Linking early determinants and cilia-driven leftward flow in left–right axis specification of Xenopus laevis: A theoretical approach

Differentiation 83 (2012) S67–S77

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Linking early determinants and cilia-driven leftward flow in left–right axis specification of Xenopus laevis: A theoretical approach Axel Schweickert a,n, Peter Walentek a, Thomas Thumberger a, Mike Danilchik b a b

University of Hohenheim, Institute of Zoology, Garbenstrasse 30, D-70593 Stuttgart, Germany Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR 97239, USA.

a r t i c l e i n f o

abstract

Available online 1 December 2011

In vertebrates, laterality – the asymmetric placement of the viscera including organs of the gastrointestinal system, heart and lungs – is under the genetic control of a conserved signaling pathway in the left lateral plate mesoderm (LPM). A key feature of this pathway, shared by embryos of all non-avian vertebrate classes analyzed to date (e.g. fish, amphibia and mammals) is the formation of a transitory midline epithelial structure. Remarkably, the motility of cilia projecting from this epithelium produce a leftward-directed movement of extracellular liquid. This leftward flow precedes any sign of asymmetry in gene expression. Numerous analyses have shown that this leftward flow is not only necessary, but indeed sufficient to direct laterality. Interestingly, however, cilia-independent mechanisms acting much earlier in development in the frog Xenopus have been reported during the earliest cleavage stages, a period before any major zygotic gene transcription. The relationship between these two distinct mechanisms is not understood. In this review we present the conserved and critical steps of Xenopus LR axis formation. Next, we address the basic question of how an early asymmetric activity might contribute to, feed into, or regulate the conserved cilia-dependent pathway. Finally, we discuss the possibility that Spemann’s organizer is itself polarized in the left–right dimension. In attempting to reconcile the sufficiency of the cilia-dependent pathway with potential earlier-acting asymmetries, we offer a general practical experimental checklist for the Xenopus community working on the process of left–right determination. This approach indicates areas where work still needs to be done to clarify the relationship between early determinants and cilia-driven leftward flow. & 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Keywords: Left–right asymmetry Leftward flow Cilia Xenopus laevis Ion-flux Early determinants

1. Introduction Most animals display distinct morphological polarizations along the three body axes: anterior–posterior (AP; head–tail), dorsal–ventral (DV; back–belly) and mediolateral (left–right; LR). The phenotypic manifestation of mirror-image asymmetries, or lateralities, along the left–right body axis varies considerably between animal groups. Observed lateralities range from shell chirality in snails to the asymmetric appearance of the pentameric adult rudiment in the echinoderm pluteus larva and to the arrangement of heart and gastro-intestinal tract in vertebrates (Basu and Brueckner, 2008; Duboc and Lepage, 2008; Grande and Patel, 2009). Despite the considerable morphological differences displayed among these organisms, asymmetric expression of a conserved gene cassette, the so-called Nodal-cascade, precedes and governs the morphogenetic events resulting in laterality in all cases. The Nodal signaling cascade consists of the TGFb growth

n

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factor Nodal (Xnr1 or nodal1 in frog), which directly activates the asymmetric gene transcription of the TGFb feedback inhibitor Lefty (Lefty2 in mouse, in fish and frog also termed Antivin) and the homeobox gene Pitx2. Whereas Nodal transcription is shut down quickly by Lefty activity, Pitx2 continues to be expressed asymmetrically in the vertebrate organ anlagen during morphogenesis (Schier, 2003; Shen, 2007; Shiratori and Hamada, 2006). The broad phylogenetic conservation of this asymmetrical signaling cassette raises the question of whether the molecular process underlying its initiation is conserved as well (Levin, 2005; Tabin, 2005). Even solely among vertebrate model systems, the extent of conservation of the mechanism by which the Nodal-cascade acquires its initial lateral bias remains an unresolved problem. Two different scenarios can be found in current literature, which differ in mechanism, factors involved, location and most importantly the developmental stage of activity. Basically, an early mechanism referred to herein as the ‘‘ion-flux’’ hypothesis of symmetry breakage (Levin, 2003), acting during early cleavage stages of development, stands in stark contrast to a well-studied, cilia based ‘‘leftward flow’’ or ‘‘Nodal flow‘‘ model, operating much later in development at neurula stages (Essner et al.,

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2002; Blum et al., 2009). In this review we explore possible relationships and interactions between these signaling mechanisms at a theoretical level using the Xenopus model in which evidence for both early and late mechanisms has been reported (Levin, 2003; Schweickert et al., 2007).

2. Cilia-driven leftward flow The leftward flow hypothesis is based on the motility of monocilia: membrane-bounded, microtubule-containing projections that extend into the extracellular space during neurulation. Motile monocilia are found in homologous embryonic structures in mouse and rabbit (posterior notochord; PNC or ventral node), fish (Kupffer’s vesicle; KV) and frog (gastrocoel roof plate; GRP) (Blum et al., 2009, 2007). Monocilia project from the posterior-facing apical surfaces of these epithelial cells and carry out a vigorous, uniformly clockwise circular beat. Because the cilia extend at an angle relative to the apical surface, the rightward portion of the rotation sweeps near the epithelial surface and the leftward portion sweeps through the extracellular medium. The viscous drag along the surface interferes with rightward flow, and a net leftward flow of extracellular fluid above the ciliated epithelium results (Marshall and Nonaka, 2006; Nonaka et al., 2005). Leftward flow was first identified in mouse by Nonaka et al. (2002) and was subsequently observed in rabbit, fish and frog by other workers (Schweickert et al., 2007; Essner et al., 2005; Kramer-Zucker et al., 2005; Okada et al., 2005). Importantly, leftward flow precedes asymmetric gene expression in the LPM, suggesting that it plays a fundamental signaling role. Numerous reports of knockouts, mutants and morphants affecting ciliogenesis, cilia motility or polarization in PNC, KV and GRP reveal the close connection between fluid flow and organ placement. In addition humans suffering from syndromes based on ciliary dysfunctions (primary ciliary dyskinesia) often show alterations in the lateral asymmetry of inner organs. Taken together, the experimental and genetic evidence demonstrate the necessity of leftward flow for the development of the LR axis (Basu and Brueckner, 2008; Afzelius, 1976; Fliegauf et al., 2007). A substantial body of literature describing nongenetic manipulations in fish, mouse and frog supports the causal connection between leftward flow and LR axis specification. For example, KV ablation experiments in medaka and zebrafish embryos resulted in lack of asymmetric gene expression and randomization of organ placement (Essner et al., 2005; Hojo et al., 2007). Corresponding dissection experiments have been performed in frog and mouse neurulae as well (Davidson et al., 1999; Ohi and Wright, 2007), reaching the same conclusion that LR patterning requires leftward ciliary flow. Laser ablation or surgical removal of the precursor cells of KV or GRP (dorsal forerunner cells in fish; superficial mesoderm (SM) in frog at gastrula stages) altered laterality specifically without impacting on the AP or DV axis (Essner et al., 2005; Blum et al., 2009). In Xenopus, leftward flow can be interfered without seriously damaging or removing cells. For example, at neurula stages, embryos were injected with methylcellulose (MC) into the gastrocoel nearby the ciliated GRP epithelium. The high viscosity of MC efficiently prevents leftward liquid flow. MC treatment prior to and at flow stages alters organ laterality and asymmetric gene expression of tadpoles. MC manipulations at later, post-flow stages has no effect on the LR axis, indicating the nontoxic nature of this treatment (Schweickert et al., 2007). Finally, the application of an artificial flow on mouse embryos provides another substantial piece of evidence that flow is indeed part of the LR pathway. Hamada and co-workers administered a strong rightward flow on wild-type embryos, thereby overriding

the endogenous leftward flow. This artificially reversed stimulus induced the Nodal-cascade on the right but not on the left side of treated embryos. Moreover the same technique could be used to direct laterality in mutants lacking ciliary motility and leftward flow. Homozygous iv (inversus viscerum) embryos show randomized Nodal expression (left, right, bilateral and absent) due to a mutation in a motor protein (left–right dynein) necessary for cilia movement. Leftward artificial flow rescued to wild-type left and rightward flow reversed Nodal expression in iv mutants (Nonaka et al., 2002). These experiments and the growing literature on the role of ciliary function in left–right determination have established that an ancient, conserved mechanism specifies the left–right axis and asymmetric organ laterality in vertebrates. While this mechanism is, by the bulk of experimental evidence and criteria, regarded as both necessary and sufficient for left–right determination, it does not specifically indicate whether other earlier pathways might be considered ‘‘necessary’’ as well. In the case of frog development, this possibility is exemplified by the ion-flux hypothesis, which posits that very early cleavage-stage localization of maternally deposited factors plays a patterning role in LR axis specification (Levin, 2003). In addition, factors reported to be active in the ionflux model have been implicated in laterality in a wide range of species including sea urchin, ciona, fish and chick (Fukumoto et al., 2005; Hibino et al., 2006; Shimeld and Levin, 2006; Adams et al., 2006), suggesting a conserved requirement for LR development. In this review, we attempt to reconcile these disparate models by considering – at a theoretical level – how early determinants might contribute, whether morphogenetically or in some signaling capacity, to the patterning consequences of leftward flow, i.e. specification of the LR axis. We hope that this approach will provide an instructive framework upon which ideas bridging these two models can be tested experimentally

3. LR pathway in Xenopus laevis The critical and conserved steps in LR development, which are considered both necessary and sufficient for the left–right axis, are illustrated in the following section by five sequential processes in Xenopus. Relevant work in fish and mouse is cited to underscore conservation. 3.1. Step 1: superficial mesoderm specification and Foxj1 expression. The superficial mesoderm (SM), the outer cell layer of Spemann’s organizer, is specified at early gastrula stages (Fig. 1A and A0 , (Shook et al., 2004)). Homologous tissues have been identified in mouse and fish as well (Shook et al., 2004). Cell labeling experiments in Xenopus have shown that after involution, SM cells are fated to form the gastrocoel roof plate (GRP) during neurula stages (Fig. 1B; Shook et al., 2004) and to express the forkhead-family transcription factor Foxj1 (Pohl and Knochel, 2004; Stubbs et al., 2008). Foxj1 is considered a master control gene for ciliogenesis in the GRP, as its overexpression is sufficient to induce the whole ciliogenesis program. Further, loss of Foxj1 function prevents cilia formation and consequently results in laterality defects (frog (Stubbs et al., 2008); fish (Stubbs et al., 2008; Yu et al., 2008); mouse (Zhang et al., 2004)). How Foxj1 expression in the SM itself is regulated is not yet fully understood. 3.2. Step 2: GRP morphogenesis. During early neurula stages the GRP cilia continuously grow (Fig. 1C, (Schweickert et al., 2007)). The motility of GRP cilia is provided by microtubule-dependent motor proteins: the

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Fig. 1. LR development in Xenopus laevis. (A,A0 ) The superficial mesoderm (SM; green) constitutes the outer cell layer of Spemann’s organizer and is located animally to the dorsal lip (dl). Mesodermal cells in the deep marginal zone are colored red. (A) dorsal view of early gastrula, (A0 ) sagittal section. (B,B0 ) Following SM invagination, the ciliated gastrocoel roof plate (GRP) differentiates at the posterior archenteron (ac) in neurula embryos. Note GRP is not covered with endoderm (yellow) and flanked by lateral endodermal cells (LEC). Ciliated GRP cells cover dorsal aspects of the circumblastoporal collar (cbc) and blastoporus (bp), as well. Note also that lateral somitic GRP cells (blue) are unique for co–expressing the secreted growth factors Xnr1 and Coco and project cilia centrally. (B) st. 17 neurula midsagittal section, (B0 ) ventral view on GRP. GRP cilia. Immunohistochemistry using anti-acetylated tubulin antibody; ventral view. (D,E) The Nodal inhibitor Coco is the target of leftward flow. Schematic dorsal explants of early (D; pre-flow) and late (E; post-flow) neurulae in ventral view. (D) GRP (green) is flanked by a bilateral symmetric expression of Xnr1 (purple) and Coco (blue) in somitic GRP cells. Lateral plate mesoderm (LPM) is devoid of asymmetric gene activity. (E) Flow dependent left asymmetric downregulation of Coco mRNA releases Xnr1 from Coco repression. Transfer of left positional information by the relieved Xnr1 protein, induces Xnr1 transcription in the left LPM. (F) Time course of events important in left–right specification. Stages indicated are according to Nieuwkoop and Faber (1967). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

axonemal dyneins. Morpholino-mediated knockdown of two core components, dynein heavy chain 5 and 9 (dnah5 and 9) prevents rotation of GRP cilia, thus blocking leftward flow and resulting in LR axis defects (frog (Vick et al., 2009); fish (Essner et al., 2005; Kramer-Zucker et al., 2005) mouse (Okada et al., 1999; Olbrich et al., 2002)). In addition to growth and motility, GRP cilia become progressively oriented toward the posterior pole of each cell, a prerequisite for leftward flow. This posterior polarization process is under the control of Wnt-PCP (planar cell polarity) signaling. Cilia which are not polarized cannot create a robust leftward flow, as evidenced by the observation that tadpoles lacking normal Wnt-PCP signaling show an altered left–right asymmetry (frog (Antic et al., 2010; Maisonneuve et al., 2009); fish (May-Simera et al., 2010; Oteiza et al., 2010); mouse (Song et al., 2010; Hashimoto and Hamada, 2010)).

3.3. Step 3: the target of leftward flow: the Xnr1/Coco interplay. It is important to note that not all GRP cilia are polarized via the Wnt-PCP pathway. Cells at the left and right margins of the GRP that are fated to integrate into the somites, project their cilia mostly inward towards the center of the cell (Schweickert et al., 2007, 2010; Shook et al., 2004). In mouse these lateral cilia are nonmotile, and are thought to have some mechanosensory function. It has been proposed that such cilia detect leftward flow produced by their motile neighbors and would thereby constitute a downstream part of the left–right signal transduction pathway (McGrath et al., 2003). Besides their lack of ciliary polarization, the bilateral somitic GRP cells co-express two key components of LR development, Xnr1 (frog (Lowe et al., 1996; Lustig et al., 1996); fish (Long et al.,

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2003); mouse (Lowe et al., 1996)) and Coco, a secreted cerberuslike inhibitor of Nodal, BMP and Wnt signaling (also termed dand5 in frog (Schweickert et al., 2010; Vonica and Brivanlou, 2007; Bell et al., 2003); charon in fish (Hojo et al., 2007; Long et al., 2003; Hashimoto et al., 2004); cerl2 or dand5 in mouse (Marques et al., 2004)). Xnr1 and Coco transcription in lateral GRP domains can be detected from early neurulation stages onwards in a bilaterally symmetric expression pattern (Fig. 1D). In late neurulae and early tailbud stage, this pattern also holds true for Xnr1 (Fig. 1E). However, at these later stages, the level of Coco mRNA is specifically reduced on the left side of the GRP. Embryos in which leftward flow was blocked displayed bilaterally symmetric Coco expression, suggesting that Coco asymmetry was flow dependent (frog (Schweickert et al., 2010); fish (Hojo et al., 2007); mouse (Kawasumi et al., 2011)). In addition, a series of loss-of-function experiments indicated the functional relevance for LR development and revealed the epistatic relationship between flow, Xnr1 and Coco activity. From these experiments the following model was elucidated: Coco expression, which is bilaterally symmetrical before flow begins, serves as the critical target of leftward flow (frog (Schweickert et al., 2010); fish (Hojo et al., 2007); mouse (Kawasumi et al., 2011); H. Hamada personal communication). By a yet unknown mechanism– possibly involving asymmetric calcium and/or signaling by a morphogen (McGrath et al., 2003; Hamada 2008) – flow downregulates Coco mRNA at the left GRP. Xnr1 is thus released from Coco repression and becomes free to diffuse to the left LPM (Step 4 transfer of left identity), where it induces the left asymmetric Nodal-cascade (Step 5). 3.4. Step 4: transfer of left positional information to the lateral plate mesoderm (LPM) In no vertebrate model organism the identity or process of how a left cue generated at the midline by leftward flow reaches the LPM has been satisfactorily elucidated. Given the potency of Nodal proteins to act as bonafide morphogens over long distances, it is worth considering a cell-signaling mechanism based entirely on Nodal diffusion (Schier, 2009). In agreement with this idea, loss of Xnr1 activity in the GRP abolishes asymmetric gene expression in the left LPM (frog (Vonica and Brivanlou, 2007); fish (Long et al., 2003; Hashimoto et al., 2004); mouse (Brennan et al., 2002)). Although a transgenic epitope-tagged Nodal, emanating from the mouse PNC has been detected in the neighboring extracellular space, an asymmetric distribution was not obvious (Oki et al., 2007). Because suitable antibodies are not available, the Nodal based transfer model and – more specifically – the routes by which it reaches the LPM, cannot yet be directly visualized nor confirmed. 3.5. Step 5: asymmetric Nodal-cascade in the left LPM After flow has ceased in late neurulation, left asymmetric Xnr1 expression can be detected in the posterior aspect of the left lateral plate mesoderm (LPM), the region nearest to the GRP. Because of a positive feedback loop, a wave of Xnr1 transcription rapidly shifts toward the heart anlage (Ohi and Wright, 2007). With a small delay, Xnr1 then activates the expression of Lefty and Pitx2 (Campione et al., 1999). The secreted TGFb feedback inhibitor Lefty binds to Xnr1 and its receptors, terminating Xnr1dependent signal transduction and thus finally silencing asymmetric Xnr1 and Lefty expression. Additionally Xnr1 induces Lefty transcription in the embryonic midline (floorplate and dorsal endoderm) thereby creating a so called midline barrier, preventing Xnr1 to cross to the right LPM (frog (Cheng et al., 2000); fish (Wang and Yost, 2008); mouse (Yamamoto et al., 2003)). The right LPM is indeed competent to respond to Nodal signaling as ectopic right misexpressed Xnr1 activated right-sided transcription of the

Nodal-cassette and consequently triggered laterality defects (Sampath et al., 1997). Members of the EGF-CFC family serve as co-receptors essential for Nodal signaling and are bilateral symmetrically expressed in the LPM. In frog three EGF-CFC genes have been identified of which one, XCR2, conveys competence to LPM. Loss of XCR2 function and thus the inability to activate the Nodalcascade alters organ laterality as well (frog (Onuma et al., 2006); fish and mouse (Yan et al., 1999; Gaio et al., 1999)). Because of the bilateral competence of the LPM and long range signaling potential of Nodal, the crucial left sided restriction of Nodal-cascade activation is exerted by the long range inhibitor Lefty. The potency of Lefty to travel over long distances has been recently shown using a tagged version of Lefty in Xenopus. Following left sided misexpression, epitope-tagged Lefty protein was detectable in the right LPM, suggesting that besides its expression along the midline, Lefty proteins synthesized in the left LPM might contribute to the sustained repression of nodal cascade activation in the right LPM (Marjoram and Wright, 2011). In this context the Nodal/Lefty interplay amplifies the asymmetric cue provided by the leftward flow and at the same time ensures the restriction of signaling to the left LPM. Mathematical modeling and experimental evidences have led to the self-enhancement and lateral inhibition (SELI) model to describe the mechanism of Nodal/Lefty activity after symmetry breakage (Ohi and Wright, 2007; Marjoram and Wright, 2011; Meinhardt, 2000, 2001, 2008; Nakamura et al., 2006). The presented model of LR axis formation in Xenopus is supported by data gathered in fish and mouse, indicating that the main morphological, biochemical, and genetic features of this pattern-specifying mechanism are well conserved. We conclude that the 5 basic steps described above and their underlying molecular processes invariably act on development of the left– right axis in these species.

4. Early asymmetric determinants and their hypothetical LR targets The question of what mechanism initiates the sidedness of the vertebrate embryo is still heavily discussed. The cilia-based leftward flow model certainly describes an inherently consistent mechanism for the symmetry breaking event. However, analysis of even more distantly related organisms like C. elegans and snails reveal unambiguously that LR axis specification can be accomplished during the earliest cleavage stages via the asymmetric parceling of maternal factors that occurs during the highly stereotypic cell division planes exhibited in these species (eutely and spiral cleavage respectively) (Pohl, 2011; Kuroda et al., 2009). In this particular regard, Nodal expression becomes positioned asymmetrically by cleavage in snail development (Grande and Patel, 2009), indicating a functional link to the vertebrate pathway. These findings suggest that a similar early mechanism could also operate in vertebrates to regulate some aspect of laterality. Thus far, Xenopus remains alone in providing any experimental evidence that the ion-flux model operates in the vertebrates. Asymmetric activity of ion pumps resulting in asymmeric localization of the neurotransmitter serotonin at early cleavage stages has been described in 2–64 cell stages. The relevance of this phenomenon with respect to left–right axis specification is underscored by pharmacological loss-of-function experiments, which result in altered asymmetric gene expression in the LPM and impaired organ placement (Fukumoto et al., 2005; Adams et al., 2006; Levin et al., 2002; Vandenberg and Levin, 2010). Similar drug treatments are effective in other species as well, although timing of activity and presence of asymmetries differ greatly. For example, asymmetries of ion-flux components operate at gastrula

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Fig. 2. Targeted tissues and LR processes of hypothetical early determinants. Xenopus 4-cell embryos could hypothetically harbor four possible asymmetries along the dorso-ventral axis; dorsal-right, dorsal-left, ventral-right and ventral-left (blue). Based on the restricted cell-lineage asymmetric early determinants could target different tissues, which are superimposed with blue color on neurula-stage embryos, respectively. GRP location inside the archenteron is shown as green triangles. The respective consequences on the LR pathway in the targeted tissue are indicated. For comparison symmetric dorsal and ventral factors that could act on left–right development are shown as well. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

stages in chick embryos (Fukumoto et al., 2005; Adams et al., 2006; Levin et al., 2002). Interestingly, chick shows no evidence of dependence on cilia-based flow in symmetry breakage. Loss of cilia was reported in talpid mutants of chick, fish and mouse, but resulted in laterality defects in fish and mouse only (Bangs et al., 2011; Ben et al., 2011; Yin et al., 2009), suggesting that birds have adopted a different mechanism of LR axis specification during vertebrate evolution. Thus, among the vertebrates, evidence for an early ion-flux and a late cilia dependent mechanism, upstream of the Nodal-cascade, coincide only in Xenopus. Hence, the question of how the two mechanisms might theoretically interact is solely addressable in the frog. In order to critically interpret published or future experiments, we therefore present a theoretical framework in which potential early asymmetries can be assumed and their potential impacts on the conserved late, cilia-based LR pathway are described. In Xenopus the site of fertilization in the animal hemisphere initiates cortical rotation, a microtubule-dependent process that superimposes the dorsoanterior–posterioventral axes onto the egg’s original animal–vegetal polarity. This rotation shifts internal components of the egg relative to the cortex by shearing the cortex 301 relative to the egg’s internal content toward the prospective dorsal midline (Gerhart et al., 1989). Rotation is macroscopically a symmetrical process (Danilchik, unpublished observations). Although the cleavage pattern is itself only loosely related to the dorsal–ventral body plan (Black and Vincent, 1988; Danilchik and Black, 1988), the restriction of cell fates to dorsal and ventral can usually be discerned by pigmentation differences resulting from the cortical rotation as early as the 4 cell stage. Consequently the blastomeres adopt identifiable left and right cell fates; their lineages do not intermingle extensively during development (Moody and Kline, 1990; Muller et al., 2003; Ramsdell et al., 2006). Therefore four different cell lineages, giving rise to distinct cell fates exist and four possible asymmetric localizations of early determinants can be envisaged:

dorsal-left, dorsal-right, ventral-left and ventral-right (Fig. 2). How can an early lineage, arising via cleavage and maintained through further development, possibly interact with the cilia-dependent LR model described above?

5. Early dorsal determinants By following identified dorsal cell lineages, it should be possible to detect putative dorsal determinants that regulate tissues involved in LR development. Such factors might be important for superficial mesoderm (SM) specification during gastrulation and/or act on morphogenesis, ciliogenesis or ciliary polarization (PCP) of the gastrocoel roof plate (GRP) at neurula stages. Additionally factors might regulate the synthesis, stability or secretion of Xnr1/Coco in the lateral somitic GRP cells (Fig. 2). In addition, Lefty expression and function along the dorsal midline (floor plate and dorsal endoderm) could constitute a putative target. Early, asymmetric dorsal determinants should act upon these tissues in an asymmetrical manner, a phenomenon which so far has not been reported. In theory, asymmetrically localized dorsal determinants should be expected to set up two functionally distinct right- and left-sided organizers, which could harbor diverging axis-inductive properties or contribute asymmetrically to gastrulation processes. However, left and right halves of an early gastrula dorsal lip were equally effective in inducing secondary axis following transplantation into ventral regions of a host embryo (Mayer, 1935). Moreover, recombinants of bisected wild-type blastula or early gastrula with organizer deprived (UV irradiated, see below) embryo halves, and thus containing only a left or right organizer developed into bilateral symmetric tadpoles. Although asymmetric organ positioning of heart and gut was not analyzed in such recombinants, the presence of bilaterally symmetric structures like eyes, somites and axial midline demonstrates equal axis-inducing potencies of the left- or

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right-sided organizer (Stewart and Gerhart, 1990). Finally, in Xenopus laevis we have neither seen any robust asymmetry in ciliation, polarization or in GRP appearance, nor in Xnr1/Coco expression prior to flow (Schweickert et al., 2007, 2010; Vick et al., 2009; Maisonneuve et al., 2009).

6. Early ventral determinants In the ventral cell lineage, only one tissue has been implicated in left–right asymmetry, the lateral plate mesoderm (LPM). The LPM, the future smooth muscle layer of the inner organs, represents an ideal signaling center for regulating asymmetric morphogenesis; and, as described above, it expresses the Nodal-cascade asymmetrically. However, both right and left LPM express the cognate Nodal receptors and co-receptors of the EGF-CFC family and are for that reason equally competent for Nodal signaling. Thus, this bilaterally symmetric signaling cascade is poised at the neurula stage to respond to an asymmetric cue. What kind of maternally based cues, which would have to depend in this case on a ventral localization mechanism, could provide such a balance-tipping cue? If we posit that a maternal, ventrally localized determinant must impinge on this Nodal cascade to regulate laterality, what would its properties be? A left-ventral localization of an early asymmetric factor could either be responsible for left asymmetric gene expression in the LPM or for repressing an unknown right signaling pathway (Fig. 2). A right-ventral activity of early asymmetric determinants should either repress Xnr1 induction or induce right identity by an unidentified gene function (Fig. 2). Although numerous screens for new asymmetrically expressed genes have been performed in various labs, so far no right pathway has been identified in Xenopus, mouse or fish. A substantial influence of early activities on the Nodal-cascade in the LPM, independent of the leftward flow process, again is very unlikely in the light of LPM explant culture experiments performed recently by Ohi and Wright (2007). An inductive property of an early determinant, independent of leftward flow, should activate left Xnr1 expression, irrespective of the presence of dorsal structures like the GRP. However the detailed analysis by Ohi and Wright (2007) on the temporal and spacial Nodal/Lefty interplay at the LPM and midline showed that left LPM explants, taken at early neurula stages into culture, prior to leftward flow failed to induce asymmetric Xnr1 transcription. In contrast, left LPM dissected from late neurula embryos (i.e during or post leftward flow) always showed Xnr1 expression (Ohi and Wright, 2007). Therefore, two conclusions can be drawn: (1) The left Nodal gene cascade is induced at a timepoint of development which correlates perfectly with leftward flow and (2) the left LPM has no pre-determined left identity. The finding that asymmetric gene expression is induced renders unlikely an active right repression mechanism that depends on early determinants. Because this scenario would predict that induction of the Nodal cascade is initially, i.e. by default bilateral, and that it only later becomes actively repressed in the right LPM. However, neither left nor right LPM explants excised prior to flow do express Xnr1 (Ohi and Wright, 2007). In summary, our theoretical analysis revealed no particular step in which early asymmetries could direct left–right specification. However, given that early asymmetries of components involved in this pathway have been observed, we suggest that their presence, but not their localization per se, may play a supporting, perhaps metabolic or physiological role.

7. The ion-flux hypothesis The ion-flux model of symmetry breakage suggests that following fertilization, mRNAs and proteins of ion pumps are

asymmetrically distributed in a microtubule and actin dependent manner between left and right blastomeres of the 2 and 4 cell embryo (Adams et al., 2006; Levin et al., 2002; Aw et al., 2008; Qiu et al., 2005; Vandenberg and Levin, 2010). These ion pumps, mainly the gastric H/K-Atpase and the vacuolar vATPase, are active at the 4–32 cell stages and subsequently generate an electric charge gradient between left and right blastomeres. This electrochemical gradient constitutes the driving force to enrich a small, charged and initially uniform distributed LR morphogen, the neurotransmitter serotonin, via gap junctional communication to the right-ventral cell (Vandenberg and Levin, 2010; Levin, 2006). Indeed, asymmetric localization of H/K-ATPase /(ATP4a) mRNA at the 2–8-cell and right-ventral accumulation of serotonin at 32–64 cell stages have been reported (Fukumoto et al., 2005; Adams et al., 2006). However, the authors themselves admit that the asymmetric distribution of ATP4a mRNA seems to be dependent on the analyzed embryo batch (Aw et al., 2008), questioning the robustness of this localization process. Downstream of serotonin, the pathway appears unclear as well: specific drug treatments indicate the involvement of receptors, but a recent report suggest a receptor independent, epigenetic serotonin activity on the Xnr1 promoter, resulting in repression in the right LPM (Fukumoto et al., 2005; Carneiro et al., 2011). So far, effects elicited by loss of function of ion pumps or serotonin signaling on superficial mesoderm (SM) specification, GRP morphogenesis, leftward flow or somitic Xnr1/Coco expression were not analyzed. However, pharmacological inhibition of vATPase in fish resulted in perturbed laterality as well as a defect in ciliogenesis in Kupffer’s vesicle (Adams et al., 2006), suggesting that leftward flow was altered. Interestingly, a function of the ion pump vATPase in Wnt signaling, a developmentally crucial pathway, has been recently demonstrated (Cruciat et al., 2010; Coombs et al., 2010). A role of canonical and non-canonical Wnt signaling on organ laterality has been shown in fish and mouse (Oteiza et al., 2010; Hashimoto and Hamada, 2010; Lin and Xu, 2009; Nakaya et al., 2005), suggesting that vATPase might be involved in similar processes in frog. Additionally, an effect on Wnt dependent dorsal organizer formation could be envisaged.

8. Is LR axis specification independent of Spemann’s organizer? Irrespective of which blastomere might harbor an early determinant and how determinants might act, the basic assumption behind any early mechanism is that Spemann organizer is not able to set up a correct LR axis on its own. The potency of the organizer to regulate anterio-posterior (AP) and dorso-ventral (DV) development has been demonstrated repeatedly since the discovery by Spemann and Mangold (1924). The concept of early determinants implies that a predetermination mechanism would exist, prior to organizer formation and that this could imprint asymmetrical positional cues onto the embryo. In Xenopus such an early process clearly exists with respect to the DV axis. The DV axis is specified at the one cell stage by cortical rotation that is initiated by sperm entry. During rotation, maternally vegetal deposited components of the Wnt pathway are transported to a position opposing the fertilization site. As a consequence, the point of sperm entry determines ventral fate as the canonical Wnt signaling induces subsequently the organizator and dorsal cell fates on the opposite side (Weaver and Kimelman, 2004). Because the outer cell layer of Spemanns organizer, the SM, develops into the gastrocoel roof plate (GRP), this process should be critical for LR formation in the cilia model. Indeed, the analysis of axis-impaired, ultraviolet (UV) irradiated embryos revealed laterality defects. UV irradiation at the one cell

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stage disrupts cortical rotation (Elinson and Pasceri, 1989). Phenotypes displayed in such specimen are, depending on the strength of exposure, shortened AP-axes, smaller heads, subsequent loss – up to complete lack – of dorsal tissues. The frequency of altered organ laterality is very high in mildly affected specimens (Danos and Yost, 1995), demonstrating that proper dorsal development is necessary for LR axis specification. However, using the UV method it is possible to test for the presence of a relevant asymmetrically acting mechanism independent of the organizer. Strong UV irradiation completely abolishes cortical rotation and thus organizer formation. A new organizer can then be introduced at desired ectopic or native locations by injecting mRNAs encoding Wnt pathway components or downstream targets like the homeoboxgene Siamois. These components have been shown to fully rescue AP and DV axes of UV treated embryos (Sokol et al., 1991; Fan and Sokol, 1997).

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If an assumed chirality of the egg and/or the site of sperm entry fixes LR identities to the respective blastomeres the effect on laterality would vary predictably in axis-rescued embryos depending on the relative position of the newly induced organizer. For simplification, we assume that the early mechanism determines some aspect of sidedness of both left and right blastomeres in the 4-cell embryo (Fig. 3). Performing an organizer rescue in UV treated embryos, opposite the side of sperm entry should thus reconstitute the position of the native dorsal organizer and generate wild-type AP and DV axes. As a consequence all rescued embryos should develop normal left-sided activation of the Nodal-cascade and wild-type organ placement (situs solitus; Fig. 3). In contrast, if organizer rescue occurs on the same side as sperm entry, the DV and AP axes switch position with respect to the initial axes. If the native asymmetrical identity of blastomeres is indeed predetermined, then all tadpoles consequently should

Fig. 3. Spemann‘s organizer independent LR axis formation. Prediction of laterality in rescued UV irradiated embryos. 4-cell stage embryos with hypothetical predetermined LR axis are shown (red, right; uncolored, left). Endogenous ventral and dorsal blastomeres are indicated by sperm symbols (entry site) and circles (former organizer), respectively. Following UV irradiation and thus elimination of endogenous organizer, rescue could be targeted to four distinct positions (yellow star) relative to initial dorso-ventral (DV) and hypothetical pre-determined left–right axes. Organizer rescues could be performed at initial dorsal or ventral (sperm entry) side or at a 901 angle to initial DV axis, thus at left or right blastomeres. Induced dorso-ventral axes are indicated (d–v). Cell fates of hypothetically predetermined left and right blastomeres are superimposed on neurula specimens (red, right; uncolored, left). If LR axis would be prefixed, UV rescued embryos should predictably display different laterality phenotypes depending on site of rescue. Asymmetric organ placements range from wild-type (situs solitus), mirror image (situs inversus) to randomization of single organs (heterotaxia). Note the differences in the frequency of wildtype situs solitus between prediction of an early prepatterned LR axis and the published experimental data by Vandenberg and Levin (2010) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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display a mirror-imaged, inverted LR axis (situs inversus; Fig. 3). Lateral organizer rescues by an angle of 901 to the sperm entry would result in two different scenarios, depending on whether left or right cells were the ones being targeted (Fig. 3). If rescue is induced on the prefixed left side, native left blastomeres would give rise to dorsal midline tissue whereas blastomeres with former right identity would differentiate into ventral fates, including the LPM. The resulting right and left LPM should behave more like right-side LPM and therefore not be able to activate the Nodal-cascade. Organ laterality in such rescuees should be completely randomized (heterotaxia; Fig. 3). In Xenopus laevis, three organs are scored for specific asymmetry (outflow-tract of the heart, gall bladder, gut coiling) with each having 50% chance of being normal or inverted in randomized specimens. A random orientation of all three organs therefore leads to 12.5% (50%  50%  50%¼ 12.5%) of specimens displaying wild-type situs solitus by chance. The same scenario would hold true for a the new organizer set up in right blastomeres. Both predetermined left blastomeres would acquire ventral fates and give rise to the bilateral LPM. In this scenario, left determinants should induce the left asymmetric Nodal-cascade in the LPM bilaterally, which would likewise result in a randomization of organ situs (Fig. 3). Even if predetermination of sidedness is restricted to the single cell linage at the 4-cell stage, the hypothetical outcome would be basically the same (not shown). Taken together, the organizer rescue in UV treated embryos with an assumed early prefixed LR axis alters laterality in a predictable manner. On the other hand, if the organizer itself has the property to align all three body axes, the site of rescue relative to sperm entry should be irrelevant for left–right development. All rescued embryos should establish a wild-type LR axis and display situs solitus. Recent work by Vandenberg and Levin (2010) have adressed this question and indeed performed such UV rescue experiments. UV irradiation was rescued by Siamois mRNA injections at cleavage stages and asymmetric organ placement was subsequently analyzed. Independently of injection site (initial ventral, dorsal or lateral) approx. 75% of tadpoles showed wildtypic situs solitus. A second method of organizer induction in UV embryos was applied by tipping embryos 20–901 along the animal–vegetal axis immediately after microtubule network destruction by UV irradiation. By mimicking the normal rotation, tipping induces a fully functional organizer (Weaver and Kimelman, 2004; Scharf and Gerhart, 1980). Vandenberg and

Levin determined that, without genetically interfering, tipping was somewhat more efficient in rescuing the LR axes to wild-type (95% situs solitus). In summary, both methods of organizer rescues regulate body axis formation with correct LR patterning in a majority of cases. Our interpretation of this experiment is that early LR asymmetries are not able to govern laterality in an organizer independent mechanism in Xenopus. Surprisingly, Vandenberg and Levin interpreted their data in a very different manner. In their view rescuing UV irradiation by tipping or Siamois mRNA injections are biologically different methods. Tipping, at the one cell stage, represents an ‘‘early’’ rescue, whereas Siamois mRNA injections consitutes a ‘‘late’’ rescue, because regulation of gene transcription is only possible after mid-blastula transition. The differences in the efficiency to induce situs solitus in rescued body axes (95% tipping vs. 75% Siamois) is interpreted as dependent on the time at which rescue was applied. The conclusion by Vandenberg and Levin was that a ‘‘late’’ rescue is not able to fully restore wt asymmetry and thus a ‘‘late’’ induced organizer lacks asymmetric positional information. On the other hand, the tipping method is effective early and thereby creates an organizer with additional early LR cues, resulting in wild-type laterality only (Vandenberg and Levin, 2010). We question the rationale that the frequency of 75% situs solitus obtained by Siamois mRNA injection is a substantial evidence for an early acting mechanisms. Even if our theoretical assumptions may not be fully applicable to a more sophisticated/ complex but unknown mechanism, a lack of a robust asymmetric input on Siamois induced organizers should at least result in complete randomization of organ placement, i.e. only 12.5% situs solitus. Additionally, varying results obtained by two very diverse experimental approaches (tipping vs. gain of Siamois function) might reflect methodological differences and may be attributed to dosage effects. Using tipping endogenous signaling factors are present at optimal levels, whereas mRNA injections face the intrinsic property to deliver sub-optimal Siamois dosage to the embryo. Slightly less or more Siamois might pattern AP and DV axes correctly but specifically lead to defects in SM specification, GRP morphology, ciliogenesis or Xnr1/Coco transcription and thereby to altered organ laterality. This notion is in agreement with the wild-type maintenance of AP and DV axis development following superficial mesoderm (SM) ablation at early gastrula stages, resulting in LR defects only (Blum et al., 2009).

Table 1 Left–right determination in Xenopus: an experimental checklist. Experimental methods to analyze different phases of Xenopus LR development and corresponding references are listed. Embryonic stages are indicated by brackets. in situ hybridization (ISH); Scanning electron microscope (SEM); immunohistochemistry (IHC). LR process

Methods

Specification of SM

ISH: Foxj1 (st.10.5)

Pohl and Knochel (2004); Stubbs et al. (2008)

GRP morphogenesis

Analysis of dorsal explants (st.13–20)

Blum et al. (2009)

ISH: Tekt2, Spag6, dnah9

Ciliogenesis

References

Polarization of cilia

IHC: ac. tubulin; SEM

Cilia motility Leftward flow

In vivo videography (fluorescence microscopy) tracking of fluorescent beads (most robust st. 17) ISH: Coco asymmetry ( Z st.19) ISH: Coco, Xnr1 (st.15–19)

Essner et al. (2002); Schweickert et al. (2007) Stubbs et al. (2008); Vick et al. (2009) Essner et al. (2002); Schweickert et al. (2007, 2010); Stubbs et al. (2008); Vick et al. (2009); Antic et al. (2010) Schweickert et al. (2007, 2010); Antic et al. (2010); Maisonneuve et al. (2009) Schweickert et al. (2007); Vick et al. (2009) Schweickert et al. (2007); Vick et al. (2009) Schweickert et al. (2010) Schweickert et al. (2007); Vonica and Brivanlou (2007)

Midline integrity

ISH: Lefty, Xbra ( Z st.19)

Cheng et al. (2000); Smith et al. (1991)

LPM competence

LPM explants & ISH: Xnr1 RT-PCR: XCR2

Ohi and Wright (2007) Onuma et al. (2006)

IHC: ac. tubulin; SEM,

Target of leftward flow

Nodal-cascade in left LPM

ISH: Xnr1, Pitx2c, Lefty (4st.22)

Lowe et al. (1996); Campione et al. (1999); Cheng et al. (2000)

Organ situs

orientation of heart, gallbladder, gut-coiling (st.45)

Nieuwkoop and Faber (1967)

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9. Conclusion We propose that LR asymmetry of Xenopus laevis cannot be established independent of the organizer’s functions, which includes development of the ciliated GRP upon which leftward flow depends. The implication of this relationship is that any damage to the organizer, whether it occurs early in development when dorsal–ventral axis specification is dependent on localizations, or later, during the expression of pattern-generating zygotic genetic programs, is likely to alter leftward flow, and hence increase the likelihood of heterotaxy and/or situs inversus. We attempted to theoretically integrate the concept of early asymmetric determinants into the conserved, flow-dependent LR pathway of Xenopus. In general, we were not able to unravel a profound necessity of an early mechanism when leftward flow is present. The greatest difficulty with this early mechanism is that its influence cannot be measured independent of the flowdependent Nodal-cascade. Thus, we cannot formally rule out the possibility that some degree of competence on the various relevant signaling pathways is provided by early determinants. However, the functional analysis of early factors in LR development is incomplete and so far restricted to asymmetric gene expression and organ laterality. Proving or disproving a connection of early asymmetries to organizer function and/or leftward flow is the only way to resolve this discrepancy. We emphasize that any experimental manipulation of Xenopus laterality should include the analysis of the conserved steps described above, irrespective of factors, methods used or timing aspects of treatments (see Table 1). The latter notion is especially relevant for pharmacological inhibitor studies, as incubations during cleavage stages may still affect late processes. The burden then falls on maternally driven models to show that early effects on lateralization can be elicited without affecting the wnt-PCP-dependent orientation of cilia, the development and motility of cilia, and the [presently poorly defined] receptor modalities that detect flow itself. Only if these major aspects of LR development in Xenopus are addressed, can progress toward reconciling these two seemingly incompatible hypotheses be achieved.

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