Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium

Current models of left-right asymmetry hold that an early asymmetric signal is generated at the node and transduced to lateral plate mesoderm in a linear signal transduction cascade through the function of the Nodal signaling molecule. The Pitx2 homeobox gene functions at the final stages of this cascade to direct asymmetric morphogenesis of selected organs including the heart. We previously showed that Pitx2 regulated an asymmetric pathway that was independent of cardiac looping suggesting a second asymmetric cardiac pathway. It has been proposed that in the cardiac outflow tract Pitx2 functions in both cardiac neural crest, as a target of canonical Wnt-signaling, and in the mesoderm-derived cardiac second lineage. We used fate mapping, conditional loss of function, and chimera analysis in mice to investigate the role of Pitx2 in outflow tract morphogenesis. Our findings reveal that Pitx2 is dispensable in the cardiac neural crest but functions in second lineage myocardium revealing that this cardiac progenitor field is patterned asymmetrically.     

Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions

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.     

Defective Nodal and Cerl2 expression in the Arl13b(hnn) mutant node underlie its heterotaxia

Specification of the left-right axis during embryonic development is critical for the morphogenesis of asymmetric organs such as the heart, lungs, and stomach. The first known left-right asymmetry to occur in the mouse embryo is a leftward fluid flow in the node that is created by rotating cilia on the node surface. This flow is followed by asymmetric expression of Nodal and its inhibitor Cerl2 in the node. Defects in cilia and/or fluid flow in the node lead to defective Nodal and Cerl2 expression and therefore incorrect visceral organ situs. Here we show the cilia protein Arl13b is required for left right axis specification as its absence results in heterotaxia. We find the defect originates in the node where Cerl2 is not downregulated and asymmetric expression of Nodal is not maintained resulting in symmetric expression of both genes. Subsequently, Nodal expression is delayed in the lateral plate mesoderm (LPM). Symmetric Nodal and Cerl2 in the node could result from defects in either the generation and/ or the detection of Nodal flow, which would account for the subsequent defects in the LPM and organ positioning.     

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

Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(-)(/)(-) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(-)(/)(-) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs; the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs; and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.     

Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.     

Left and right contributions to the<Emphasis Type="Italic"> Xenopus</Emphasis> heart: implications for asymmetric morphogenesis

The left-right asymmetry of the vertebrate heart is evident in the topology of the heart loop, and in the dissimilar morphology of the left and right chambers. How left-right asymmetric gene expression patterns influence the development of these features is not understood, since the individual roles of the left and right sides of the embryo in heart looping or chamber morphogenesis have not been specifically defined. To this end, we have constructed a bilateral heart-specific fate map of the left and right contributions to the developing heart in the Xenopus embryo. Both the left and right sides contribute to the conoventricular segment of the heart loop; however, the left side contributes to the inner curvature and ventral face of the loop while the right side contributes to the outer curvature and dorsal aspect. In contrast, the left atrium is derived mainly from the original left side of the embryo, while the right atrium is derived primarily from the right side. A comparison of our fate map with the domain of expression of the left-right gene, Pitx2, in the left lateral plate mesoderm, reveals that this Pitx2-expressing region is fated to form the inner curvature of the heart loop, the left atrioventricular canal, and the dorsal aspect of the left atrium. We discuss the implications of these results for the role of left-right asymmetric gene expression in heart looping and chamber morphogenesis.     

Left-right axis development: examples of similar and divergent strategies to generate asymmetric morphogenesis in chick and mouse embryos

Left-right asymmetry of internal organs is widely distributed in the animal kingdom. The chick and mouse embryos have served as important model organisms to analyze the mechanisms underlying the establishment of the left-right axis. In the chick embryo many genes have been found to be asymmetrically expressed in and around the node, while the same genes in the mouse show symmetric expression patterns. In the mouse there is strong evidence for an establishment of left-right asymmetry through nodal cilia. In contrast, in the chick and in many other organisms left-right asymmetry is probably generated by an early-acting event involving membrane depolarization. In both birds and mammals a conserved Nodal-Lefty-Pitx2 module exists that controls many aspects of asymmetric morphogenesis. This review also gives examples of divergent mechanisms of establishing asymmetric organ formation. Thus there is ample evidence for conserved and non-conserved strategies to generate asymmetry in birds and mammals.     

Molecular basis of left-right asymmetry

In vertebrates visceral asymmetry is conserved along the left-right axis within the body. Only a small percentage of randomization (situs ambiguus), or complete reversal (situs inversus) of normal internal organ position and structural asymmetry is found in humans. A breakdown in left-right asymmetry is occasionally associated with severe malformations of the organs, clearly indicating that the regulated asymmetric patterning could have an evolutionary advantage over allowing random placement of visceral organs. Genetic, molecular and cell transplantation experiments in humans, mice, zebrafish, chick and Xenopus have advanced our understanding of how initiation and establishment of left-right asymmetry occurs in the vertebrate embryo. In particular, the chick embryo has served as an extraordinary animal model to manipulate genes, cells and tissues. This chick model system has enabled us to reveal the genetic pathways that occur during left-right development. Indeed, genes with asymmetric expression domains have been identified and well characterized using the chick as a model system. The present review summarizes the molecular and experimental studies employed to gain a better understanding of left-right asymmetry pattern formation from the first split of symmetry in embryos, to the exhibition of asymmetric morphologies in organs.     

Multiple pathways in the midline regulate concordant brain, heart and gut left-right asymmetry

The embryonic midline in vertebrates has been implicated in left-right development, but the mechanisms by which it regulates left-right asymmetric gene expression and organ morphogenesis are unknown. Zebrafish embryos have three domains of left-right asymmetric gene expression that are useful predictors of organ situs. cyclops (nodal), lefty1 and pitx2 are expressed in the left diencephalon; cyclops, lefty2 and pitx2 are expressed in the left heart field; and cyclops and pitx2 are expressed in the left gut primordium. Distinct alterations of these expression patterns in zebrafish midline mutants identify four phenotypic classes that have different degrees of discordance among the brain, heart and gut. These classes help identify two midline domains and several genetic pathways that regulate left-right development. A cyclops-dependent midline domain, associated with the prechordal plate, regulates brain asymmetry but is dispensable for normal heart and gut left-right development. A second midline domain, associated with the anterior notochord, is dependent on no tail, floating head and momo function and is essential for restricting asymmetric gene expression to the left side. Mutants in spadetail or chordino give discordant gene expression among the brain, heart and gut. one-eyed pinhead and schmalspur are necessary for asymmetric gene expression and may mediate signaling from midline domains to lateral tissues. The different phenotypic classes help clarify the apparent disparity of mechanisms proposed to explain left-right development in different vertebrates.     

A role for BMP signalling in heart looping morphogenesis in Xenopus

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.