 50°C for 24 hours without shaking.

India Ink Injections

Embryos were dissected and placed in ice cold PBS. Individual embryos were placed in warm PBS to facilitate ventricular contractions. Using a pulled glass pipette, India Ink was injected into ventricles until ink penetrated small vessels. Embryos were post fixed in 10% formalin and cleared in benzyl alcohol:benzyl benzoate (2:1).

RESULTS

Pitx2c is asymmetrically expressed in splanchnic and branchial arch mesoderm and outflow tract myocardium

Previous studies have shown that the Pitx2c isoform is asymmetrically expressed in the developing embryo while the Pitx2a and Pitx2b isoforms are co-expressed with Pitx2c in symmetrical regions of the embryo (Kitamura et al., 1999; Liu et al., 2001; Schweickert et al., 2000; Yu et al., 2001). For example, Pitx2c is expressed in left lateral plate mesoderm and in the left side of most organ primordial such as developing guts, heart and lungs. The three Pitx2 isoforms in mice are co-expressed in periocular mesenchyme, oral and dental epithelium, as well as anterior body wall. A fourth Pitx2 isoform, Pitx2d, has recently been described in humans (Cox et al., 2002).

Because of the correct dextral looping in Pitx2 null embryos, we hypothesized that Pitx2 functioned in a cell population that contributed to the heart during or after cardiac looping. Experiments performed in chick and mouse embryos have revealed that cells outside the primary heart field contribute to conotruncal development. One cell population originates in the splanchnic and branchial arch mesoderm, and migrates into the OFT and right ventricle of the looped heart (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). We found that cells within this presumptive secondary heart field express Pitx2c asymmetrically at 9.5 dpc both as they migrate and after populating the OFT and right ventricle, revealing that this cell population has laterality.

Whole-mount in situ using a Pitx2c-specific probe on 9.5 dpc embryos revealed left-sided Pitx2c expression in splanchnic mesoderm at the level of and just caudal to the OFT (Fig. 1A,B). Serial sectioning showed that Pitx2c expression in left splanchnic mesoderm was continuous with expression in left aortic sac mesothelium and OFT myocardium (Fig. 1C-F). Expression of the Pitx2a and Pitx2b isoforms was not detected in the presumptive secondary heart field or developing OFT (not shown).

Fig. 1.

Expression of Pitx2c in the branchial arch mesoderm and aortic sac. (A,B) Whole-mount images of 9.5 dpc wild-type embryos hybridized to a Pitx2c-specific probe showing Pitx2c in left branchial arch mesoderm (arrow in B). (C-E) Transverse serial sections through a 9.5 dpc wild-type embryo hybridized to Pitx2c probe. Pitx2c expression is denoted by arrows. (F,G) Coronal sections through a 9.5 dpc embryo hybridized to a Pitx2c probe showing Pitx2c expression in left branchial arch mesoderm near junction of the aortic sac with the branchial arch artery (arrow in F). Pitx2c expression within left aortic sac myocardium is denoted by arrowhead. In G, arrow designates Pitx2c expression in dorsal branchial arch mesoderm in proximity to forming branchial arch arteries. (H,I) Parasagittal section through a 10.5 dpc embryo hybridized to Pitx2c probe. Arrow denotes ventral Pitx2c expression while arrowhead in I indicates less abundant dorsal Pitx2c expression. (J-M) Transverse sections through hearts of 10.5 dpc embryos hybridized to Pitx2c probe. Arrows in J,K,L indicate Pitx2c expression in dorsal mesocardium, while arrowhead in L indicates Pitx2c expression in left atrium. Arrows in M indicate Pitx2c expression in right ventricular and interventricular myocardium. as, aortic sac; ba, branchial arch; baa, branchial arch artery; da, dorsal aorta; oft, outflow tract.

We examined coronal and parasagittal sections through 9.5 and 10.5 dpc embryos to investigate in more detail the Pitx2c expression pattern during these timepoints prior to remodeling of the BAA. Ventral coronal sections through 9.5 dpc embryos showed left-sided Pitx2c expression in aortic sac mesothelium near the junction of the aortic sac and the BAA (Fig. 1F). More dorsal coronal sections revealed low levels of Pitx2c expression in left branchial arch and splanchnic mesoderm in proximity to the third BAA (Fig. 1G). Serial parasagittal sections through 10.5 dpc embryos showed Pitx2c expression in ventral branchial arch and splanchnic mesoderm that was continuous with Pitx2c expression in left branchial arch mesoderm evident on more lateral sections (Fig. 1H,I). The parasagittal sections at this timepoint also revealed the diminished intensity in Pitx2c expression dorsally towards the dorsal aorta (Fig. 1I). Our expression studies also showed Pitx2c expression in the left atrium, primary interatrial septum, left dorsal mesocardium, and right ventricular and interventricular myocardium (Fig. 1J-M). From these studies, we conclude that Pitx2c is asymmetrically expressed in a subpopulation of the presumptive secondary heart field that contributes to the OFT and right ventricular myocardium after cardiac looping, suggesting that Pitx2c provides laterality to the OFT and right ventricle myocardium. Moreover, asymmetric Pitx2c expression in ventral branchial arch and splanchnic mesoderm, with higher levels ventrally near the junction of the BAA and aortic sac, suggests a role for Pitx2c in formation of the BAAs.

Pitx2c mutants survive gestation and turn normally

The Pitx2c isoform is encoded by exons 4, 5 and 6, and uses a distinct promoter from that of the Pitx2a and Pitx2b isoforms (Shiratori et al., 2001). The Pitx2 exon 5 and exon 6, which encode the homeodomain, are common to all Pitx2 isoforms in mice (Fig. 2A,B). To directly investigate Pitx2c function using a loss-of-function approach in mice, we constructed a targeting vector that replaced the Pitx2c-specific exon 4 with a PGKneomycin LoxP cassette, the Pitx2 δc neo targeting vector (Fig. 2B,C). Upon germline transmission, the Pitx2 δc neo allele was crossed to the cmv cre recombinase deletor strain to remove the PGK neomycin cassette and generate the final Pitx2 δc allele (Fig. 2C,D). Both Pitx2 δ c neo and Pitx2 δc-/- mutants were obtained at the Mendelian ratio at 18.5 dpc. Mutant neonates were born alive but quickly became cyanotic and died a few minutes after birth. We noted that Pitx2 δc-/- mutants turned normally, suggesting that the Pitx2a and Pitx2b isoforms have redundant function with Pitx2c in turning or body wall closure as Pitx2a, Pitx2b homozygous mutant embryos also turn normally (Liu et al., 2001) (Fig. 2E).

Fig. 2.

Gene targeting strategy to generate the Pitx2 δc allele. (A) Summary of exon use by Pitx2 isoforms. (B) Pitx2 genomic structure and Pitx2c-specific targeting strategy. Boxes represent exons and straight lines introns. P1 and P2 indicate two promoters that regulate expression of different isoforms. (C) Pitx2 δc targeted allele before and after PGKneomycin removal. At the bottom, the Pitx2 null allele (δabcnull) and cre knock-in alleles (δabccreneo) also used in this study are shown. (D) Southern blot with flanking probes: left panel shows a Southern blot of tail DNA probed with 5′ flanking probe and center panel shows Southern blot probed with 3′ flanking probe. Right panel shows a Southern blot probed with 3′ flanking probe after crossing to CMV cre recombinase deletor strain to generate Pitx2 δ c+/- mice. After recombination, the 3′ flanking probe hybridizes to an 8 kb fragment and a 10 kb fragment in mice retaining PGKneomycin. The `+' above lanes denotes mice retaining PGKneomycin and `-' indicates a mouse that deleted PGKneomycin. (E) Lateral view of wild-type and Pitx2 δ c-/- 18.5 dpc embryos.

Pitx2c patterns the aortic arch vessels

To determine if Pitx2c had a role in patterning the great vessels of the aortic arch, we performed casting dye and corrosion cast experiments on 18.5 dpc Pitx2 δc-/- embryos. Among the 21 mutant embryos examined, 57% (12 out of 21) had the wild-type pattern of left aortic arch with right innominate artery while 29% (six out of 21) had right aortic arch with left innominate artery (Fig. 3A-D). In addition, 14% (three of 21) showed double aortic arch without innominate artery (Fig. 3E-J). Of the double aortic arches, two were right dominant and the other left dominant. Thus, of all arches examined, 62% (n=13) were left dominant and the remaining 38% (n=6) were right dominant. In addition, all Pitx2 δc-/- embryos had double outlet right ventricle (DORV) in which both the aorta and pulmonary artery drain the right ventricle (Fig. 3O,P). Blood exited the left ventricle of the Pitx2 δ c-/- embryos through a ventricular septal defect.

Fig. 3.

Pitx2c in remodeling the great vessels and outflow tract. (A) Remodeling of aortic arch arteries and derivation of mature aortic arch vessels (adapted from Moore, 1982). (B-D) Corrosion cast of wild-type (B) and Pitx2 δ c-/- embryos with correct direction of aortic arch (C) and reversed orientation (D). (E-G) Corrosion cast of a Pitx2 δ c-/- embryo with double aortic arch showing ventral (E), ventral oblique (F) and dorsal (G) views. (H-J) Diagrams of the corrosion cast to more clearly show changes in vessel morphology. Each diagram is associated with the cast directly above. (K-N) India ink injection into wild-type and Pitx2 δc-/- embryos at 11.5 dpc. Right and left oblique views are shown. (O,P) Casting dye injection into 18.5 dpc wild-type and Pitx2 δc-/- embryos showing DORV in mutant (P). ao, aorta; baa, branchial arch arteries; cc, common carotid; d, ductus arteriosus; in, innominate artery; lcc, left common carotid; lpa, left pulmonary artery; lsa, left subclavian artery; pa, pulmonary artery; pt, pulmonary trunk; rcc, right common carotid artery; rpa, right pulmonary artery; rsa, right subclavian artery; s, subclavian artery.

The corrosion casting experiments revealed that Pitx2c had an important role in patterning of the BAAs. To determine if Pitx2c had a role in the initial formation of the BAAs or was important in BAA remodeling, we performed India ink injections at 11.0 and 11.5 dpc at the initiation of BAA remodeling. At these timepoints, all Pitx2c mutant embryos (n=6) formed symmetric BAAs that were indistinguishable from wild type littermates (Fig. 3K-N). From this, we conclude that Pitx2c functions in remodeling of the BAA. The very discrete, asymmetric Pitx2c expression pattern within the region of the forming BAAs also supports the idea that Pitx2c would have a role in modulating BAA remodeling rather than in the initial endothelial tube assembly.

Cardiac neural crest migrates normally in Pitx2c mutants

Great vessel remodeling and patterning of the conotruncal region, both defective in Pitx2c mutants, require normal development of the cardiac neural crest. To determine if cardiac neural crest contributed to the conotruncal region of Pitx2 δc-/- embryos, we performed a fate-mapping experiment with the wntl cre transgenic line that directed cre expression to the precursors of the cardiac neural crest and the Rosa26 reporter line (Jiang et al., 2000; Soriano, 1999). cre expression will induce recombination at the Rosa26 locus resulting in expression of lacZ in all descendents of Wntl-expressing cells that include the cardiac neural crest. At both 11.5 and 12.5 dpc, we found that cardiac neural crest contributed normally to the conotruncal region and aortic and pulmonic valves of Pitx2 δc-/- embryos suggesting that Pitx2 function in conotruncal cushion morphogenesis occurred subsequent to neural crest migration into the Pitx2 mutant heart (Fig. 4A-F). Analysis of sections through the branchial arch arteries of 10.5 dpc wild type and Pitx2 δc-/- embryos showed similar amounts of mesenchyme surrounding the arteries further supporting the idea that cardiac neural crest was correctly deployed in Pitx2 δ c-/- embryos (Fig. 4G-J).

Fig. 4.

Fate mapping with Wnt1 cre transgenic and the Rosa26 reporter. (A,B) Ventral view of whole-mount lacZ staining of 12.5 dpc wild-type and Pitx2 δc-/- mutant embryo. (C-F) Rostral (C,D) and caudal (E,F) transverse section through the conotruncus of lacZ stained embryos showing lacZ-labeled cardiac crest derivatives contributing to valves and cushions of OFT (denoted by arrows). (G-J) Parsagittal sections through 10.5 dpc wild-type (G,I) and Pitx2 δ c-/- (H,J) mutant embryos at different mediolateral planes of section. Branchial arch arteries are numbered. ao, aorta; pt, pulmonary trunk.

To determine how loss of Pitx2 affected development of OFT myocardium, we examined the expression of semaphorin 3c (Sema3c), an OFT myocardial marker, in wild type and Pitx2c mutants (Brown et al., 2001; Feiner et al., 2001). Although at 10.5 and 11.5 dpc, Sema3c was expressed normally in Pitx2c mutant OFT myocardium (Fig. 5A-D), this expression was downregulated by 12.5 dpc (Fig. 5E,F). These data suggested that OFT myocardium was correctly specified and that migration of OFT myocardial precursors was intact in Pitx2c mutants.

Fig. 5.

Whole-mount in situ with markers of outflow tract myocardium. (A,B) 10.5 dpc wild-type (A) and Pitx2 δc-/- (B) embryos hybridized with semaphorin 3c probe. (C,D) 11.5 dpc wild-type (C) and Pitx2 δc-/- (D) embryos hybridized with semaphorin 3c probe showing expression in outflow tract myocardium (arrows). (E,F) 12.5 dpc wild-type (E) and Pitx2 δc-/- (F) embryos hybridized with semaphorin 3c probe showing that expression of semaphorin 3c is reduced in the mutant (n=3) (arrows). (G,H) Whole-mount views of 12.5 dpc wild-type (G) and Pitx2 δ abccreneo;δabcnull (H) null mutant embryos. Pitx2 δabccreneo;δabcnull embryos demonstrate embryonic rotation, anterior body wall closure defects and eye anomalies typical of Pitx2 null embryos. (I,J) 12.5 dpc wild-type (I) and Pitx2 δabccreneo;δabcnull (J) embryos hybridized with cre probe showing expression in outflow tract and right ventricular myocardium (arrows).

To establish this more firmly, we used a Pitx2 cre knock-in allele (Pitx2 δabccreneo), an allele of Pitx2 that expresses cre in the endogenous Pitx2 expression domain, to mark cells fated to express Pitx2. The Pitx2 δ abccreneo allele has a cre recombinasePGKneomycin cassette introduced into Pitx2 exon 5 to generate a null Pitx2 allele, removing function of all Pitx2 isoforms (see Materials and Methods, and Fig. 5G,H). Moreover, expression of cre from the Pitx2 δabccreneo qualitatively recapitulates the endogenous Pitx2 spatiotemporal expression pattern (not shown). At both 10.5 and 12.5 dpc, spatial expression of cre was similar in the OFT of the control δabccreneo heterozygotes and δabccreneo;δabcnull Pitx2-null mutant embryos, supporting the idea that Pitx2 patterns the OFT myocardium after it is established (Fig. 5I,J and not shown). Taken together, these data support the notion that Pitx2 functions in branchial arch and splanchnic mesoderm, a developmental field that is distinct from cardiac neural crest. Moreover, downregulation of Sema3c expression in Pitx2 mutants suggests that Pitx2 has a role in maintenance of gene expression in OFT myocardium.

Pitx2 daughter cells contribute to OFT, inner curvature myocardium and valves

In addition to cardiac neural crest, OFT and inner curvature myocardium invades the cardiac cushions (van den Hoff et al., 1999; van den Hoff et al., 2001). To determine if descendents of Pitx2-expressing myocardium populated the cardiac cushions, we used the Pitx2 δabccreneo allele and the Rosa26 reporter allele to follow the fate of Pitx2 daughter cells after Pitx2 expression had been extinguished. At timepoints when Pitx2c is actively expressed in the heart, 9.5 dpc until 12.5 dpc, distinctions between Pitx2 daughter cells and newly labeled Pitx2-expressing cells can be made in regions of the heart that never express Pitx2c. For example, at 9.5 and 10.5 dpc Pitx2-expressing cells are restricted to the left side of the forming OFT (Fig. 1C-E). By contrast, lacZ-positive cells were detected on both sides of the OFT tract myocardium, suggesting that labeled Pitx2 daughter cells, found on the right side of the OFT, had moved from the left side (Fig. 6A). The distribution of lacZ-positive cells in the OFT tract in Pitx2 null embryos was similar to that of the wild type, suggesting that Pitx2 is not required for movement of the myocardial precursors from branchial arch mesoderm into the OFT (Fig. 6A,B). We noted that the number of lacZ-labeled cells in the OFT myocardium of 10.5 dpc embryos was less than what would be expected from the Pitx2c expression pattern. This may reflect the delay between cre transcription and Cre-mediated excision that requires the accumulation of adequate levels of Cre protein. Moreover, the delay in the readout is also lengthened by the need for transcription and translation of lacZ from the Rosa26 locus (Nagy, 2000).

Fig. 6.

Fate mapping with Pitx2 δabccreneo and Rosa26 reporter allele. (A,B) lacZ staining of 10.5 dpc Pitx2 δabccreneo+/- (A) and Pitx2 δ abccreneo;δabcnull (B), and Rosa26 reporter trans-heterozygous embryo. Arrow indicates the lacZ-positive cells in the OFT that have crossed the midline (broken line). (C,D) lacZ staining (C) and Pitx2c whole-mount in situ (D) of 12.5 dpc Pitx2 δ abccreneo+/- and Rosa26 reporter trans heterozygous embryos. Signal is indicated by the arrows. (E-H) lacZ staining (E,G) and Pitx2c whole-mount in situ (F,H) of 14.5 dpc (E,F) and 16.5 dpc (G,H) Pitx2 δ abccreneo+/- and Rosa26 reporter trans heterozygotes. lacZ-positive cells are indicated by arrows (E,G). Pitx2c expression has been extinguished in Pitx2 daughter cells that would be lacZ positive and are marked with and asterisk (F,H). (I,J) Coronal sections through a 16.0 dpc Pitx2 δabccreneo+/- and Rosa26 reporter trans-heterozygotes at slightly different dorsoventral planes. Arrows indicate lacZ-positive cells in myocardium (I) and in interatrial and interventricular septum (J). (K,L) Whole-mount lacZ staining of 14.5 dpc wild-type (K) and Pitx2 δabccreneo;δc Pitx2 mutant (L), and Rosa26 reporter trans-heterozygous embryos. Arrows indicate lacZ-positive cells. Circled area in L indicates region with fewer lacZ-positive cells. (M,N)Whole-mount lacZ staining of 16.5 dpc wild-type (M) and Pitx2 δ abccrenoe;δc Pitx2 mutant (N), and Rosa26 reporter trans-heterozygotes. Arrows indicate lacZ-positive cells. Circled area in N indicates region with fewer lacZ-positive cells. (O-R) Transverse sections through a 16.5 dpc Pitx2 δabccreneo+/- (O,Q) and Pitx2 δ abccreneo;δc (P,R) and Rosa26 reporter trans-heterozygous embryo. More rostral sections show lacZ-positive Pitx2 daughter cells (arrow) in myocardium of both Pitx2 δ abccreneo+/- (O) and δabccreneo;δc mutants (P), while more caudal sections near cardiac apex show lacZ-positive cells in δabccreneo+/- (Q) but not in δabccreneo;δc mutants (R) as denoted by the asterisk. (S,T) Transverse sections through a 12.5 dpc Pitx2 δ abccreneo+/- (S) and Pitx2 δ abccreneo;δabcnull (T) and Rosa26 reporter trans-heterozygote showing lacZ expression in AV cushion in heterozygote (arrow) but absent in the mutant (asterisk). (U) Coronal section through a 14.5 dpc Pitx2 δ abccreneo;δabcnull and Rosa26 reporter trans-heterozygote showing exclusion of lacZ-positive cells from AV cushion (asterisk). (V,W) Coronal section through a 16.5 dpc Pitx2 δabccreneo+/- (V) and δabccreneo;δc (W) and Rosa26 reporter trans-heterozygote showing lacZ-positive cells within valve leaflet in the Pitx2 δabccreneo+/- embryo (arrows) but exclusion of lacZ-positive cells from the AV valve leaflets of Pitx2 mutants(asterisk). rv, right ventricle; lv, left ventricle; ivs, interventricular septum; scv, superior caval vein.

At 12.5 dpc, when Pitx2c is still expressed in the heart, the Pitx2 δabccreneo allele cannot distinguish between newly labeled lacZ-positive cells and Pitx2c descendents that are no longer expressing Pitx2c. At this timepoint, lacZ-labeled cells were predominantly found in the myocardium overlying the interventricular groove with some cells found in the proximal OFT (Fig. 6C,D). By 14.5 dpc and 16.5 dpc, when Pitx2c expression is extinguished (Fig. 6F,H), there was an increase of lacZ-positive Pitx2c descendents over the medial aspect of the heart, suggesting an outward expansion of Pitx2c descendents from the right ventricular and inner curvature myocardium (Fig. 6E,G). Sections through 16.5 dpc hearts, after Pitx2c expression had been extinguished, demonstrated that lacZ-positive Pitx2 daughter cells populated the myocardium of the proximal OFT, as well as the remodeled membranous and muscular ventricular septum and atrial septum (Fig. 6I,J).

Analysis of the fate of Pitx2 daughter cells in the Pitx2 δ abccreneo; δc mutant embryos, that turned normally and survived longer than Pitx2 null embryos, revealed that fewer lacZ-positive cells were found in the right ventricular and inner curvature myocardium of Pitx2 mutant embryos at both 14.5 dpc (Fig. 6K,L) and 18.5 dpc (Fig. 6M,N). Serial transverse sections through the 18.5 dpc hearts revealed that in Pitx2 δ abccreneo heterozygous hearts lacZ-labeled cells were found at the inferior border of the heart near the cardiac apex (Fig. 6O,Q). By contrast, in the Pitx2 δabccreneo; δc mutants lacZ-labeled cells were not found at the inferior boundary of the heart (Fig. 6P,R). Moreover, sections through 12.5 dpc hearts revealed that lacZ-positive cells were found in the central AV cushion of Pitx2 δ abccreneo heterozygous embryos, revealing that Pitx2 descendents contributed to the cushion mesenchyme (Fig. 6S). By contrast, in both 12.5 dpc and 14.5 dpc Pitx2-null mutant embryos, lacZ-positive cells were excluded from the central AV cushion mesenchyme suggesting that Pitx2 function was required for invasion of Pitx2 daughters into the AV cushion (Fig. 6T,U). As Pitx2c expression is never detected in endocardium, this fate mapping data suggests a myocardial source for the lacZ-labeled cells in the AV cushion.

Defective valve morphogenesis is a common feature in human patients with laterality defects and Pitx2-null embryos (Brown and Anderson, 1999; Icardo and Sanchez de Vega, 1991; Liu et al., 2001). At 16.5 dpc, lacZ-positive Pitx2 descendents were detected in the AV valve leaflets of Pitx2 δ abccreneo heterozygotes but were excluded from the valve leaflets of Pitx2 δabccreneo; δc mutants (Fig. 6V,W).

Defective pulmonary and caval vein morphogenesis in Pitx2 mutants

Corrosion casting and scanning electron microscopy was used to analyze the morphology of the pulmonary veins in Pitx2 mutant embryos. The left superior caval vein (LSCV) normally flows into the coronary sinus, while the right superior caval vein (RSCV) and inferior caval vein (ICV) are connected to the right atrium (RA) by thin strips at the valves. The left and right pulmonary veins join to a common pulmonary vein (PV) that drains into the left atrium (LA) (Fig. 7A,C). In most Pitx2 δc-/- embryos, morphology of these structures was defective, with all these veins running together into a common medial venous sinus (Fig. 7B,D). Consistent with this phenotype, fate mapping with the Pitx2 δabccreneo allele showed that lacZ-positive Pitx2 daughter cells were observed bilaterally in the pulmonary veins of Pitx2 δabccreneo heterozygous embryos but were severely reduced in the pulmonary veins of Pitx2 δabccreneo; δc mutant embryos (Fig. 7E-H).

Fig. 7.

Analysis of pulmonary vein morphology in Pitx2 δ c-/- embryos and fate mapping with Pitx2 δ abccreneo allele. (A-D) Scanning electron microscopy of corrosion casts of wild type (A,C) and the Pitx2 δ c-/- (B,D) embryos. In wild-type embryos, the LSCV drains into the coronary sinus, guarded by the valve of the coronary sinus. The RSCV and ICV are connected to right atrium by thin strips at the valves. The left and right pulmonary veins join to a common pulmonary vein that drains into left atrium. White stars indicate left atrium, just superior to entry of common pulmonary vein (A,C). By contrast, in Pitx2 δ c-/- embryos, all these veins converge into a common, medial venous sinus (B,D). Stars indicate inferior caval vein, just inferior to entry of pulmonary vein and the left superior caval vein. This embryo also has bilateral inferior caval veins. (E-H) Fate mapping with Pitx2 δ abccreneo allele. Transverse sections through lungs of 16.5 wild-type (E) and Pitx2 mutant (F). lacZ positive cells marking Pitx2 daughter cells are present in wild type but are severely reduced in mutant (arrows). (G,H) Whole mounts of 16.5 dpc lungs from wild type (G) and Pitx2 mutants (H), showing lacZ-positive cells in pulmonary veins of wild type and reduced staining in mutant (arrows).

DISCUSSION

In this work, we provide evidence that Pitx2c patterns a presumptive secondary heart field, the branchial arch mesoderm, that invades the heart after looping and contributes to the OFT and right ventricular myocardium. This finding is consistent with the phenotypes of the Pitx2-null embryos that have correct dextral looping of the heart tube but severe defects in cardiac morphogenesis (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Liu et al., 2001; Lu et al., 1999b). We also show that Pitx2c has an important role in asymmetric remodeling of the BAAs. Moreover, our data reveal that Pitx2 daughter cells invade the AV cushions and valves and that this cellular movement into the cushions requires Pitx2 function. The data presented here provide insight into the phenotypes observed in humans with laterality syndromes and demonstrate a direct causal link between the genetic pathways regulating left right asymmetry and complex cardiac morphogenesis.

Pitx2 functions in the presumptive secondary heart field derived from branchial arch and splanchnic mesoderm

Recent advances have revealed that the primary heart field receives contributions from a number of secondary fields. Functional studies have implicated the cardiac neural crest in patterning of the aortic arch vessels and conotruncus of the heart. For example, mice with mutations in components of the endothelin signaling pathway (Yanagisawa et al., 1998), forkhead genes (Iida et al., 1997; Kume et al., 2001; Winnier et al., 1999) and splotch mutant mice (Epstein et al., 2000) have defective arterioventricular connections secondary to cardiac neural crest abnormalities. Moreover, inactivation of Sema3c and neuropilin 1 leads to faulty conotruncal cushion formation as a result of aberrant cardiac neural crest migration (Brown et al., 2001; Feiner et al., 2001; Kawasaki et al., 1999). By contrast, our data reveal that Pitx2c has an important role in patterning a separate heart field derived from the branchial arch and splanchnic mesoderm.

We found that Pitx2c is expressed asymmetrically in the left branchial arch and splanchnic mesoderm within cells that will contribute to the OFT myocardium. Moreover, Pitx2c is also expressed in OFT myocardium and right ventricular myocardium, regions of the heart that are populated by cells derived from branchial arch and splanchnic mesoderm, but not by cardiac neural crest (Jiang et al., 2000; Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). Our fate-mapping studies with the Wnt1 cre and Rosa26 reporter mice also show that cardiac neural crest migrates normally in Pitx2 mutants.

Our data suggest that Pitx2c is not required for the initial migration of branchial arch mesoderm into the outflow tract. Analysis of Sema3c expression in Pitx2c mutants suggests that the defect in Pitx2c mutants occurs relatively late in conotruncal development. Moreover, cre expression in Pitx2 δabccreneo; δ abcnull mutants was similar to that observed in the δabccreneo heterozygous OFT. One idea to explain these data is that Pitx2 functions to maintain signaling between the outflow tract myocardium and underlying endothelium and forming conotruncal cushions. This model would be similar to what has been proposed for Pitx2 in craniofacial development where Pitx2 has a role in epithelial-mesenchymal signaling important for tooth organogenesis (Lin et al., 1999; Lu et al., 1999b). Another possibility, based on the ventricular myocardial defect observed in Pitx2 mutant embryos (see below), is that Pitx2 regulates local expansion of OFT myocardium. We favor the first hypothesis as we do not detect differences in the number or localization of lacZ-labeled cells in the OFT of wild-type and Pitx2 mutant embryos. Nonetheless, it is still formally possible that subtle differences in OFT myocardial expansion could be responsible for the conotruncal defect observed in Pitx2 mutants. Taken together, our findings support the idea that Pitx2 patterns the branchial arch mesoderm and OFT myocardium to support normal development of the conotruncus.

Pitx2c and asymmetric remodeling of the branchial arch arteries

Development of the branchial arch arteries and subsequent remodeling into the mature aortic arch arteries involves a series of paracrine signaling events (Fishman and Kirby, 1998; Hanahan, 1997; Yancopoulos et al., 2000). The forming BAA endothelial tubes are located within the branchial arch mesoderm in close proximity to surface ectoderm and the endoderm-derived epithelium of the branchial pouches. Signaling from endothelium to mesenchyme is thought to be important for recruitment of supporting cells, such as smooth muscle precursors and pericytes, which are important for stabilization of the forming endothelial tubes (Hanahan, 1997).

Vascular remodeling involves local disruption of the critical interaction between endothelium and support cells with resulting regression of the endothelium. In one system, endothelial regression occurs by programmed cell death secondary to loss of survival factors (Meeson et al., 1996; Meeson et al., 1999). The mechanisms underlying asymmetric remodeling of the BAAs, resulting in left-sided aortic arch, are poorly understood.

Cell ablation studies in chick embryos and loss-of-function experiments performed in mice have defined a role for cardiac crest in maintaining the integrity of branchial arch arteries (Brown et al., 2001). However, recent fate mapping experiments using Wnt1 cre and Rosa26 reporter mice, while confirming the importance of the cardiac crest in mouse BAA formation, suggest that cardiac neural crest does not provide the signal for asymmetric remodeling of the BAAs (Jiang et al., 2000).

The important role of branchial arch endoderm in BAA development has been illustrated by phenotypes of individuals with DiGeorge syndrome and mouse models of this syndrome that include severe defects in aortic arch artery formation (Lindsay et al., 2001; Merscher et al., 2001). Importantly, defects were observed more commonly in the right fourth BAAs of a haploinsufficent mouse model for DiGeorge Syndrome (Lindsay and Baldini, 2001). The gene implicated in these events, Tbx1, is expressed in branchial arch endoderm, suggesting that endoderm-derived signals may have a role in asymmetric remodeling of BAA.

Pitx2c is expressed asymmetrically in a very discrete population of cells in proximity to the left aortic sac and left BAAs. Despite this restricted expression, there is a strong BAA phenotype in Pitx2c mutants. These observations suggest that Pitx2c may have a role in recruitment or maintenance of supporting cells to the left BAAs and aortic sac. In wild-type embryos, Pitx2c may be important for stabilization of left-sided BAAs, such as the sixth BAA, that will form the left-sided ductus arteriosus. In the absence of Pitx2c function, maintenance of the sixth BAA would be impaired, resulting in formation of a right-sided ductus arteriosus in some embryos. This alteration would initiate a cascade, perhaps resulting from the altered hemodynamics of the persistent right-sided sixth BAA, to alter remodeling of the other BAAs. Although these ideas will need to be verified in future experiments, our data provide new information about the role of Pitx2 in asymmetric remodeling of the BAA.

Pitx2 in cushion and valve morphogenesis

Our data suggest that Pitx2 has a greater role in AV cushion morphogenesis when compared with formation of the conotruncal cushions. Pitx2-null embryos have severe defects in the central mesenchymal mass that forms the AV cushions and valves resulting in complete AV canal (Kitamura et al., 1999; Liu et al., 2001). The conotruncal phenotype is a failure of rotation of the truncus arteriosus and conotruncal cushion dysmorphology (Kitamura et al., 1999; Liu et al., 2001). Genetic evidence from mice implicates Bmp-signaling in conotruncal cushion morphogenesis (Kim et al., 2001). Noggin overexpression experiments performed in chick embryos revealed that Bmp signaling in conotruncal cushion formation functioned through a mechanism involving regulation of cardiac neural crest migration (Allen et al., 2001). Less is known about the signaling pathways that regulate AV cushion morphogenesis, although recent experiments suggest that Bmp-signaling has a central role (Gaussin et al., 2002).

Data from zebrafish suggest that composition of matrix is of crucial importance in the initial formation of valves and implicate Wnt and Bmp signaling in these events (Walsh and Stainier, 2001). In vitro studies suggest that the action of matrix metalloproteases on cushion mesenchyme is required for migration of mesenchyme into the forming cushions (Song et al., 2000). This epithelial-mesenchymal transition that leads to cushion deposition requires Tgfβ signaling (Brown et al., 1996; Brown et al., 1999). In addition, Tgfβ2 null mice have multiple defects in valve and septal morphogenesis, implicating this signaling pathway in cushion morphogenesis (Bartram et al., 2001; Sanford et al., 1997). Our data reveal that Pitx2 has a role in regulating cellular movement into the formed AV cushion, a late step in cushion morphogenesis. One idea to explain these data is that Pitx2c is required for the myocardial invasion of AV cushion mesenchyme. Pitx2c is expressed in the inner curvature myocardium that surrounds the AV cushion and these myocardial cells have been shown to invade the AV cushion mesenchyme (van den Hoff et al., 2001). However, another possible source of cells that invade the AV cushion is dorsal mesocardium that also expresses Pitx2c. Further experiments are currently under way to elucidate the exact source of invading Pitx2 daughter cells.

Pitx2 function in the venous pole

The data presented here extend our previous understanding of Pitx2 function in development of the venous pole of the heart. Previous studies have demonstrated an important role for Pitx2 in patterning of the atrial appendages and atrial septation (Kitamura et al., 1999; Liu et al., 2001). Analysis of the Pitx2c mutants also reveal a role for Pitx2 in morphogenesis of the pulmonary and caval veins. Fate mapping suggests a direct role for Pitx2 in vein morphogenesis as Pitx2 daughters populate pulmonary and caval veins. Moreover, diminished contribution of Pitx2 daughters to the Pitx2 mutant pulmonary vein suggests a role for Pitx2 in cell movement or cell sorting that may be similar to Pitx2 function in AV cushion morphogenesis. Alternatively, Pitx2 may function to regulate proliferation or survival of pulmonary vein and AV cushion progenitors.

Pitx2 function in expansion of ventricular myocardium

Pitx2c is expressed in the right ventricular and inner curvature myocardium (Campione et al., 2001; Schweickert et al., 2000). Our fate mapping experiment revealed that Pitx2 daughter cells expand to extensively populate both right and left ventricular myocardium. In Pitx2 mutant embryos, fewer Pitx2 daughters are observed contributing to ventricular myocardium. Moreover, analysis of cre expression in Pitx2 mutants, that marks the right ventricle, suggested that the size of the right ventricle was reduced in Pitx2 mutants. One interpretation of these data is that Pitx2 functions in growth of the right ventricular myocardium. Further experiments will be required to distinguish between defective movement of precursors into the right ventricle and failure of the right ventricular myocardium to proliferate.

Acknowledgments

We thank R. Behringer, A. Bradley and P. Soriano for reagents; J. Epstein for sema3c in situ probe; A. McMahon and D. Rowitch for wnt1 cre transgenic line; and A. Baldini for critical comments and insightful discussions. C. L. was supported in part by Harry S. and Isabel C. Cameron Foundation. Supported in part by a grants from NIDCR (R29 DE12324 and R01DE013509), by grant number 5-FY00-135 from March of Dimes to J. F. M., and by the British Heart Foundation (RG/98004 to N. A. B.).

Footnotes

  • * These authors contributed equally to this work

    • Accepted July 24, 2002.

References

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