Capdevila J , Vogan KJ , Tabin CJ , Izpisúa Belmonte JC .

	Figure 1. Asymmetric Disposition of Visceral Organs in Humans The normal disposition of organs is called situs solitus (A). The right lung has three lobes, whereas the left lung (indicated in a different color for clarity) has two. In addition, the apex of the heart points to the left side, the liver is on the right side, and the stomach and spleen are on the left side. Although not shown in the figure, the gut coils counterclockwise in the abdominal cavity. In right isomerism (B), also called asplenia syndrome, the heart and lungs are double-right (as indicated by the structure of the heart chambers and by both lungs having three lobes), as is the liver, which is generally found in a midline position. The stomach may be located on either side or in the midline, and the spleen is absent. In left isomerism (C), also called polysplenia syndrome, the heart and lungs are double-left, the liver may be double-left, located in a midline position, or normal, and the stomach is usually found in a midline position. There is always more than one spleen (termed splenules), although multilobulated single spleens may also occur. Situs inversus refers to the complete mirror-image reversal of organ asymmetry (D). Since laterality defects are highly variable, the figure depicts simplified cases, and is not intended to portray accurately the whole range of possible defects. The terms situs inversus and right or left isomerism can also be used to describe laterality defects in individual organs, even if they are not included in specific syndromes. Adapted from Brown and Anderson, 1999.
	Figure 2. Breaking Symmetry and Establishing Asymmetric Gene Expression in and around the Node In the mouse embryo (A), between E7.5 and E8.5, Shh is expressed in the axial mesendoderm (future notochord, in dark blue), while Fgf-8 is expressed in the primitive streak (in yellow). Shh, Fgf-8, Activin βB, and Activin receptor IIA (ActRIIA) are all expressed in the mouse node without any obvious asymmetry, and it is postulated that a leftward nodal flow (indicated by the green arrow, see main text for details) activates left-specific gene expression, represented by asymmetric expression of Nodal in and around the node. In the mouse, Shh activity is required for lefty-1 expression in the left side of the node and midline (specifically, in the left prospective floor plate; PFP). In the chick embryo (B), between stages 4 and 6, the initially symmetric expression of Shh in the node is restricted to the left side (in light blue) by an inferred Activin-like signal (possibly Activin βB), which also causes upregulation of ActRIIA on the right side (in light yellow). Shh is also expressed in the head process or nascent notochord (in dark blue). Shh acts upstream of lefty-1 expression in the left node and left side of the midline, and also acts upstream of Nodal in a domain adjacent to the left side of the node. The inferred Activin-like signal simultaneously activates Fgf-8 in the right side of the node (in light yellow); Fgf-8 is also expressed in the primitive streak (in yellow). The red squares indicate the regions of the embryos shown in detail to the right of the panels.
	Figure 3. Relay of L/R Information from the Node to the LPM and Establishment of Side-Specific Gene Expression in the LPM In the chick, the Cerberus-related gene Car (indicated in red) is initially expressed bilaterally in the anterior region of the embryo, including the cells immediately adjacent to the node (A). At stage 5, Car begins to fade on the right and it is maintained in the left, concomitant with the appearance of lefty-1 expression (in green) in the left side of the node and midline, and of Nodal (in purple) in a left domain adjacent to lefty-1 (see detail in [A]). Left and right sides are as indicated in (A). By stage 7- (B), Car is completely absent from the right paraxial mesoderm, and in the left its expression coincides with the appearance of a second Nodal domain in the LPM; the medial domains of lefty-1 and Nodal also expand anteriorly at this stage as the node regresses. At stage 7 (C), BMPs (in light purple) are expressed bilaterally in the LPM, and the second domain of Nodal (in purple) consolidates in the cells that also express Car (in red). Nodal represses SnR (in orange) on the left side, so that Pitx2 (in light blue) can be expressed. The lower panel in (C) shows a scheme of the cascade of negative interactions that establishes side-specific gene expression in the LPM (see main text for details). Briefly, Car protein on the left side antagonizes the repression of Nodal transcription by BMPs. In turn, Nodal represses SnR, which is itself a repressor of Pitx2, so that SnR is expressed on the right and Pitx2 in the left LPM. The green arrow indicates positive autoregulation, since Nodal activates its own expression in the presence of EGF-CFC cofactors (see main text and Figure 4). The red asterisk indicates antagonism at the protein–protein level, to distinguish it from the rest of the interactions, which act at the transcriptional level. Some of the genes depicted are also expressed in structures other than the LPM, such as the somites, but this is not indicated for simplicity.
	Figure 4. A Proposed Model of Genetic Interactions that Determine Development of the L/R Axis in Mice and Humans In the mouse embryo, and most likely in humans as well, the function of several genes, including iv, inv, KIF-3A, and KIF-3B, is required for the establishment of a normal leftward flow in the node region. This nodal flow (represented by the green arrows in the node) is thought to cause the accumulation of some key extracellular determinant in the left side of the node and/or perinodal region, so that left-specific Nodal expression is induced. Expression of lefty-1 in the left side of the midline is likewise downstream of these initiating signals, but also depends on the integrity of the midline and on the activity of several genes (Brachyury, HNF-3β, Shh, and others) expressed within midline cells. In the LPM, a pathway that requires Smad-5 function (probably triggered by BMPs) acts to suppress Nodal transcription. The presence of some BMP antagonist in the left side (similar to Car in the chick) presumably acts to relieve this repression so that Nodal can be transcribed in the left LPM. Nodal, in turn, activates lefty-2 and represses SnR, in a process that requires extracellular cofactors of the EGF-CFC family, ActRIIB (not depicted here), and the intracellular factors Smad-2 and FAST2. This repression of SnR in the left side is thought to result in activation of Pitx2. At the midline, the Lefty-1 protein might act as a barrier preventing the contralateral diffusion of the proposed BMP antagonist, thus preventing ectopic expression of left-specific genes in the right side of the embryo. Note that this is a simplified model that is not intended to depict all possible interactions. For example, EGF-CFCs may be involved in mediating some earlier functions of Nodal (i.e., in the left side of the node), and the possible roles of RA and Fgf-8 as left determinants are not depicted here since their precise functions are still unclear (see main text).
	Figure 5. Left–Right Determination in Fish and Frogs Studies in zebrafish and Xenopus have greatly contributed to our understanding of L/R development in vertebrates. Specifically, a variety of zebrafish mutants and surgical manipulations in Xenopus have been invaluable in demonstrating a key role for midline structures in L/R determination. It is becoming increasingly clear that the basic mechanisms and the molecular players are conserved among vertebrates, but interesting variations on the general L/R theme occur in fish and frogs. For example, in Xenopus (A), the L/R axis appears to develop significantly earlier than in other vertebrates. A “left–right coordinator” (LRC; in light blue), mediated by the activity of the TGF-β factor Vg1 ( 31 and 69), has been proposed to exist in the left side of the early blastula (at the 16-cell stage), establishing a mutual antagonism with a BMP activity in the right side (in light orange). The L/R coordinator would subsequently influence the Spemann organizer (depicted in black), which in turn relays the L/R positional information to other tissues. Interestingly, a transient array of microtubules during the first cell cycle appears to be essential for L/R development, suggesting that the L/R coordinator may be established as early as the D/V axis. Expression of Nodal (and, hence, Pitx2) can be induced by Vg1 and is antagonized by BMPs, which again stresses the conservation of the mechanism of control of Nodal and its targets. The midline is indicated in dark blue. (This figure has been adapted from Hyatt and Yost 1998 and Ramsdell and Yost 1999). Less is known about L/R development in the zebrafish embryo (B). None of the four hedgehog (hh) genes (Shh, twhh, ehh, and hhc) has been described to be asymmetrically expressed in zebrafish, but overexpression of Shh randomizes the situs of the heart and other visceral organs, most likely through the activity of the Nodal-related gene Ndr2. BMPs are expressed asymmetrically in the heart field (in orange), and BMP misexpression only affects the heart, pointing toward a specific role for BMPs in the development of this organ in zebrafish. It remains to be seen whether some other TGF-β (or other molecule) plays the role of repressing Ndr2. In zebrafish and Xenopus, unlike in chick and mouse, asymmetry of Nodal expression around the node has not been detected ( 48, 75 and 70).