Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism

Pitx2, a bicoid type homeobox gene, was first identified as the gene responsible for Rieger’s syndrome (Semina et al., 1996). Rieger’s syndrome, defined as a genetic disorder is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma in more than 50% of those affected, dental hypoplasia, mild craniofacial dysmorphism and umbilical stump abnormalities (Rieger, 1935; Jorgensen et al., 1978). We also found that Pitx2 (our old name: Brx1) is a transcription factor expressed in the ventral diencephalon, zona limitans intrathalamica and midbrain (Kitamura et al., 1997). Recently, Pitx2 was reported to participate in the L-R asymmetrical pattern formation in body plan (Ryan et al, 1998; Yoshioka et al., 1998; Logan et al., 1998; Piedra et al., 1998; St. Amand et al., 1998). Pitx2 is expressed on the left side of the axis shortly after nodal is detected in the lateral plate mesoderm. The Pitx2 expression domain is larger than that of nodal, persists longer, and includes not only the left lateral plate mesoderm but also the left side of the developing gut and heart. Both nodal and lefty- 2 can induce ectopic Pitx2 in chick embryos. If the right side of the axis of chick embryos is infected with retroviruses expressing Pitx2, both the heart and derivatives of the gut become left isomerized. Pitx2 is repressed in FGF8-deficient mice, while Shh-deficient mice show expression of Pitx2 in the bilateral mesoderm (Meyers and Martin, 1999). Hence, Pitx2 seems to act as a global executor of L-R patterns downstream of the genetic cascade that regulates asymmetry.

Pitx2 has three isoforms, although it is not yet known which of the isoforms are expressed symmetrically and which asymmetrically (Kitamura et al., 1997; Gage and Camper, 1997). We tried to determine the genomic structure of the Pitx2 gene and then to characterize the expression pattern of each isoform. Further, we generated Pitx2-deficient mice by homologous recombination in order to elucidate the in vivo function of Pitx2 in various expression sites. Homozygous Pitx2^−/− embryos fail to close the ventral body wall and show cardiac malformation, anomalies of extra- and periocular mesoderm and right pulmonary isomerism, and finally die around days 14-15.

The Pitx2 gene was cloned from a 129SVj mouse genomic library (Stratagene) using Pitx2 cDNA as a probe (Kitamura et al., 1997). The 4 independent genomic clones were isolated and the exon/intron structure was confirmed by sequence analysis (Fig. 1A). The reporter gene cassette ‘IRES-T-lacZ’ derived from TV2 vector was used to construct the targeting vector (Takeuchi et al, 1995). The stop codon leading to termination of translation of Pitx2 was inserted in front of IRES. The STOP-IRES-T-lacZ cassette and a 3 kb NruI-SalI fragment located at the 3′-position of the NruI site in a homeobox and an EcoRI-XhoI fragment of pKJ2 were ligated and subcloned into pBluescript II (Fig. 2A; Boer et al., 1990). Further a 2.6 kb HindII-NruI fragment located at the 5′-position of the NruI site in the homeobox and the TK gene cassette were inserted in this order into the construct (Fig. 2A).

In order to obtain the targeted ES cells, we used a positive-negative selection strategy in the 129/Sv derived ES-cell line E14TG2a. The ES colonies were screened by Southern blot analysis with a 5′ and a 3′ probe external to the genomic sequences that were contained in the targeting vector and a Neo (Fig. 2A,B). Thirteen ES clones from 168 clones were found to have undergone homologous recombination (Fig. 2B).

The positive ES clones were injected into C57BL blastocysts and transferred into pseudopregnant female recipients. The resulting chimeric mice were bred with C57BL females. Transmission of the targeted Pitx2 locus was confirmed from the Southern blot analysis. Pitx2^−/− embryos used in the following analysis were obtained by crossbreeding F₂Pitx2+/− mice (Fig. 2C). The Pitx2−/− homozygotes derived from the two independent targeted ES cell lines showed indistinguishable phenotypes.

Noon of the day after copulation was considered as day 0.5 p.c. In situ hybridization was performed as described previously (Kitamura et al., 1997). For X-gal staining, embryos were fixed with 0.2% glutaraldehyde/1% formalin/0.02% NP-40 for 2 hours, and if necessary, cryosections prepared from each embryo, and stained with X-gal. Skeletal preparation and staining was conducted essentially as described by Hogan et al. (1994).

Tissue for electron microscopy was fixed, stained and embedded using a conventional method. Thin sections were stained with uranyl-lead citrate before viewing in a JEOL 1200EX electron microscope.

We previously found two Pitx2 isoforms, Pitx2b (old name: Brx1b, accession number: AB006321) and Pitx2c (old name: Brx1a, accession number: AB006320) from a mouse embryonic library (Kitamura et al., 1997). In the present study, we found another Pitx2 isoform, Pitx2a, which is the same as Rieg and otlx2 (Semina et al., 1996; Mucchielli et al., 1996; Gage and Camper, 1997). The structure of the Pitx2 gene was examined in order to elucidate the exon/intron structure of the gene and assign each exon into one of the three isoforms. An open reading frame region of the three Pitx2 isoforms was found to consist of at least five exons (Fig. 1A). Exon I was specific to Pitx2a and Pitx2b, exon II only to Pitx2b, and exon III only to Pitx2c, and further exons IV and V were common to all Pitx2 isoforms (Fig. 1A). Thus, Pitx2a, Pitx2b and Pitx2c were alternative splicing products from one Pitx2 gene. The human RIEG gene has a non-coding exon at the 5′-end, however, we were unable to find it in the present study (Semina et al., 1996).

We examined whether the complicated expression of Pitx2 could be regionally assigned to the expression pattern of each Pitx2 isoform. cDNA corresponding to exons I and II was too short to function as a specific probe for in situ hybridization of Pitx2a or Pitx2b, and therefore the expression of Pitx2a/Pitx2b and Pitx2c was examined in ICR mouse embryos at 9.5 dpc. Interestingly, asymmetric expression in the heart and gut was attributed to Pitx2c (Fig. 1C-E, compared with C′-E′), while the symmetric expression in the head mesoderm and the lateral body wall was common to both the Pitx2a/Pitx2b and Pitx2c genes (Fig. 1F,F′,G,G′). Thus, only Pitx2c is expressed asymetrically.

To suppress the expression of all Pitx2 isoforms, we disrupted exon IV of the Pitx2 gene by inserting the lacZ gene (Fig. 2A). The F2 heterozygous crosses resulted in normal Mendelian ratios of homozygote, heterozygote and wild type (87:162:93) until 14.5 dpc, and thereafter no Pitx2^−/− embryos were found. Pitx2^−/− embryos at 10.5 dpc exhibited a lack of ventral closure (Fig. 3B, compared with A). Further, at 13.5 dpc, the typical morphology of Pitx2^−/− embryos was extrusion of visceral organs (evisceration) to the left side, anticlockwise bending of the body axis at a thoracic/abdominal site and depression of the oculus (Fig. 3C-F). However, the morphology of the Pitx2^+/− embryos was similar to that of the Pitx2^+/+ embryos.

Pitx2 is expressed in the left lateral plate mesoderm at 8-9 dpc and begins to be expressed in both the left and right distal ends of the lateral body wall mesoderm at 9.0-9.5 dpc (Fig. 4A,B). The right distal end extends towards the midline, while the left distal end extends towards the exterior at 9.0-9.5 dpc (Fig. 4C2). Along with development, the left distal end also comes close to the midline umbilical artery and ventral closure of the left and right body walls begins from the posterior end of the body axis (Fig. 4C1, F). In order to elucidate the differences in the orientation of the left distal ends of Pitx2^+/− and Pitx2^−/− embryos, we analyzed the number of lacZ(+) cells in the distal ends at 9.25-9.5 dpc. Cell counts were done in the two caudal regions, P-1 and P-2, because a change in the orientation of the left distal ends and the closure of both lateral walls are started from the caudal end of the body axis. The right distal ends in the P-1 and P-2 regions of both Pitx2^+/− and Pitx2^−/−embryos extend internally (Fig. 4C1, C2, D1, D2), and the numbers of lacZ(+) cells of the right distal ends in the P-1 and P-2 region of Pitx2^−/− embryos were each about 1.2 times those in Pitx2^+/− embryos (Fig. 4E). The left distal ends in P-1 of Pitx2^+/− embryos orient toward the axis, while those in Pitx2^−/− embryos orient to the exterior (Fig. 4C1, D1). The number of lacZ(+) cells in the left P-1 of Pitx2^−/− embryos was about 2.0 times that in Pitx2^+/− embryos (Fig. 4E). Further, in the left P- 2 region, although Pitx2^+/− embryos have not yet finished changing the orientation of the distal end towards the axis (Fig. 4C2), the number of lacZ(+) cells in left distal end in P-2 of Pitx2^−/− embryos was also about 1.8 to 1.9 times that in Pitx2^+/− embryos (Fig. 4E).

A lot of lacZ(+) cells were found on the left half of the amnion ball of Pitx2^−/− embryos (Fig. 4J, K, compared with H,I), and furthermore, the electron microscopic observations showed that the mesodermal cell layers of Pitx2^−/− embryos are composed of 4 to 6 layers, while that of Pitx2^+/+ embryos was only one layer (Fig. 4M, compared with L). The increase in the number of lacZ(+) cells in the left distal end and the thickening of the lacZ(+) left amnion in the Pitx2^−/− embryos are thought to result in a change of normal impetus, physical constraints and direction of the extending body wall. The left lateral body wall in Pitx2^−/− embryos actually did not turn inwards, resulting in failure of the ventral body wall closure (Fig. 4G, compared with F).

Anticlockwise bending of the body axis was found at the thoracic/abdominal site in the Pitx2^−/− embryos. In these embryos when the body wall failed to close there was extrusion of not only abdominal but also thoracic organs toward the left side (Fig. 3E,F). The left ribs splayed outwards, while the right ribs remained in the cartilage primordium of the proximal part of each rib and did not form the left part of the rib cage (* in Fig. 5B). The number of ribs was unchanged on both sides. In Pitx2^+/+/Pitx2^+/− embryos at 11.0-11.5 dpc the loop of the midgut is formed in the abdominal cavity when the midgut extends and then rotates in an anticlockwise direction (−90°), but this primary rotation does not takes place in Pitx2^−/− embryos (Fig. 5D, compared with C).

In the wild type, Pitx2 was expressed only in the left bud of lung rudiment at 9.5 dpc (Fig. 5E,F). The right lung bud forms four lobes while the left lung bud only forms one. The asymmetric pattern of lung lobation was, however, altered in the Pitx2^−/− embryos in which the left lung also had four lobes, in a mirror image to the right lung (Fig. 5H, compared with G). This condition is known as right pulmonary isomerism.

Development of the cardiovascular system in the Pitx2^−/− embryos showed various patterning defects. At 10.5 dpc, the hearts of the Pitx2^−/− embryos showed the same d-ventricular loop as normal hearts (Fig. 6A,B). However, the mutant hearts showed hypoplasia of the right ventricle and enlargement of the left atrium. It seemed that the mutant right ventricles were sometimes positioned on the left ventricle (not shown). In Pitx2^−/− hearts at 11.5 dpc, a prominent swelling was seen at the atrioventricular (AV) canal region between the left atrium and left ventricle (arrowheads in Fig. 6D, compared with C). Inside the swelling of the Pitx2^−/− heart, the AV cushions exhibited pronounced growth (Fig. 6E′). Expression of lacZ was seen in the prominent swelling at the AV canal region (Fig. 6D). In serial sections of Pitx2^−/− embryos at 10.5 dpc, lacZ expression in the AV canal region was limited to the myocardium adjacent to the AV cushion but was not seen in the cushion tissues (arrows in Fig. 6F). Pitx2^−/− hearts at 13.5 dpc failed to develop the tricuspid valve within the right ventricle and the mitral valve within the left ventricle, and formed a common AV valve on the left ventricle (arrows in Fig. 6H, compared with G). The right ventricle in the Pitx2^−/− hearts was connected to the common AV valve through a ventricular septal defect (not shown).

The Pitx2^−/− hearts had the morphological right atrial appendage juxtaposed with the morphological left atrial appendage at 13.5 dpc (Fig. 6J, compared with I. Arrowheads indicate left-sided juxtaposition of the atria). The atria of the Pitx2^−/− hearts formed common atrium with an ostium primum atrial septal defect (not shown). In Pitx2^+/+/Pitx2^+/− hearts at 13.5 dpc, both the left and right superior venae cavae and the inferior vena cava were connected to the right atrium, and the common pulmonary vein was connected to the left atrium (Fig. 6K,L). In contrast, the common atrium of the Pitx2^−/− hearts was connected to the bilateral right and left superior venae cavae, inferior vena cava, and common pulmonary vein (Fig. 6M,N).

The aorta is located posteriorly and to the right of the pulmonary trunk, and the aorta originates from the left ventricle and the pulmonary trunk from the right ventricle in Pitx2^+/+/Pitx2^+/− hearts (Fig. 6G, I). In Pitx2^−/− hearts, the aorta was located anteriorly and to the right of the pulmonary trunk, and both great arteries arose from the right ventricle (double outlet right ventricle) and were in parallel positions with the aorta located to the right of the pulmonary trunk (Fig. 6H,J).

Pitx2 is expressed in extraocular muscles, as shown by the coexpression of Pitx2 with myogenin and myf5 (Fig. 7A-C). In Pitx2^−/− embryos, no expression of myogenin or myf5 was found at 12.5 dpc (Fig. 7D,E) and no extraocular muscle was formed, while Pitx2-expressing tongue muscles were formed normally (Fig. 7H,I). Further, it is noted that no lacZ(+) cells were detected in the extraocular muscle forming region (Fig. 7G, compared with arrowheads in F). Along with the deletion of extraocular muscles, in Pitx2^−/− embryos there was a 5- to 10-fold increase in thickening of the lacZ(+) mesothelial layer of the cornea between cuboidal epithelium constituting the anterior part of the lens and cornea ectoderm at 12.5-13.5 dpc (Fig. 7G, compared with F; Kaufman, 1998). The thick mesothelial layer was invaded by the cuboidal epithelium of the anterior lens after 12.5 dpc (Fig. 7G). The extraocular muscle dysgenesis and corneal thickening resulted in enophthalmos.

Deficiency of FGF8, an upstream factor in the genetic cascade that participates in L-R asymmetry in the vertebrate body plan, results in asymmetrical abnormalities at the organ level in the lung, heart and digestive tube (Meyers and Martin, 1999). The present study has shown that Pitx2, a downstream factor, also participates in asymmetry of lung at the organ level (right pulmonary isomerism), while asymmetrical abnormalities of the heart and digestive tube at the organ level (reversed looping and situs) were not observed in the Pitx2 deficient embryos. However, the present results indicated that Pitx2 plays important roles in the various phases of asymmetric morphogenesis of heart and gut and symmetric morphogenesis of lateral body wall and extra- and periocular mesoderm. The latter anomalies in the morphogenesis are closely related to Rieger’s syndrome.

We have shown that three isoforms of Pitx2 are derived by an alternative splicing of the Pitx2 gene, and that only Pitx2c is expressed asymmetrically. As Pitx2c is also expressed symmetrically in the head and lateral body wall, it is under dual control. Thus, it is expected that ectopic expression of upstream genes of Pitx2 will result in the induction of Pitx2c in the right lateral plate mesoderm. Pitx2 has been ectopically expressed in chick embryos using Pitx2a (Logan et al., 1998).

Nevertheless, the ectopic expression resulted in the asymmetrical effects on the morphology of the heart and gut. Thus, this shows that the asymmetrical effects of Pitx2 are carried by exons IV and V which are part of the homeobox and the 3′ region downstream to the homeobox.

Pitx2 is expressed in the left lateral plate mesoderm of mouse and chick embryos (Ryan et al, 1998; Yoshioka et al., 1998; Logan et al., 1998; Piedra et al., 1998; Campione et al., 1999). Ectopic Pitx2 expression in chick embryos at stage 4 was shown to result in a reversal in the direction of embryonic rotation (Ryan et al., 1998), while in the present study, Pitx2- deficient mouse embryos showed no reversal of axial rotation at 8-9 dpc and thus were different from situs inversus (iv/iv) mutant embryos (Hummel and Chapman, 1959). Although the reason for this is unknown, the deficiency of Pitx2 may not result in the activation of cell proliferation in right lateral plate mesoderm and in right extraembryonic membrane at 8.0-9.0 dpc (Miller and White, 1998).

The effects of Pitx2 deficiency were first found in the left lateral body wall of the embryos at 9.25-9.5 dpc. An increase in the Pitx2^−/− cells (lacZ positive) was followed by the exterior bending of the distal end of the wall and thickening of the amnion. Pitx2^−/− and Pitx2^+/− mesodermal cells in the distal end also spread throughout the lateral body wall as development progressed after 9.5 dpc. However, the thickening of the amnion was not observed in Pitx2^+/+/Pitx2^+/− embryos. Thus, the thickening of the amnion associated with the early increase of Pitx2^−/− mesodermal cells in the left distal end at 9.25-9.5 dpc is thought to be very important for the exterior bending of the left lateral body wall. We attempted to detect the changes in expression of genes that are expressed in the body wall (BMP1, BMP2, BMP4, Prx1, Prx2), however, no changes in expression were observed. This strongly suggests that the changes in the left lateral body wall are due to physical constraints. This is supported in the case of iv/iv mutant embryos in which asymmetrical cell proliferation in the embryonic body and extraembryonic membrane results in axial rotation (Miller and White, 1998). Lack of ventral closure has not been reported in mice deficient in genes related to asymmetry, such as FGF8, Shh, lefty-1 and ActRII. Furthermore, not only asymmetrically expressed Pitx2c but also symmetrically expressed Pitx2a/Pitx2b are present in the lateral body wall after 9.25 dpc. Thus, Pitx2 that causes a failure of ventral closure is under another genetic cascade which is different from the FGF8-derived genetic cascade for asymmetry.

Lung anatomy is a good indicator of asymmetry. Mice mutant in Shh and lefty-1, which are midline signals, have left lung isomerism, while mice mutant in FGF8 and ActRIIB, which are left sided signals, have right lung isomerism (Chiang et al., 1996; Oh and Li, 1997; Meno et al., 1998; Meyers and Martin, 1999). Deficiency of Pitx2 also resulted in right pulmonary isomerism. Thus, Pitx2 is involved in lung asymmetry at the organ level. In early development of the lung, the right bud of the lung rudiment is larger than the left one, and FGF10 is expressed in the right bud earlier than in the left one (Bellusci et al., 1997). The expression of Pitx2 in lung is stage and site specific, and Pitx2 is expressed only in the left lung bud. This suggests the possibility that a property of “left” in the left lung rudiment may be endowed with the repressive regulation of proliferation in the left endoderm by Pitx2 expression, compared with active proliferation in the right endoderm in the absence of Pitx2.

Cardiac looping has been useful as the earliest morphological marker for determining asymmetry in vertebrate embryos. Mice mutant in FGF8 and SIL showed reversed cardiac looping (Meyers and Martin, 1999; Izraeli et al., 1999). On the other hand, deficiencies of Shh, lefty-1, ActRIIB, and Pitx2 in the present study did not result in reversed cardiac looping, although the mice mutant in lefty-1, ActRIIB and Pitx2 showed alteration in the development of the cardiovascular system (Chiang et al., 1996; Oh and Li, 1997; Meno et al., 1998). Thus, it is thought that determination of lung asymmetry and cardiac looping may occur via different gene pathways.

Congenital cardiovascular anomalies are the most common form of human birth defects and are well characterized anatomically and physiologically. However, there is still little information on the genetic basis for most of these anomalies. In this study the Pitx2^−/− hearts were characterized by a common AV valve with ventricular septal defect and a common atrium showing an ostium primum atrial septal defect. In humans the morphology of the heart in the Pitx2^−/− embryos is similar with a complete type of AV septal defect (AVSD; Feldt et al., 1995). After cardiac looping, it is generally assumed that the AV canal shifts to the right side and the outflow tract shifts to the left side (Lamers et al., 1992), following which the right and left atria connect to the right and left ventricles, respectively, to establish the four chambered heart. The pulmonary trunk and aorta then connect to the right and left ventricles, respectively, to establish the normal ventriculoarterial connections. Since the AV cushions showed prominent growth in the Pitx2^−/− hearts, it is speculated that the right-ward shift of the AV canal is inadequate and causes the left sided juxtaposition of the right atrium appendage. The left-ward shift of the outflow tract is not sufficient to form the normal ventriculoarterial connection. During development in the Pitx2^−/− embryos, the AV canal region in the heart exhibited prominent growth. The myocardium of this region adjacent to the AV cushion tissues showed lacZ expression. It is likely that the myocardium in the AV canal region plays an important role in regulating the transformation of endothelial cells into the cushion mesenchyme (Eisenberg and Markwald, 1995). Therefore, it is thought that disruption of Pitx2 in the myocardium of the region results in hyperplasia of the cushion tissues. Pitx2, although it is one of the genes that mediate asymmetry in vertebrates, seems to be an important gene for cardiac morphogenesis. The Pitx2 mouse model will prove valuable for understanding the developmental mechanism of AVSD.

Pitx2 and Pax3 colocalize in trunk myotome, while only Pitx2 is expressed in the extraocular muscle region (not shown, Tajbakhsh et al., 1997). Muscle differentiation was seen to proceed normally in trunk myotome and tongue of Pitx2^−/− embryos, while no extraocular muscle was formed. This suggests two possibilities: Pitx2 is a key upstream regulatory gene in extraocular muscle differentiation in place of Pax3, or there is no redundancy with another related gene for Pitx2 functions in the extraocular muscle differentiation. In either case, the extraocular muscle-forming cells in Pitx2^−/− embryos lose the muscle traits and get mixed with lacZ(+) Pitx2^−/− mesoderm in the anterior periocular region. Another quite different possibility is that the progenitor cells of the extraocular muscles, per se, were lost in Pitx2^−/− embryos, although the origin of the progenitor cells has not yet been precisely identified.

The human Pitx2 gene, RIEG, which is responsible for Rieger’s syndrome, is the human homolog of Pitx2a that is expressed symmetrically (Semina et al., 1996). The mutations in Rieger’s syndrome include C-terminal truncations and point mutations in the homeobox as well as splice mutations. The mutated genetic structure of Pitx2a in the Pitx2^−/− embryos was partially similar to that in Rieger’s syndrome. Therefore, common defects were found in the Pitx2^−/− embryos and Rieger’s syndrome patients, such as defects in the umbilical cord and peri- and extraocular mesoderm. Enophthalmos in Pitx2^−/− embryos is seen in Rieger’s syndrome. Furthermore, the thickening of the mesothelial layer of the cornea is referred to as the prestage in iris dysplasia, macrocornea, goniodysgenesis and congenital cataract in Rieger’s syndrome. Thus, the present Pitx2^−/− embryos are useful for understanding the developmental mechanisms of Rieger’s syndrome. On the other hand, the contribution of the human homolog of Pitx2a to Rieger’s syndrome explains the fact that laterality defects have not yet been reported in the syndrome. The interesting cardiac defects in the Pitx2^−/− embryos suggest that a part of Rieger-associated abnormalities may possibly be caused by a deficiency of human homologs of other Pitx2 isoforms (Kulharya et al., 1995).

The involvement of Pitx2 in craniofacial, tooth, pituitary and heart morphogenesis has been described very recently (Lu et al., 1999; Lin et al., 1999; Gage et al., 1999).

The authors express their appreciation to Dr Austin G. Smith for the ES-cell line E14TG2a, Dr Kazunori Hanaoka for the TK gene cassette, Dr Takashi Takeuchi for the IRES-T-lacZ cassette, Dr Naohiro Hashimoto for the myogenin and myoD clones, Drs Shosei Yoshida and Kohji Uchiyama for the myf5 clone, Dr Ayumi Miyake for helpful discussions and Mr. Shinichi Kamijo for providing the mouse embryos.