First Printed in R&D Systems 2003 Catalog
The vertebrate body plan develops along three geometric axes: anterior-posterior, dorsal-ventral, and left-right (LR). Bilateral symmetry observed on the outside of an organism belies a characteristic asymmetric distribution of internal organs with respect to the LR axis.
||Figure 1. Asymmetric Organization of Visceral Organs in Humans The normal arrangement (see text for description) of organs is called situs solitus. In right isomerism, also called asplenia syndrome, the heart, lung and liver are double-right, and the spleen in absent. Isomerism can also be used to describe LR defects of individual organs. Situs inversus totalis is the complete mirror image reversal of all organ asymmetry. This figure represents only a portion of the whole range of possible LR defects
The cardiac apex points to the left, the aorta arches to the left, the liver and gallbladder are situated on the right, while the stomach and spleen are on the left. During embryogenesis, the LR asymmetry of both position and morphology of several organs transpire from initial symmetry. First, unpaired organs such as the heart, stomach, liver, gallbladder, and pancreas begin development in the midline and subsequently orient to their adult positions. Second, large arteries and veins first arise as symmetrically-paired structures and later remodeling occurs by regression of one of the mirror-image counterparts. Finally, the paired lungs manifest LR differences in having three lobes on the right and two on the left. Development of LR asymmetry is important, as malformations of normal LR asymmetry are associated with cardiovascular defects, causing significant morbidity and mortality in human newborns (reviewed in reference 1).
The process of normal LR patterning of the vertebrate embryo can be described in three different stages. First is the establishment of LR asymmetry with respect to the dorsal-ventral and anterior-posterior axes in the embryo. This results in a global LR axis for the embryo such that asymmetries are consistently oriented in the body. Next, this global LR patterning information must be transmitted to developing organ primordia, via signaling molecules. Finally, the organ primordia must correctly interpret the positional cues and execute appropriate morphogenetic responses.2 When perturbations of LR development occur (Figure 1), numerous defects result including:
- bilateral symmetry (lack of asymmetry in organ physiology)
- isomerism (duplicated left- or right- sided identity)
- heterotaxia (heart and visceral organs are oriented independently of each other)
- situs inversus totalis (total LR mirror image inversion of the heart and viscera)
- situs inversus (reversal of a particular organ's orientation).
A reversal rate of 50% indicates that the LR determination is "randomized;" half the time orientation is normal and half the time it is reversed (reviewed in reference 3). It turns out that altered expression patterns of asymmetrically-expressed signaling molecules can randomize cardiac orientation. Several members of the TGF-beta superfamily (TGF-beta SF) are vital components for vertebrate LR development. Although other signaling molecules, such as FGF-8 and Sonic hedgehog (Shh), are also involved in specifying vertebrate LR asymmetry, this review will focus on the components of the TGF-beta signaling pathway.
TGF-beta Signaling Pathway
The TGF-beta SF consists of over 30 structurally related proteins including subfamilies such as bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), Activins, and Inhibins, along with more distantly related members such as Nodal and Müllerian Inhibiting Substance (MIS). These ligands are synthesized as prepropeptides of approximately 400-500 amino acids (aa). The N-terminal variable length pro-region is cleaved at a consensus RXXR site prior to secretion. The secreted C-terminal mature segment has 6 - 7 spatially conserved cysteines that form a cysteine knot structure in the monomer. It is the conserved dimeric structure with two opposing "hands," however, that give specificity for receptor binding and biological function.4 Small secondary structural elements arising from the non-conserved regions give family members their specificity for ligand-receptor binding.4
TGF-beta SF members transmit signals to the nucleus by signaling through type II and I serine-threonine kinase receptors and intracellular effectors known as Smads. Ligands bind to type I and II receptors on the cell surface, promoting activation of the type I receptor via phosphorylation. This activated complex in turn activates intracellular Smads, which assemble multi-subunit complexes that regulate transcription. Members of the TGF-beta SF are divided into two signaling subgroups: those functionally related to TGF-beta/Activin and those related to the BMP/GDF subfamily. The TGF-beta/Activin ligands bind to TGF-beta and Activin type II receptors and can activate Smad-2 and -3. Nodal and Lefty signal through this Activin-type pathway. The BMP/GDF ligands bind to BMP type II receptors and can activate Smads 1, 5, and 8 (reviewed in reference 5).
Nodal is a distant member of the TGF-beta SF, sharing only 25-39% aa identity to other superfamily members in the mature region. It was first identified for its role in specification of mesodermal and endodermal cell fates in the mouse, and named for its strong expression in the node (a dorsal mesoderm organizing center that gives rise to the dorsal midline structures).6 Similar to Activin pathways, Nodal signaling utilizes Smad-2, and -3.7 Nodal signaling is also mediated by the EGF-CFC family of cofactors, which are not involved in Activin signaling.8, 9 This early role and expression during gastrulation and the mechanism of signaling is conserved in other vertebrate species such as frog (Xenopus), chick and zebrafish.10 Likewise, Nodal and its homologs have a consistent role in LR development. Nodal is expressed asymmetrically after gastrulation, in the left lateral plate mesoderm (LPM) before the onset of morphologic asymmetry.11, 12, 13, 14, 15 In mice, abnormal expression of Nodal genes is correlated with alterations of cardiac orientation.14, 15 Interventions that randomize cardiac orientation in frog or chick are also associated with altered expression patterns of Nodal-related genes.13, 16 In addition, misexpression of Nodal on the right side of the embryo after gastrulation is associated with a high incidence of reversed hearts or bilaterally symmetric hearts.17, 18 Although the mechanisms that induce left-sided Nodal expression is different in different organisms, left-sided Nodal expression is thought to be the convergence point for molecular LR development.19 Thus Nodal is a left-side determining signal, and often used as a marker of LR asymmetry.
Lefty-1 and -2 are highly homologous murine proteins that are distant members of the TGF-beta SF. Lefty-related genes have also been identified in other vertebrates including humans, frogs, chickens, and zebrafish.20, 21, 22, 23, 24, 25 By aa sequence, mouse Lefty-1 and -2 are more similar to each other (90%) than to either Lefty-A or -B in humans (81 - 82% identical).20 Lefty contains six cysteine residues that are conserved among TGF-beta related proteins and necessary to form the cysteine-knot structure. Lefty is distinct from other family members, however, as it has two cleavage sites and lacks the seventh cysteine residue required for intermolecular disulfide linkage. Lefty is differentially processed depending on the cells in which it is synthesized, and both cleavage sites can be utilized.26 The missing cysteine residue is the one required for covalent dimerization, indicating that Lefty either acts as a monomer or forms non-covalently linked dimers. Like Nodal, early symmetric expression during embryogenesis gives way to left-sided expression after gastrulation.27 Lefty-1 is expressed primarily on the left side of the prospective floor plate at the midline, whereas Lefty-2 is expressed predominantly in the left lateral plate mesoderm, in a pattern overlapping with Nodal.27 Unlike Nodal, however, Lefty does not play a role in determining left side identity. Instead, Lefty proteins function in patterning left-right asymmetry of the developing organ systems such as the heart and lung by antagonizing Nodal. Loss of Lefty-1 function leads to bilateral expression of left-side markers, such as Nodal and Lefty-2, and bilateral left isomerism.28 The role of Lefty-1, expressed in the middle of the embryo at the floorplate, is to prevent Nodal and Lefty-2 expression on the right side of the embryo.28 Similarly, loss of Lefty-2 function also leads to bilateral left isomerism along with various other LR defects. The role of Lefty-2 in the lateral plate mesoderm (LPM) is to prevent Nodal from diffusing over long distances.29 Thus in both instances, Lefty acts as an antagonist to Nodal signaling, helping to make Nodal expression transient in the LPM.
||Figure 2. TGF-beta Signaling in Vertebrate Left-Right Development This simplified schematic represents key interactions involved in vertebrate LR development, as discussed in the text. Note the convergence of signaling on Nodal. Nodal is thought to act through Activin Receptors and Smads 2,3 to regulate transcription. Nodal expression and activity are affected by other TGF-ßSF members: BMPs, Leftys, GDFs, and Vg1, and the BMP antagonist, Caronte.
BMPs represent a large subset of the TGF-beta SF and were first identified as regulators of bone and cartilage formation. BMPs are involved in early embryonic development as well as in growth, differentiation and morphogenesis of a variety of tissues and organs. Although BMPs are also implicated in LR development, they act differently depending on the species concerned. In chick, BMP-4 signaling has dual roles. The first is maintenance of asymmetric expression of Shh in the node, since BMP-4 and Shh negatively regulate each other's transcription.30 Later, BMP signaling in the lateral plate mesoderm acts to inhibit Nodal signaling. A BMP antagonist, Caronte is induced on the left side by Shh, which allows the activation of Nodal on that side.31, 32, 33 Hence BMP-4, remaining active on the right, turns on a right-sided program, including FGF-8.30 In zebrafish, mutants that disrupt the normal, early asymmetric "jogging" of the heart tube, all reveal a perturbed pattern of BMP-4 expression in the primitive heart tube. BMP-4 normally shows predominant expression on the left side of the heart tube.34 In addition, misexpression of BMP-4 on the right side results in cardiac reversals, providing more direct evidence that left-sided BMP signaling in zebrafish is involved in LR development of the heart.35 In the early Xenopus blastula, however, it is ectopic, left-sided expression of BMP-2 or -4 that randomizes cardiac asymmetry.36 This result is likely due to antagonism of an endogenous Vg1 pathway (discussed below) and the altered expression of a Nodal-related gene in the lateral plate mesoderm.36,37 BMP signaling may have an even more direct effect on LR development of the heart. In Xenopus, BMP-4 is also expressed predominantly on the left side of the linear heart tube, and transgenic embryos overexpressing BMP-4 randomizes heart orientation.38
Vg1 was one of the first TGF-beta related factors to be identified in vertebrate embryos, as a molecule localized to the vegetal pole of Xenopus embryos and involved in mesoderm induction. It is most closely related to the BMP/GDF subgroup of the TGF-beta SF and its activity is regulated by post-translational processing to cleave off the N-terminus pro region (reviewed in reference 39). A homolog in mouse, originally identified as GDF-1 (Growth Differentiation Factor-1), is antigenically related to Xenopus Vg1.40, 41 Mouse GDF-1 shows more sequence homology to Vg1 from frog, chick and zebrafish than to other proteins of the TGF-beta SF.41 In both mouse and Xenopus, this protein plays a role in regulation of LR development. In early Xenopus embryos, misexpression of Vg1/GDF-1 on the right side causes cardiac and visceral reversals and altered nodal expression.36, 41, 42 A targeted mutation of GDF-1 in mouse embryos reveals a spectrum of LR defects including visceral situs inversus, right pulmonary isomerism, and morphologic anamolies of the heart. GDF-1 acts upstream of Nodal and Lefty-1 and -2, since the GDF-1 mutant mice exhibit aberrant expression patterns of these genes.43
GDF-3, known as Derriere in Xenopus, is also implicated in LR development. It was initially isolated based on its expression and involvement in mesodermal patterning, particularly in the posterior region of the embryo.44 In the mature regions of the protein, Derriere has 79% aa identity to Xenopus Vg-1 and 62% aa identity to mouse GDF-3.44 Misexpression of this protein has a similar effect as Vg1 on LR development in the embryos, including similar alterations in Nodal expression patterns.45 In mesoderm induction, Derriere and Nodal are involved in a mutual positive feedback loop, and have been shown to interact through the formation of heterodimeric ligands.46 Whether these activities are also active in LR development remain to be seen. Interestingly, asymmetric expression of either of the GDFs described here has not been detected in embryos. Asymmetric post-translational processing or highly localized and transient asymmetric expression patterns, however, have not been ruled out.
Evidence from chick, mouse, frog and fish all help to model vertebrate LR development. The roles these TGF-beta signaling molecules play in mesoderm development also provide clues for components of the pathways and how the pathways may interact.2 While there are differences in these individual organisms, a generalized model can be resolved from the similarities (Figure 2). A GDF-1/Nodal/Activin-like pathway acting through Smad-2 and -3 characterizes the left side. On the right, an antagonistic pathway involving BMPs and Smad-1 and -5 is implicated. Loss of function experiments in mice and gain of function experiments in Xenopus and chick support this model. Trans-heterozygotes for Smad-2 and Nodal mutations display defects in left-right patterning, along with other phenotypes associated with the role these genes play in mesoderm induction and patterning.47 A targeted mutation of Smad-5 results in defects in heart morphogenesis and orientation. These mutant mice also display bilateral expression of left-sided genes such as Nodal and Lefty-2, indicating that a Smad-5 signaling pathway is involved in preventing expression of left-sided genes on the right side.48 In Xenopus, a BMP/ALK2 (Activin type I receptor)/Smad1 pathway antagonizes the Vg1/Nodal pathway active on the left. In chick, Caronte, an antagonist of BMP signaling, is active on the left side (resulting in Nodal expression), while it is turned off on the right.31, 32, 33 Hence, TGF-beta signaling is a cornerstone of vertebrate LR development, interacting with other early developmental signaling pathways and participating on both sides of the embryo.
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