SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

Xenopus, an Ideal Model System to StudyVertebrate Left-Right AsymmetryMartin Blum,* Tina Beyer, Thomas Weber, Philipp Vick, Philipp Andre, Eva Bitzer, andAxel Schweickert

Vertebrate organ laterality is manifested by the asymmetric morphogenesis and placement of inner organs.Asymmetric induction of the Nodal signaling cascade in the left lateral plate mesoderm (LPM) precedes andis essential for asymmetric organ morphogenesis. While the Nodal cascade is highly conserved, symmetrybreakage is considered to vary between the different classes of the vertebrates. In Xenopus, earlydeterminants at cleavage stages were thought to break symmetry, opposed to cilia-driven leftward flow inmammals and fish. The main objectives of this review are to emphasize the conserved nature of symmetrybreakage, and to demonstrate the power of Xenopus embryology to analyze and manipulate flow. Inaddition, mutant phenotypes described in other model organisms can easily be mimicked in frog by singleor multiple knockdowns in combination with experimental manipulations and flow analysis. Xenopus,therefore, is ideally suited to address the major open questions in the field. Developmental Dynamics 238:1215–1225, 2009. © 2009 Wiley-Liss, Inc.

Key words: Xenopus; left-right asymmetry; cilia; leftward flow; gastrocoel roof plate

Accepted 10 December 2008

A CENTURY OFEXPERIMENTAL ANALYSISOF LATERALITY INAMPHIBIANS

Hans Spemann and his co-workersinitiated experimental analysis ofbody plan specification in the early20th century. To date, probably one ofthe best-cited embryological works isthe famous 1924 Spemann andMangold paper, “On the Induction ofEmbryonic Primordia by Implanta-tion of Heterologous Organizers”(“Uber Induktion von Embryonalanla-gen durch Implantation artfremderOrganisatoren”), the molecular analy-sis of which is still keeping countlessscientists busy in the different verte-

brate model organisms (Spemann andMangold, 1924). It is less well known,however, that Spemann, before heturned to the organizer, was the firstto manipulate the left-right (LR) axisin a defined and predictable manner,i.e., that experimental analysis of lat-erality was established in the amphib-ian embryo.

In 1904, Spemann reported inver-sions of the normal asymmetric ar-rangement of inner organs (situs in-versus) in twinned embryos, which hehad created by partial or complete lig-ature of newt embryos (Triturus tae-niatus, Triturus cristatus; Spemann,1904). His observation was in agree-ment with the then-known fact thatsitus inversions occur frequently in

identical and Siamese human twins.Fifteen years later, Spemann andFalkenberg concluded this work withan in-depth study, in which they de-scribed that it was always the righttwin that was affected, and that situsinversions occurred in about 50% ofcases (Spemann and Falkenberg,1919). A second experimental ap-proach was to rotate the mid-dorsalpart of neurula embryos by 180°, in-cluding all three germ layers, i.e., neu-ral plate, notochord, somites (includ-ing perhaps intermediate and someLPM as well), and the archenteronroof (Fig. 1A, B). These experiments,initiated by Spemann and followed upby his students Kurt Pressler and Ru-dolph Meyer using yellow-bellied toad

Additional Supporting Information may be found in the online version of this article.University of Hohenheim, Institute of Zoology, Stuttgart, GermanyGrant sponsor: Deutsche Forschungsgemeinschaft.*Correspondence to: Martin Blum, University of Hohenheim, Institute of Zoology, Garbenstrasse 30, 70593 Stuttgart, Ger-many. E-mail: mblum@uni-hohenheim.de

DOI 10.1002/dvdy.21855Published online 10 February 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 238:1215–1225, 2009

© 2009 Wiley-Liss, Inc.

(Bombinator pachypus, renamedBombina variegata), common waterfrog (Rana esculenta), and commontoad (Bufo bufo) embryos, provided anamazing result: a few days later, op-erated tadpoles revealed a completeinversion of organ placement in mostcases (�80%; Fig. 1C; Spemann, 1906;Pressler, 1911; Meyer, 1913).

Hilde Wilhelmi followed up on Falk-enberg’s ligature experiments to cre-ate twins and occasionally found em-bryos with alterations of organ situs,which were not the result of twinningbut of tissue ablations. Her carefulanalysis revealed that laterality de-fects only occurred when she inducedablations on the left side of the embryoat mid-neurula stages. Wilhelmi con-cluded from her experiments and fromearlier ones from Spemann and col-leagues that “. . .situs inversion ingeneral was explained by the fact thatthe left side of the germ has some-thing that the right half does nothave” (Wilhelmi, 1921). For a good de-cade now we have known that this“something” is represented by theNodal signaling cascade in the leftLPM, which was uncovered over a4-year period in chick, frog, mouse,and zebrafish (Levin et al., 1995; Loweet al., 1996; Logan et al., 1998; Re-bagliati et al., 1998; Ryan et al., 1998;Yoshioka et al., 1998; Campione et al.,1999).

CILIA, FLOW, ANDSYMMETRY BREAKAGE

Evidence that motile cilia are involvedin symmetry breakage has accumu-lated from 1976 onwards, when Afze-

lius reported the absence of dyneinarms on axonemes of human Kartage-ner patients (Afzelius, 1976). Thissyndrome comprises, among other cil-ia-related malfunctions, inversion oforgan situs (Fliegauf et al., 2007).Schoenwolf and colleagues first spec-ulated about cilia at the distal pit ofthe mouse egg cylinder as a determi-nant of symmetry breakage (Sulik etal., 1994). We have previously arguedthat this indentation in fact is distinctfrom the organizer/node, and repre-sents the posterior-most aspect of thenotochordal plate (posterior noto-chord, PNC; Blum et al., 2007). Thedistinguishing features of node vs.PNC are summarized in Table 1. Hi-rokawa and colleagues in theirground-breaking work on the Kif3Bknockout mouse provided compellingevidence that (1) cilia at the PNC aremotile, (2) cilia produce a leftwardflow of extracellular fluid, and (3) lackof cilia and/or flow results in lateralitydefects (Nonaka et al., 1998). Mean-while, knockout mice and spontane-ous mutants have proven that inmouse cilia-driven leftward flow pre-sents the decisive step upstream ofthe Nodal cascade (Okada et al., 1999;Murcia et al., 2000).

The chick, in which the Nodal cas-cade was initially characterized(Levin et al., 1995), presents an excep-tion in that no flow has been reportedthus far. We have previously hypoth-esized that chick embryos may havelost flow secondarily, as they lack su-perficial mesoderm and represent arather derived taxon (Shook et al.,2004; Blum et al., 2009). Flow was,however, present in basal vertebrates;

in Kupffer’s vesicle (KV) of teleost fishembryos, motile cilia produce a vecto-rial fluid flow from right to left, andthis flow is instrumental for symmetrybreakage (Essner et al., 2005;Kramer-Zucker et al., 2005). Evolu-tionary conservation of this mecha-nism was demonstrated by the recentfinding that amphibian embryos alsopossess a ciliated epithelium at thegastrocoel roof (gastrocoel roof plate,GRP), where flow develops duringneurula stages shortly before the left-sided onset of asymmetric gene ex-pression (Essner et al., 2002; Shook etal., 2004; Schweickert et al., 2007;Blum et al., 2009). PNC, GRP, and KVshould represent homologous struc-tures, as they share a great number ofstructural, molecular, and functionalfeatures, which are summarized inTable 2. In particular, these epitheliaare always monociliated, derived fromsuperficial mesoderm, bordered by bi-lateral expression of Nodal, and pro-duce a leftward flow of extracellularfluids (Blum et al., 2007, 2009).

Given the evolutionary conserva-tion of flow, the frog offers the oppor-tunity to tackle the major open ques-tions in the field in a highly accessibleand potent system, namely (1) What istransferred by flow? (2) How andwhere is such a determinant per-ceived? (3) How and by what route isan asymmetric signal transportedfrom the midline to the left LPM? (4)How is asymmetric organ morphogen-esis executed downstream of theNodal cascade? (5) How do early (pre-flow) determinants of laterality con-nect to flow?

TABLE 1. Criteria Distinguishing Between the Primary Embryonic Organizer (Spemann’s Organizer) and theCiliated Epithelium, Which Produces a Vectorial Leftward Flow (Left-Right Coordinator)a

Spemann’s organizer Left-right coordinator

Morphology Indistinguishable germ layers Distinguishable germ layersNo cilia Monocilia

Indicative genes goosecoid left-right dynein (Lrd)Function Embryonic organizer Leftward flow 3 symmetry

breakageNomenclature mouse Node Posterior notochord (PNC)

frog Dorsal lip Gastrocoel roof plate (GRP)zebrafish Embryonic shield Kupffer’s vesicle (KV)rabbit Hensen’s node Posterior notochord (PNC)

aSummarized from Blum et al. (2007).

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TABLE 2. Determinants and Models of Symmetry Breakage in Frog, Mouse, Zebrafish, and Chick

Frog Mouse Zebrafish Chick References

Leftward flow � � � � Schweickert et al. (2007); Nonakaet al. (1998); Essner et al.(2005); Kramer-Zucker et al.(2005)

Structuralcomponents

Cilia (GRP) Cilia (PNC) Cilia (KV) � Shook et al. (2004); Sulik et al.(1994); Essner et al. (2002)

Posteriorlocalization

Posteriorlocalization

� � Schweickert et al. (2007); Nonakaet al. (2005)

Rotationalmovement

Rotationalmovement

Rotationalmovement

Schweickert et al. (2007); Nonakaet al. (2005); Kramer-Zucker etal. (2005)

Cilia-relatedproteins

Lrdr Lrd Lrdr Lrdr Schweickert et al. (2007); Essneret al. (2002); Supp et al. (1997);Essner et al. (2005)

� IFT IFT � Nonaka et al. (1998); Murcia etal. (2000); Bisgrove et al. (2005)

PC-2 PC-2 PC-2 PC-2 Schweickert et al. (2007);Pennekamp et al. (2002);Bisgrove et al. (2005); Qiu etal. (2005)

Inversin Invs � � Schweickert et al. (2007); Morganet al. (1998);

FoxJ1 FoxJ1 FoxJ1 � Aamar and Dawid (2008); Stubbset al. (2008); Zhang et al.(2004); Yu et al. (2008)

Flanking expressionpattern

Xnr-1 Nodal spaw cNr-1 Lowe et al. (1996); Zhou et al.(1993); Long et al. (2003);Levin et al. (1995)

Coco Dand5 Charon Caronte Vonica and Brivanlou (2007);Marques et al. (2004);Hashimoto et al. (2004);Rodriguez Esteban et al. (1999)

Derriere Gdf1 � � Vonica and Brivanlou (2007);Rankin et al. (2000)

Asymmetric markergenes

Xnr1 Nodal spaw cNr-1 Lowe et al. (1996); Long et al.(2003); Levin et al. (1995)

antivin Lefty-2 Lft-1/2 Lefty-1 Thisse and Thisse (1999); Menoet al. (1996); Bisgrove et al.(1999); Ishimaru et al. (2000)

Pitx2 Pitx2 Pitx2 Pitx2 Yoshioka et al. (1998); St. Amandet al. (1998); Ryan et al. (1998);Campione et al. (1999)

Model for symmetrybreakage

ion flux � � ion flux Levin (2004, 2005);

leftward flow leftward flow leftward flow � Nonaka et al. (1998); Hirokawaet al. (2006)

Early determinants Vg1 � � � Hyatt et al. (1996); Hyatt andYost (1998)

Claudin � � Claudin Brizuela et al. (2001); Simard etal. (2006)

Syndecan-2 � � Syndecan-2 Kramer et al. (2002); Kramer andYost (2002); Fukumoto andLevin (2005)

Gap junctions � � Gap junctions Levin and Mercola (1998); Levinand Mercola (1999);

Serotonin � � Serotonin Fukumoto et al. (2005)H�/K�-ATPase � � H�/K�-

ATPaseLevin et al. (2002); Aw et al.

(2006)V-ATPase � V-ATPase V-ATPase Adams et al. (2006)

–, Not reported thus far.

LEFT-RIGHT ASYMMETRY IN XENOPUS 1217

FAST AND RELIABLEASSESSMENT OF FLOWPARAMETERS IN XENOPUS

Flow in frog was detected many yearslater than in mouse and fish. The rea-son for this delay was the hidden na-ture of the ciliated epithelium in thedorsal gastrocoel roof, although Ess-ner et al. (2002) and Shook and col-leagues (2004) had already pinpointedthe potential of the GRP and theircilia for LR development. Once ex-posed in dorsal explants, which can beeasily prepared (Fig. 2), flow is readilydetected upon addition of fluorescent

beads. To better visualize flow, weprocess time-lapse videos of beadmovements to yield gradient timetrails (GTTs), i.e., color-coded tracksof beads that reveal direction of trans-port and velocity of particles (fromgreen to red; 25 sec). Undirected par-ticle movement is eliminated from theanalysis to filter out particles movedby Brownian motion. When GTT tra-jectories are compared using frog,mouse, and rabbit (Fig. 3G–I; and seeSuppl. Movie S1, which is availableonline), flow is clearly directed to theleft in all cases. As a qualitative mea-sure, we have introduced the dimen-

sionless number rho, which is themean resultant length of flow. Rho iscalculated by performing a Rayleigh’stest of uniformity over the mean an-gles of bead trajectories. In lay terms,when rho equals 1, all beads projectinto the same direction. A rho value of0 reflects movement of beads into alldifferent directions. A robust leftwardflow in frog (i.e., at stage 17) is typi-cally characterized by rho values be-tween 0.7–0.9, with similar character-istics in mouse and rabbit. Toillustrate the directedness of all beadsin an explant, we draw wind rosessuch as the ones depicted in Figure

Fig. 1. Inversion of organ situs by 180° rotation of dorsal tissue. A,B: Classical experiment by Spemann and Meyer in which the dorsal region ofamphibian embryos comprising neural plate, notochord, and archenteron roof was rotated during neurula stages. C: Fifteen days after the operation,most tadpoles revealed a complete inversion of organ situs. b, bulbus (outflow tract); e, endoderm; g, gut; l, liver; m, mesoderm; n, notochord; np,neural plate; po, post operation; v, ventricle of heart; y, yolk. Panels reproduced from Meyer (1913).

Fig. 2. Preparation of dorsal explants to visualize and image flow at the GRP. Embryos at stage 17 (A) are cut in half (B), followed by dissection ofthe posterior part along the red dashed lines (C). Ventral and dorsal parts are pulled apart (arrows in C), and the dorsal explant is completed by removalof the yolk ventral of the circumblastoporal collar (D; red dashed line). a, anterior; cbc, circumblastoporal collar; d, dorsal; GRP, gastrocoel roof plate;l, left; LEC, lateral endodermal crest; p, posterior; r, right; v, ventral.

1218 BLUM ET AL.

3G�–I�, in which bead trajectories aregrouped into sectors of 45° each in acircular histogram, and the size of pet-als reflects the relative frequency with

which trajectories are found in a givensector.

Parameters, which are not directlyaccessible in the frog, are beat pattern

and beat frequency of cilia. This is dueto the high yolk content of cells, whichresults in scattering of polarized light.In contrast, mouse and rabbit cilia canbe directly observed and video-graphed. This apparent disadvantageof the frog system can be overcome byfluorescent labeling of axonemal pro-teins. Park et al. (2008), for example,injected a tau-GFP fusion constructtargeted to the epidermis, which la-beled the axonemes of epidermal ciliain vivo. In summary, the basic charac-teristics of leftward flow in frog areconserved compared to mouse, rabbit,and zebrafish embryos.

A number of differences, however,apply as well. In frog, rabbit, andmouse, the epithelia themselves arebasically flat, while the fish KV maybe dome- (medaka) or sphere-shaped(zebrafish). In mouse, the margins ofthe ciliated epithelium, i.e., the tran-sit zone to the flanking endoderm, iselevated towards the ventral side (Fig.3E), a feature not seen in Xenopus. Ithas been argued that this elevationmay be of functional relevance, i.e., toprovide a barrier, at which vesiclestransported by flow would be smashed(Nakamura et al., 2006). The GRP to-pology, however, does not support thisnotion (see Fig. 3D). A second note-worthy feature is the great variabilityof dimensions of the respective struc-tures. While the PNC in mouse mea-sures 50 �m from left to right, thisamounts to about 100 �m in rabbit,and approximately 200 �m across theXenopus GRP, and KVs in fish rangebetween 50–150 �m (Cooper andD’Amico, 1996; Melby et al., 1996;Okada et al., 2005; Blum et al., 2007,2009; Schweickert et al., 2007). Thelarge size of the GRP not only allowsfor easier imaging, but also simplerpreparation and handling comparedto the PNC in mouse and rabbit. TheKV in teleost fish embryos, represent-ing the remnant of the archenteron(Cooper and Virta, 2007), presents it-self as a closed sphere that very muchlimits access to cilia, requiring techni-cally challenging 3D imaging equip-ment.

Comparing all systems used to date,the frog GRP offers distinct advantag-es: (1) a great number of explants canbe prepared and analyzed in a shorttime, facilitating statistical analysis;(2) qualitative and quantitative pa-

Fig. 3. Comparison of ciliated epithelia and leftward flow in frog, mouse, and rabbit. A–C: Scanningelectron micrographs of monociliated epithelia (dashed lines) at the GRP (A), and PNC in mouse (B) andrabbit (C). A’–C’: Blow-ups of cilia. Note that the dimensions of the respective structures vary consid-erably. D–F: Topology of GRP/PNC shown in transverse sections (levels indicated in A–C) of embryoshybridized in whole-mounts for the expression of the flanking Nodal gene. Please note that while theGRP appears flat (D), the PNC in mouse is a concave structure (E), and the PNC in rabbit bendsconvexly (F). G–I: Comparison of leftward flow depicted as GTTs (G–I) and wind roses (G’–I’). Note thatthe flat GRP allows for the simultaneous imaging of the whole structure, while due to the bent natureof the PNC only parts can be imaged and sampled in focus. Please note also that the quality of flow(rho-value) is highest in frog. a, anterior; d, dorsal; fp, floor plate; GRP, gastrocoel roof plate; l, left; n,notochord; p, posterior; PNC, posterior notochord; r, right; s, somite; v, ventral. Bar graphs � 50 �m inA–C and D–I, 2 �m in A’–C’. Color gradient bar in G and I � 25 sec, and 4.3 sec in H.

LEFT-RIGHT ASYMMETRY IN XENOPUS 1219

rameters can be determined with sta-tistical significance, such as velocity,directionality, and the ratio of di-rected (flow) vs. random (Brownian)movements; and (3) stages unequivo-cally predict the status of flow, withvery little variability. Stage 17/18neurulae will in all cases reveal robustleftward flow in dorsal explants. Incontrast, the impossibility to stagemouse and rabbit embryos in uterowith the required accuracy results inthe frequent recovery of pre- or post-flow embryos, necessitating largenumbers of animals to be processed inorder to receive statistically validdata. Taken together, the frog systemoffers a highly standardized assay sys-tem for the analysis of cilia-drivenleftward flow.

EXPERIMENTALMANIPULATIONS OF GRPAND FLOW

A straight-forward approach to ma-nipulate flow is its direct eliminationby placing a droplet of methylcellulose(MC), i.e., a highly viscous liquid, ontothe ciliated epithelium of the GRP byinjection into the archenteron at stage14–17. In such experiments, weachieved on average 50% of embryoswithout induction of Xnr1 or Pitx2 inthe left LPM (Schweickert et al., 2007;and our unpublished data). This ma-nipulation established leftward flowas the decisive step in setting up em-bryonic laterality.

Shook et al. (2004) described thederivation of the GRP from superficialmesoderm (SM), i.e., surface epithelialcells that give rise to the outer celllayer of the dorsal lip of the blastoporeat stage 10–10.5. These cells invagi-nate during gastrulation and form adistinct epithelium of small ciliatedcells from stage 12/13 onwards. Theyare positioned in the posterior roof ofthe archenteron, from where they foldoff successively from stage 15 onwardsto integrate into deep mesoderm (no-tochord, hypochord, and somites;Shook et al., 2004). In order to analyzewhether SM is required for lateralitydevelopment, we used classical micro-surgery to remove this tissue at stage10 (Fig. 4A). Ablated embryos devel-oped through gastrulation and neuru-lation without apparent dorso-ante-rior axis defects (DAI � 5); however,

organ situs at stage 45 was random-ized. Positioning of the gall bladderand heart and gut looping were in-verted (situs inversus) or developeddiscordantly (heterotaxia; Fig. 4B).This rather simple experiment dem-onstrates that the SM-derived GRP isstrictly required for LR development.In zebrafish, a comparable experimenthas been reported, in which the ma-ture KV was mechanically destroyedby injection of water (Bajoghli et al.,2007). In these experiments, no otherdefects except laterality disturbancesresulted, strongly suggesting that KVand GRP indeed are functionally ho-mologous.

As a prerequisite of further manip-ulations, the GRP lineage needs to beknown. For this purpose, we injectedLacZ mRNA at the 4–32 cell stage.The dorsal-marginal region of the4-cell embryo (Fig. 5A,B) or the dor-sal-marginal blastomere C1 of the 32-cell embryo define the GRP lineagemost accurately (Fig. 5C, D). Depend-ing on the stage of injection, the floorplate is targeted with a certain likeli-hood as well. This should be kept inmind when ciliary determinants areaddressed by experimental manipula-tions, as in these cases neural tubeclosure defects may ensue, possibly re-sulting in misjudgment of embryonicstages. A unique possibility of the Xe-nopus system is the opportunity totarget the GRP in a sided manner,such that the left or right half is spe-cifically hit (Fig. 5E, F). Such manip-ulations are not available in any othervertebrate model organism in whichflow was characterized.

We have started to use these injec-tion schemes to specifically knockdown axonemal proteins and otherknown left-right determinants. As anexample, we have targeted a dyneinheavy chain gene, because the humanKartagener syndrome and the mouseiv mutant both are caused by muta-tions in axonemal dyneins. In agree-ment with the genetic mutants, weobserved laterality defects and abnor-mal flow (our unpublished results),demonstrating that frog morphantscan effectively mimic mutations gen-erated in mouse or zebrafish. The frogsystem offers the opportunity to per-form multiple tissue-specific geneknock-downs as well as combinationsof loss- and gain-of-function experi-

ments, schemes that are much morecomplicated to set up in mouse or ze-brafish. In addition, epistasis experi-ments can be designed by combiningknock-downs and experimental ma-nipulations such as MC treatment ormicrosurgery. Finally, ablations andtransplantations (Spemann andMangold, 1924; Borchers et al., 2000;Ohi and Wright, 2007) can be com-bined with knock-downs and drugtreatments, to avoid deleterious ef-fects due to collateral damage (i.e.,targeting of unwanted tissues alongthe same lineage).

The perspective for such approachesis bright, as every new player publishedin any system can be assessed directlyin the frog. The recent advent of trans-genic frog technologies in Xenopustropicalis, which has a GRP and flowjust like Xenopus laevis (Blum et al.,2009), provides further options for gen-erating and analyzing mutant lines us-ing all of the above-mentioned tech-niques. The first three examples ofmutants with LR defects have alreadybeen published (Noramly et al., 2005),and more should become available asmutagenesis projects proceed (Amaya,2005; Grammer et al., 2005; http://tropicalis.berkeley.edu/home).

THE POTENTIAL OF THEFROG TO ADDRESSPRESSING LEFT-RIGHTISSUES

Symmetry Breakage

Two models are currently being dis-cussed for flow-mediated symmetrybreakage. The morphogen model pos-tulates that a factor is transported byflow to the left side of the ciliated ep-ithelium (Nonaka et al., 1998; Hiro-kawa et al., 2006). At the left margin,it is perceived by an unknown recep-tor, from where the signal is trans-ferred by an unknown route to the leftLPM. A morphogen could either be re-leased from the margins, or arise fromthe ciliated epithelium itself (Okadaet al., 1999), for example via FGF-trig-gered extrusion of factor-bearing ves-icles, as has been described in mousewhere vesicles were shown to harborretinoic acid and SHH (NVP model;Tanaka et al., 2005). The alternativetwo-cilia hypothesis postulates thatcilia-driven flow results in left-sided

1220 BLUM ET AL.

bending of a second class of cilia, i.e.,mechanosensory cilia, which in turn viasecondary signals such as calciumwould lead to left-sided Nodal tran-scription in the LPM (McGrath et al.,2003; Tabin and Vogan, 2003). Whilenone of the components of these twomodels have been analyzed in the frogthus far, the experimental repertoireoutlined above should have a great po-tential to address both models. For ex-ample, unilateral knock-down of flowcould answer the question whether theright side of the GRP is necessary forsymmetry breakage, i.e., whether amorphogen enters the GRP from theright side. Methylcellulose should blocktransport of vesicles, provided they ex-ist in frog. Combinations of MC-treat-ment and left- or right-sided supplemen-tation with retinoic acid and/or SHHshould potentially restore the Nodal cas-cade on the left or right side. Such exper-iments may thus have the power to dis-tinguish between the two models.

Transfer of Asymmetric Cue(s)From the GRP to the LeftLPM

The sole readout of flow upstream ofthe induction of Nodal in the left LPM

Fig. 4. Superficial mesoderm is required for correct left-right axis development. Superficialmesoderm (highlighted in red) ablation at stage 10 (A) causes situs defects at stage 45 (B), includingheterotaxia (ht) and situs inversion (si). Heart looping is outlined by black dots, position of gallbladder is shown by green auto-fluorescence, gut coiling is indicated by red dots. d, dorsal; l, left;r, right; ss, situs solitus; v, ventral.

Fig. 5.

Fig. 6.

Fig. 5. Determination of the GRP lineage. A–D:LacZ staining of dorsal explants at stage 17 (B,D) following injections into the dorsal-marginalzone (DMZ) at the 4-cell stage (A), or blas-tomere C1 at the 32-cell stage (C). Specimensshown in B, D, and F’ are equivalent to theupper half of the preparation shown in Figure2D. B’,D’: Planes of sections are indicated bydashed white lines in B, D. Note that the simul-taneous targeting of the floor plate (B’) can becircumvented by injecting 32-cell embryos (D’).E, F: Specific targeting of the left and right halfof the GRP (F) by sided injection of rhodaminedextran into the left and GFP mRNA into theright DMZ at the 4-cell stage (E). External dorsalview at stage 16 demonstrates targeting of thefloor plate (F), while dorsal explant (F’) revealssided fluorescence at the GRP. Note the strictseparation of left and right sides. a, anterior;cbc, circumblastoporal collar; d, dorsal; fp,floor plate; GRP, gastrocoel roof plate; l, left;no, notochord; p, posterior; r, right; v, ventral;VMZ, ventral marginal zone.

Fig. 6. Possible routes of signal transfer fromthe GRP to the left LPM. Signal(s) generated byleftward flow at the GRP could travel throughthe endoderm (yellow), mesoderm (red), noto-chord and ectoderm (blue), archenteron (black),or between endo- and mesoderm (white) toreach the left LPM (green, outlined by dashedline). bp, blastoporus; n, notochord; s, somite.

LEFT-RIGHT ASYMMETRY IN XENOPUS 1221

is an asymmetric calcium wave at theleft margin of PNC in mouse and KVin zebrafish (McGrath et al., 2003;Sarmah et al., 2005; Hadjantonakis etal., 2008). How this calcium signalacts on LPM Nodal is currently un-known. An attractive alternative wayhas been proposed by Hamada andcolleagues (Oki et al., 2007). They sus-pect that Nodal itself reaches the LMPin a flow-dependent manner from theleft margin of the PNC via the extra-cellular space between endoderm andmesoderm (Fig. 6). In support of thisnotion, Brivanlou and colleagues haveshown that Nodal at the left GRPmargin is required for induction ofasymmetric genes in the LPM (Vonicaand Brivanlou, 2007). In principle, asignal in the frog could also travelthrough the endoderm or outside ofthe endoderm through the gastrocoel,through the somitic and intermediatemesoderm, or even through the noto-chord and ectoderm (Fig. 6). All ofthese options can be tested in the frog,for example by combining MC-medi-ated blockage of flow with sided re-lease of calcium (caged calcium; iono-phore), or by using a tagged Nodalconstruct.

Asymmetric OrganMorphogenesis

A prerequisite for asymmetric organmorphogenesis is the sustained main-tenance of the Nodal cascade on theleft side. A model was proposed inmouse and Xenopus to account for thisfact, involving positive (Nodal) andnegative (Lefty) feedback loops (Lohret al., 1998; Meinhardt and Gierer,2000; Nakamura et al., 2006; Ohi andWright, 2007). As the Nodal cascadehas been known for �10 years in frog,it is surprising that organ morphogen-esis has been investigated only rarely(Breckenridge et al., 2001; Gormleyand Nascone-Yoder, 2003; Muller etal., 2003). The heart lineage in froghas been well studied and described indepth, as well as morphogenesis of thegastrointestinal tract. The molecularmechanisms of asymmetric morpho-genesis, for example how the Nodal-induced transcription factor Pitx2 ex-erts its function, i.e., what thetranscriptional targets are, havelargely remained unresolved. It maybe worthwhile to explore the experi-

mental toolbox for the analysis of or-gan morphogenesis, which in frog asin all other vertebrates involves bend-ing of linear tubes in the first place.Thus, despite the three-chamberednature of the adult frog heart, the ini-tial steps of asymmetric morphogene-sis should be conserved (Ramsdell etal., 2006).

Early Determinants

A number of additional componentswith influence on the LR pathwayhave been identified in Xenopus with-out obvious link to cilia and flow. Briefpharmacological disruption of corticalactin during the first cell cycle ran-domizes the LR orientation of tadpoleheart and gut, representing the earli-est acting process affecting lateralityreported thus far (Danilchik et al.,2006). The maternal growth factorVg1 has been described as a left-rightcoordinator, as its overexpression in asingle right ventro-lateral blastomereat the 16-cell stage can (unlike otherTGF� growth factors) fully invert theorgan situs in � 80% of cases (Hyatt etal., 1996; Hyatt and Yost, 1998). Theproteoglycan Syndecan-2, initially ex-pressed in the animal cap of Xenopusblastula and gastrula embryos, isthought to act as a co-receptor for leftasymmetric Vg1 signaling in the invo-luting mesoderm. Supporting this no-tion, asymmetric phosphorylation ofSyndecan-2 in the right involutingmesoderm was reported during earlygastrulation, and thus still upstreamof flow stages. This phosphorylation,which depended on protein kinase C �(PKC�), was instrumental for lateral-ity determination, as both loss- andgain-of-function of both factors effec-tively altered heart looping and asym-metric marker gene transcription(Kramer et al., 2002; Kramer andYost, 2002). It should be noted thatnone of these three determinants wasreported to be relevant for LR asym-metry in any other vertebrate systemso far.

Gap junctional communication(GJC) was originally suspected in theLR pathway due to connexin 43 muta-tions in human laterality patients(Britz-Cunningham et al., 1995).Gain- and loss-of-function experi-ments have indicated that GJC mayact during cleavage (frog) or gastrula-

tion (chick) (Levin and Mercola, 1998,1999). In rabbit embryos, however, werecently showed that GJC act down-stream of flow in the transfer of asym-metric cue(s) from the midline to theLPM (Feistel and Blum, 2008). Ourpreliminary experiments confirm thisobservation in Xenopus as well (T.B.and M.B., unpublished). It is currentlyunclear whether GJC in addition actat earlier stages, and if and how suchearlier functions impact on GRP andflow. Another component of cell-cellinteractions has been identified withthe tight-junction protein claudin,originally identified in a screen forproteins enriched in microsomes inthe blastula (Brizuela et al., 2001).Upon Xcla overexpression, Xnr1 wasbilaterally expressed in about 50% ofcases, and the visceral situs was ran-domized (Brizuela et al., 2001). It willbe interesting to analyze whether flowis affected in these experiments, par-ticularly as Xcla may be expressed inthe GRP (see fig. 2G,H in Brizuela etal., 2001). Claudin may be a particu-larly important factor, because it hasbeen involved in laterality determina-tion in the chick upstream of theNodal cascade as well (Simard et al.,2006).

Other presumably early acting deter-minants (i.e., during cleavage) comprisethe ion pumps H�-K�-ATPase and V-ATPase as well as the monoamine Se-rotonin and the 14-3-3 family memberE (Levin et al., 2002; Bunney et al.,2003; Adams et al., 2006). These factorshave been identified using pharmaco-logical inhibitors. In addition, H�-K�-ATPase mRNA and Serotonin were re-ported to be asymmetrically localized inthe 4- and 64-cell embryo, respectively(Levin et al., 2002; Fukumoto et al.,2005). The relevance of asymmetric lo-calization of H�-K�-ATPase mRNAseems questionable, however, as Levinand colleagues recently reported signif-icant variabilities of in situ signals inembryos from different females (Aw etal., 2008). Given the robustness of theLR pathway, occasionally encounteredasymmetries in mRNA localization can-not possibly account for the asymmetricinduction of the Nodal cascade. How-ever, H�-K�-ATPase activity certainlyinfluences LR asymmetry, particularlybecause pharmacological inhibitors alsoaffect laterality in chick (Levin et al.,2002). As the specificity of pharmaco-

1222 BLUM ET AL.

logical drugs may be limited due to sol-ubility, half-life, diffusion rate, and ac-cessibility of target tissue (e.g., SMfollowing invagination), to name just afew critical parameters, all of these fac-tors should be addressed by gene-spe-cific knock-downs in the tissue of inter-est in the future. GRP morphogenesisand flow as readout should be givenspecial emphasis, as these stages arereached several hours before markergenes can be assessed, and 3–4 daysbefore organ situs becomes evident. Insummary, linking up early determi-nants to GRP cilia and flow will uncoverwhether some or all of these factors actin parallel pathways, or, as we wouldexpect from our preliminary experi-ments, play important roles during flowstages by influencing different kinds offlow-related steps: GRP morphogenesis,ciliogenesis, ciliary beat frequency, andcilia polarization.

PERSPECTIVES

Over the past decade, experimentalinvestigation of LR asymmetry in thefrog Xenopus was governed by the con-viction that mechanisms differ greatlyfrom mouse and fish. With the discov-ery of flow as the evolutionary con-served common denominator betweenthese three species and, thus, betweenfish, amphibians, and mammals, fu-ture work should clearly have the po-tential to develop a unifying hypothe-sis for symmetry breakage. If thishope comes true, this will be particu-larly satisfying because it was difficultin the past to assume radically differ-ent modes of symmetry breakage up-stream of a highly conserved module,i.e., the Nodal cascade. We, therefore,expect a new focus on Xenopus as amodel organism to study LR asymme-try, particularly with respect to themost pressing issues of symmetrybreakage, the transfer of asymmetriccues to the left LPM, and the link-ing-up of early determinants with flowstages and flow as mechanism. Maybeeven Spemann’s experiments, whichin the light of our present day knowl-edge still await molecular elucidation,can be tackled anew using the reper-toire of modern Xenopus technologies.

ACKNOWLEDGMENTSWe apologize to those colleagueswhose work we could not cite due to

space limitations. We thank all mem-bers of the Blum lab for lively discus-sions, valuable suggestions, and criti-cal reading of the manuscript. T.B.,T.W., and P.V. were recipients ofPh.D. fellowships from the Landes-graduiertenstiftung Baden-Wuert-temberg. Frog work in the Blum labwas funded by a grant from the Deut-sche Forschungsgemeinschaft.

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