1 Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA
2 Department of Anatomy and Developmental Biology, St. Georges Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, UK
*Author for correspondence (e-mail: jmartin{at}ibt.tamu.edu)
Accepted March 7, 2001
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SUMMARY |
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Key words: Homeobox, Left-right asymmetry, Morphogenesis, Mouse
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INTRODUCTION |
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Insight into this problem has been obtained by the demonstration that Pitx2, a paired-related homeobox gene that was identified as the gene mutated in Rieger syndrome type I (Semina et al., 1996), plays an important role in the local generation of asymmetry within organs. Overexpression studies performed in chick and Xenopus embryos suggested that Pitx2 functioned in handed organs to interpret the asymmetric signals that originate in the pre-somitic embryo (Campione et al., 1999; Logan et al., 1998; Piedra et al., 1998; Ryan et al., 1998). Loss-of-function experiments in mice supported the idea that Pitx2 played an important role in asymmetric morphogenesis of multiple organs (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999). However, it remained unclear how a single transcription factor functioned in different contexts to direct both asymmetric organ morphogenesis and the development of symmetric organs such as the teeth and eyes.
Recently, it has been recognized that the Pitx2 gene encodes three isoforms, Pitx2a and Pitx2b that are generated by alternative splicing mechanisms, and Pitx2c that uses an alternative promoter located upstream of exon 4 (Fig. 1K,L; Kitamura et al., 1999; Schweickert et al., 2000). Although overexpression of any Pitx2 isoform alters left-right asymmetric organ morphogenesis (Essner et al., 2000; Logan et al., 1998; Ryan et al., 1998), other experiments suggest that Pitx2a, Pitx2b and Pitx2c have distinct expression profiles and target genes (Essner et al., 2000; Kitamura et al., 1999; Schweickert et al., 2000). Thus, different Pitx2 isoforms may have distinct roles in left-right asymmetry and symmetric organogenesis.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as previously described (Lu et al., 1999). The Pitx2c-specific probe was a 1 kb genomic fragment containing exon 4 that was linearized with XhoI and transcribed with T7 polymerase. The Pitx2a and Pitx2b-specific probe was a genomic fragment containing exons 2 and 3 that was linearized with NotI and transcribed with T3. The probes for bone morphogenetic protein 4 and sonic hedgehog have been previously described (Echelard et al., 1993; Winnier et al., 1995).
Histology
Embryos were fixed overnight in Bouins fixative, dehydrated through graded ethanol and embedded in paraffin. Sections were cut at 7 µm and stained with Hematoxylin and Eosin.
Ribonuclease protection assays
Whole embryo RNA was harvested with triazol reagent (Gibco) according to the manufacturers instructions. Ribonuclease protection assays were performed using the RPA II kit (Ambion). The probe for nuclease protection assays was a NcoI/NotI subclone from a Pitx2c cDNA. Pitx2c-protected fragments were quantitated with a phosphoimager and relative values analyzed for statistical significance (ANOVA). Standardization for differences in loading was performed separately using ß-actin. Four experiments were performed and the difference in Pitx2c levels between abcnull +/- and the
abcnull;
abhypoc alleles was statistically significant (P<0.05). However, the difference in Pitx2c mRNA levels between the
abcnull +/- and
abcnull;
ab and between the
abcnull;
abhypoc and
abcnull;
ab allelic combinations did not reach statistical significance.
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RESULTS |
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In developing guts, Pitx2a, Pitx2b and Pitx2c were expressed in the stomach, cecal diverticulum, duodenum and midgut (Fig. 1D,F). With the exception of the cecal diverticulum, expression of Pitx2a and Pitx2b was a minor component of overall Pitx2 gut expression, and was most readily detectable in mice homozygous for a Pitx2 lacZ knock-in allele by X-gal staining (see below and Fig. 1F). Pitx2c, Pitx2a and Pitx2b were expressed symmetrically in oral ectoderm, body wall, umbilical structures and the developing eye at later developmental stages (Fig. 1G,H and not shown).
Isoform-specific deletion of Pitx2a and Pitx2b
To dissect the functions of the Pitx2 isoforms, we used gene targeting in ES cells to generate the ab and
abhypoc alleles that removed the Pitx2a and Pitx2b isoforms by introducing lacZ into exon 2 while deleting the coding region of exon 2 and all of exon 3 (Fig. 1K-N). The
abhypoc allele contained a LoxP-flanked PGKneomycin cassette that was removed with Cre recombinase to create the
ab allele (Fig. 1M,N).
We intercrossed ab+/- mice and found that a proportion of
ab;
ab mice were viable and fertile. Genotyping of weanling progeny from crosses between
ab;
ab and
ab+/- mice showed that 29% were homozygous mutant, suggesting a loss (21%) of homozygous mutant mice in the postnatal period (n=58). Analysis of neonatal
ab;
ab mice revealed that postnatal lethality was secondary to cleft palate (9%; n=30) or midgut malrotation (87%; n=30) that in some cases resulted in volvulus and bowel infarction. In addition, we found that
ab;
ab mutants had eye defects (Fig. 1I,J).
We examined the expression pattern of Pitx2c in 10.5 dpc ab;
ab mutant embryos. Pitx2c expression in left lung bud and left gut in
ab;
ab mutants was identical to wild-type littermate controls (Fig. 1D,E) suggesting that spatial expression of Pitx2c from the
ab allele is similar to the wild-type allele. We also performed ribonuclease protection assays on whole 12.5 dpc embryo mRNA using a probe that distinguishes between Pitx2 isoforms (Fig. 1O,P) to measure Pitx2c levels in
abcnull heterozygotes and
abcnull;
ab and
abcnull;
abhypoc allelic combinations. As the
abcnull allele does not express Pitx2c (Fig. 1P, lanes 11 and 12; Fig. 1Q, lane 5), this analysis measured Pitx2c expression from the wild-type, the
ab and the
abhypoc alleles.
We found that expression of Pitx2c in the abcnull heterozygotes was 58±11% compared with embryos with two wild-type Pitx2 alleles (Fig. 1P, lane 6; Fig. 1Q, compare lanes 1 and 2). Expression of Pitx2c in the
abcnull;
ab embryos was 50±14% (not statistically significant when compared with
abcnull+/-; Fig. 1P lanes 7,8; Fig. 1Q compare lanes 2 and 3); in the
abcnull;
abhypoc embryos, Pitx2c expression was 37±11% (P<0.05, compared with
abcnull +/-; Fig. 1P, lanes 9,10; Fig. 1Q compare lanes 2 and 4). These results show that the
abhypoc allele encodes significantly reduced levels of Pitx2c mRNA compared with the wild-type allele in the
abcnull heterozygous embryos. Moreover, the ribonuclease protection assay data suggest a trend in which the
ab allele encodes levels of Pitx2c intermediate between that of the
abhypoc and wild-type alleles.
The abhypoc and
ab alleles encode different degrees of Pitx2c function
Although the ribonuclease protection assay analysis suggests that Pitx2c levels are comparable in the abhypoc and
ab alleles, it is possible that this analysis has missed subtle, but biologically significant differences between the two alleles. Moreover, as it has been reported that a retained PGKneomycin cassette in an intron can interfere with Pitx2 function (Gage et al., 1999), we suspect that the PGKneomycin in the
abhypoc locus might also have a deleterious effect on Pitx2c function.
In order to test this idea and determine if the abhypoc and
ab alleles encode equivalent Pitx2c function, we performed a genetic experiment and intercrossed these Pitx2 alleles with the
abcnull allele. The
abcnull;
abcnull mutant embryos, that lack all Pitx2 function, died by 14.5 dpc (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999). In contrast, intercrosses between the
abcnull and
abhypoc heterozygous mice showed that about half of
abcnull;
abhypoc embryos were still alive at 16.5 dpc (Table 1). Moreover, the
abcnull;
ab mutants survived 2 days longer, as embryo loss was first detected at 18.5 dpc (Table 2). These genetic data suggest that the
abcnull;
abhypoc and
abcnull;
ab embryos survive longer than
abcnull;
abcnull embryos because of residual Pitx2c function. In addition, as a result of the retained PGKneomycin cassette, the
abhypoc allele encodes less Pitx2c function than the
ab allele. Other genetic evidence, obtained from analysis of lung phenotypes, also suggests that the
ab allele encodes slightly less Pitx2c than the wild-type allele (see below).
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Pitx2c provides left identity to atrial primordia
Although abcnull;
abcnull mutant mice have correct rightward ventricular looping, they have numerous defects in cardiac development (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999). Normal right atrial structures include the coronary sinus and the venous valves, while the left atrium has the pulmonary vein. Histological analysis showed that
abcnull;
abcnull mutants had no coronary sinus, bilateral venous valves, anomalous pulmonary venous drainage, and a deficiency in the primary interatrial septum (compare Fig. 2A,B,I with 2E-H,N,O). The outflow tract cushions of
abcnull;
abcnull mutants were symmetric and the trunks of the great arteries were malaligned and unseptated (compare Fig. 2B with 2G,H,N,P). In addition, all had defects in ventriculoarterial connections, usually double outlet right ventricle (DORV), and some had a common atrioventricular canal (Fig. 2J,P). This spectrum of morphological defects is specifically associated with right isomerism in human hearts (Brown and Anderson, 1999), and strongly suggests that the
abcnull;
abcnull embryos had right atrial isomerism.
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Requirement for Pitx2c, Pitx2a and Pitx2b in lungs
Primary lung buds and mature lungs, which develop as an outpouching of the foregut, have left-right asymmetric morphology (Hogan, 1999; Fig. 3A,B). It has previously been shown that abcnull;
abcnull embryos have complete right pulmonary isomerism (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999). Consistent with the expression of Pitx2c in left splanchnopleure and primary lung buds, we found that pulmonary right isomerization was evident at the primary lung bud stage in
abcnull;
abcnull embryos (Fig. 3C,D).
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We investigated the secondary lung bud branching pattern in Pitx2 allelic combinations using whole-mount in situ hybridization with probes for sonic hedgehog (Shh), which marks lung bud endoderm, and bone morphogenetic protein 4 (Bmp4), which marks both endoderm and mesoderm (Bellusci et al., 1996; Bellusci et al., 1997). In abcnull;
abcnull mutant embryos, the left-sided branching pattern was identical to that of the right lung bud or right isomerized (Fig. 3J,K,O,P). In both
abcnull;
abhypoc and
abcnull;
ab embryos, the left-sided branching pattern was also right isomerized (Fig. 3L,M,Q,R). This suggested that these Pitx2 allelic combinations fail to express adequate Pitx2c for normal left-sided branching morphogenesis. The initial aspects of branching morphogenesis in
ab;
ab mutant embryos were similar to wild-type, suggesting a later, minor function for Pitx2a and Pitx2b in pulmonary morphogenesis (Fig. 3N). Based on these phenotypes, and the expression pattern of Pitx2c in left splanchnic mesoderm and left primary lung bud, we conclude that high levels of Pitx2c in the lung primordia are necessary for left-specific lung morphogenesis.
Pitx2c cooperates with Pitx2a and Pitx2b to regulate gut morphogenesis
The duodenum, the most rostral part of the small intestine, forms from distal foregut and rostral midgut. The early gut tube loops to the left, then as the duodenum develops, it rotates to the right to form a C-shaped structure with stereotypical relationships to the liver, pancreas and biliary tree (Moore, 1982). In all Pitx2 allelic combinations, the initial bending of the gut tube to the left was unaffected (Fig. 4A-E). However, in abcnull;
abcnull embryos, rotation of the duodenum failed to occur in the majority (72%, n=18) of embryos examined (Fig. 4F,G). A small percentage had correct or reversed rotation at the duodenum (Table 3).
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We examined duodenal Pitx2c expression, that is normally left-sided, in abcnull;
ab and
abcnull;
abhypoc embryos. Pitx2c was bilaterally expressed in the duodenum of these mutant embryos, suggesting the existence of a regulatory mechanism within the developing gut that normally restricts Pitx2c expression to the left side (Fig. 4K-M).
As midgut develops, it forms a cranial limb that gives rise to small bowel and a caudal limb that develops into large intestine. The midgut limbs rotate through a 270° counterclockwise movement that results in the final positioning of the small and large bowels (Moore, 1982). In 87% (n=30) of ab;
ab neonates, the midgut failed to rotate resulting in a right-sided midgut mass (Fig. 4N,O). In the remainder (n=4) of
ab;
ab neonates midgut rotation arrested midway through rotation. In addition, 21% of
ab;
ab neonates showed annular pancreas (Fig. 4O).
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DISCUSSION |
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Cardiac development requires only low Pitx2c levels
The cardiac atria were right isomerized in abcnull;
abcnull mutant embryos. We found right sino-atrial isomerism, including symmetry of sinus horns and bilateral paired venous valves, and abnormalities of atrioventricular and ventriculoarterial connections. In contrast,
abcnull;
abhypoc and
abcnull;
ab embryos, which encode the next highest levels of Pitx2c, had almost normal atria. From these data, we conclude that only low levels of Pitx2c are necessary to provide left identity to most of the atrium. These results also provide insight into the observation that cardiac anomalies, although described in families with Rieger syndrome, are uncommon (Bekir and Gungor, 2000; Cunningham et al., 1998; Mammi et al., 1998). In contrast, the atrial pectinate pattern requires higher levels of Pitx2c. In this respect, the atrial appendages resemble the lungs, although higher Pitx2c levels are required by the lung primordia. This fits well with the clinical observation that isomeric atrial appendages are virtually always associated with isomeric lung lobation (Brown and Anderson, 1999). In addition, the lung and atrial appendage primordia develop in close proximity.
Lungs need the highest doses of Pitx2c
Forming lungs require high levels of Pitx2c for normal morphogenesis. The abcnull;
abhypoc and
abcnull;
ab allelic combinations, which had nearly normal hearts, showed strong right pulmonary isomerism phenotypes. Branching morphogenesis involves a reiterative branching mechanism in which an initial pattern is established and modified at successive steps in a stereotypical fashion (Hogan, 1999; Metzger and Krasnow, 1999). Our finding that the primary lung buds of
abcnull;
abcnull mice are right isomerized suggests that Pitx2c functions prior to or at the initial stages of the branching process. The lung phenotypes of the
ab;
ab mutants suggest that Pitx2a and Pitx2b has a later, more restricted function in lung morphogenesis.
Tight control of Pitx2c in duodenal organogenesis
The progression of duodenal phenotypes with increasing Pitx2c dosage revealed an organ-intrinsic mechanism to distinguish between randomization and reversal. The biasing model of asymmetric organ morphogenesis suggests that absence of biasing would result in organ randomization, as in the iv;iv mouse (Brown and Wolpert, 1990; Capdevila et al., 2000). Our data suggest that an intermediate level of biasing can result in reversed organ morphogenesis.
Bilateral duodenal Pitx2c expression in abcnull;
abhypoc and
abcnull;
ab embryos suggests a mechanism within the duodenum that inhibits right-sided Pitx2c expression. This raises the possibility that relative left- versus right-sided Pitx2c levels determine the direction of gut rotation. In support of this idea, studies in Xenopus have shown that overexpression of Pitx2 on the left or right resulted in defective asymmetric morphogenesis (Essner et al., 2000). Moreover, these studies also showed that gut development was more susceptible to right-sided misexpression of Pitx2 than the heart (Essner et al., 2000), further supporting the notion of organ-specific requirements for Pitx2 function.
Our data also demonstrate that different regions of the gut are regulated by distinct mechanisms. Duodenal morphogenesis was most sensitive to changes in Pitx2c levels, while morphogenesis of the stomach was unaffected. In addition, midgut development appears to be regulated by the Pitx2a and Pitx2b isoforms, although it is possible that very high levels of Pitx2c are required for midgut development and that a slight decrease in Pitx2c expression from the ab allele is sufficient to disrupt midgut looping. Overexpression studies performed in Xenopus have also demonstrated that regional gut asymmetry can be unlinked (Bisgrove et al., 2000).
An organ-specific response to biasing
Current models propose that a biasing signal, originating at the node in mice, provides a cue that each organ uses to initiate correctly oriented asymmetric morphogenesis (Brown and Wolpert, 1990; Capdevila et al., 2000). The implication of these ideas is that asymmetric morphogenesis is all or none, either reversed or correct morphogenesis. Isomerism or loss of asymmetry would result from defects in interpretation of biasing at the organ level. Under this paradigm, uncoupling of asymmetry would be a result of defective biasing. This is illustrated by the iv and lefty1 mouse mutants that show heterotaxy (Brown and Anderson, 1999; Brown et al., 1989; Meno et al., 1998; Supp et al., 1997), implicating events occurring during the initial breaking of symmetry (iv mice) and the stabilization of left-sided gene expression by a midline barrier (lefty1 mice) in the etiology of heterotaxia. In contrast, our data suggest that left-right asymmetry within each organ is regulated not as an all-or-none decision but rather in stages. Thus, defects at the organ level, acting after biasing, can also result in heterotaxia.
Concluding remarks
We have shown that a central component of the local generation of asymmetry within an organ is a differential response to Pitx2c. These different requirements for Pitx2c dose may reflect the different morphogenetic processes, ranging from rotation of a tube to reiterated budding morphogenesis, that occur during asymmetric morphogenesis. It is conceivable that Pitx2c regulates different target genes in each organ. For example, in the atrium, Pitx2c may regulate target genes with high-affinity binding sites, while in the lung, Pitx2c target genes would have low-affinity regulatory elements. This model has the advantage of providing an understanding for how Pitx2c may regulate different morphogenetic events. Alternatively, there may exist organ-specific mechanisms to limit Pitx2c activity on a common set of target genes. This would include differential regulation of Pitx2c transcriptional levels, a mechanism that would be supported by our quantitation of Pitx2c mRNA levels. However, Pitx2c activity may also be regulated at the protein level by tight control of translation, by post-translation modification of Pitx2c or by the function of organ-specific co-factors that can modulate Pitx2c function. Further experiments are required to distinguish between these possibilities, however, the observation of a protein-protein interaction between Pitx2 and the pituitary-specific pit1 support the idea that organ-specific co-factors have a role in modulating Pitx2 function (Amendt et al., 1999).
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ACKNOWLEDGMENTS |
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