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.     

左右不对称信号分子Pitx2

同型框基因Pitx2在鸡、小鼠和爪蟾胚胎中不对称地表达在左侧板中胚层和衍生器官(如心脏、肠等)中. 转录因子Pitx2看来是Shh和Nodal等信号分子的下游效应子. Pitx2的错误表达足以产生器官逆位和身体旋转逆向,人类若有Pitx2表达缺陷就可能导致Rieger综合征. Pitx2看来是脊椎动物介导左右不对称的关键且保守的信号分子.     

Directionality of heart looping: effects of Pitx2c misexpression on flectin asymmetry and midline structures

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of beta-catenin. Thus, the IGF pathway is required for head formation during embryogenesis.     

Establishment of vertebrate left-right asymmetry

The generation of morphological, such as left-right, asymmetry during development is an integral part of the establishment of a body plan. Until recently, the molecular basis of left-right asymmetry was a mystery, but studies indicate that Nodal and the Lefty proteins, transforming growth factor-beta-related molecules, have a central role in generating asymmetric signals. Although the initial mechanism of symmetry breaking remains unknown, developmental biologists are beginning to analyse the pathway that leads to left-right asymmetry establishment and maintenance.     

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.     

Pitx homeobox genes in Ciona and amphioxus show left-right asymmetry is a conserved chordate character and define the ascidian adenohypophysis

All vertebrates have directional asymmetries in the organization of their internal organs. In jawed vertebrates, development of asymmetry is controlled by a conserved molecular pathway that includes Pitx2, which is expressed by lateral plate mesoderm cells on the left side of the embryo. Pitx2 is a member of the Pitx homeobox gene family, the expression of which also marks stomodeal ectoderm and the adenohypophysis. Here we report the characterization of Pitx genes from Branchiostoma floridae (an amphioxus) and Ciona intestinalis (a urochordate), representatives of two basal chordate lineages and successively deeper outgroups to the vertebrates. Expression of B. floridae Pitx is similar to that reported from B. belcheri, a different amphioxus species. Expression of the Ciona Pitx ortholog in the embryonic primordial pharynx and adult neural complex leads us to propose the Ciona primordial pharynx and ciliated funnel are homologous to the adenohypophyseal placode and adenohypophysis, respectively. Additionally, in both species we identify asymmetrical left-sided expression of Pitx genes during embryonic development. This shows that asymmetrical Pitx gene expression, and by inference directional asymmetry, evolved before the radiation of living chordates and should be considered a chordate character.     

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.     

Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed

Splanchnic mesoderm in the region described as the second heart field (SHF) is marked by Islet1 expression in the mouse embryo. The anterior part of this region expresses a number of markers, including Fgf10, and the contribution of these cells to outflow tract and right ventricular myocardium has been established. We now show that the posterior region also has myocardial potential, giving rise specifically to differentiated cells of the atria. This conclusion is based on explant experiments using endogenous and transgenic markers and on DiI labelling, followed by embryo culture. Progenitor cells in the right or left posterior SHF contribute to the right or left common atrium, respectively. Explant experiments with transgenic embryos, in which the transgene marks the right atrium, show that atrial progenitor cells acquire right-left identity between the 4- and 6-somite stages, at the time when Pitx2c is first expressed. Manipulation of Pitx2c, by gain- and loss-of-function, shows that it represses the transgenic marker of right atrial identity. A repressive effect is also seen on the proliferation of cells in the left sinus venosus and in cultured explants from the left side of the posterior SHF. This report provides new insights into the contribution of the SHF to atrial myocardium and the effect of Pitx2c on the formation of the left atrium.