Is the Early Left-Right Axis Like a Plant, a Kidney, Or a Neuron? the Integration of Physiological Signals in Embryonic Asymmetry

Birth Defects Research (Part C) 78:191–223 (2006) REVIEW

Is the Early Left-Right Axis like a Plant, a Kidney, or a Neuron? The Integration of Physiological Signals in Embryonic Asymmetry

Michael Levin*

Embryonic morphogenesis occurs along three orthogonal axes. While the Developmental noise often patterning of the anterior-posterior and dorsal-ventral axes has been results in pseudorandom character- increasingly well-characterized, the left-right (LR) axis has only relatively istics and minor stochastic devia- recently begun to be understood at the molecular level. The mechanisms tions known as fluctuating asymme- that ensure invariant LR asymmetry of the heart, viscera, and brain involve try (Klingenberg and McIntyre, fundamental aspects of cell biology, biophysics, and evolutionary biology, 1998); however, the most interest- and are important not only for basic science but also for the biomedicine of a wide range of birth defects and human genetic syndromes. The LR axis ing phenomenon is invariant (i.e., links biomolecular chirality to embryonic development and ultimately to consistently biased) differences behavior and cognition, revealing feedback loops and conserved functional between the left and right sides. For modules occurring as widely as plants and mammals. This review focuses brevity, as well as because these on the unique and fascinating physiological aspects of LR patterning in a are likely to be secondary to embry- number of vertebrate and invertebrate species, discusses several profound onic asymmetries, this review mechanistic analogies between biological regulation in diverse systems largely neglects behavioral/sensory (specifically proposing a nonciliary parallel between kidney cells and the LR asymmetries (Harnad, 1977; axis based on subcellular regulation of ion transporter targeting), high- Bisazza et al., 1998). lights the possible importance of early, highly-conserved intracellular events that are magnified to embryo-wide scales, and lays out the most important open questions about the function, evolutionary origin, and conservation of mechanisms underlying embryonic asymmetry. Birth The Unique Fascination Defects Research (Part C) 78:191–223, 2006. VC 2006 Wiley-Liss, Inc. of the LR Axis The establishment of LR asymmetry Key words: embryogenesis; left-right asymmetry; physiology; modeling raises a number of interesting biological questions. Why does asymmetry exist at all? What are the INTRODUCTION consistently asymmetric place- implications of asymmetry for the The geometrical invariance known ment of various internal organs normal structure and physiology of as symmetry is a prominent aspect such as the heart, liver, spleen, the heart, gut, and brain? Why are of developmental morphology dur- and gut, or the asymmetric devel- all normal individuals not only ing embryogenesis. Animal body- opment of paired organs (such as asymmetric, but asymmetric to the plans occur in a wide variety of sym- brain hemispheres and lungs). A same direction (i.e., why a consist- metries: spherical (e.g., volvox), fascinating atlas of such morpho- ent bias and not a 50%/50% race- radial (e.g., sea anemone), chiral logical left-right (LR) asymmetries mic population, given that individu- (e.g., snails, ciliates), bilateral (e.g., throughout the animal kingdom is als with full inversion are not obvi- housefly), and pseudobilateral (e.g., given in Neville (1976), and evolu- ously impaired)? While it is possible man). Vertebrates have a generally tionary surveys have analyzed as- to devise plausible evolutionary rea- bilaterally-symmetrical body plan, ymmetries in diverse phyla (Palmer, sons for why organisms might be but this symmetry is broken by the 1996). asymmetric in the first place (op-

Michael Levin is from the Forsyth Center for Regenerative and Developmental Biology, The Forsyth Institute, and the Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts.

This manuscript was written in a Forsyth Institute facility renovated with support from Research Facilities Improvement Grant Number CO6RR11244 from the National Center for Research Resources, National Institutes of Health. Grant sponsor: National Institutes of Health (NIH); Grant number: GM-06227; Grant sponsor: March of Dimes; Grant number: 6-FY04-65; Grant sponsor: NHTSA; Grant number: DTNH22-06-G-0001. *Correspondence to: Michael Levin, Department of Developmental Biology, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115. E-mail: [email protected] Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20078

VC 2006 Wiley-Liss, Inc. 192 LEVIN timal packing, fluid dynamics, maxi- feature of the world that distin- phology (Delhaas et al., 2004). This mizing surface area of tubes, etc.) guishes left from right? suggests that the gross asymmetry (Kilner et al., 2000), there is no ob- Answers to these questions of the organs is separable from the vious reason for why they should all require a detailed understanding, at chirality of subcellular components, be asymmetric to the same direc- the molecular, genetic, and bio- and also potentially explains why tion. It is, after all, much easier to chemical levels, of the formation of 50-50 racemic populations are not imagine a developmental mecha- biased asymmetry in embryos. seen: discordance between subcel- nism for generating antisymmetry lular asymmetries and large-scale (such as local amplification and Not Just Basic Biology: structures in full situs inversus indi- long-range inhibition of stochastic Asymmetry and Human viduals may sufficiently contribute biochemical differences resulting in Medicine to lower fitness in the reversed indi- a morphologically biphasic popula- viduals (over evolutionary scales) to tion), than for biasing the LR axis to Errors of LR patterning during em- explain the existence of a consistent a given direction. When, during evo- bryogenesis are relevant to the clini- and robust biasing mechanism. lution, did handed asymmetry ap- cal considerations of several fairly LR asymmetries contribute to pear, and were there true bilater- common human birth defects: syn- human physiology involving many ally-symmetrical organisms prior to dromes such as Kartagener’s and organs in addition to the heart. For the invention of oriented asymme- Ivemark’s (Winer-Muram, 1995), example, proper propulsion of the try (Cooke, 2004)? Is it connected dextrocardia, situs inversus (a com- digesta appears to depend on diges- to chirality in lower forms (such as plete mirror-image reversal of the tive tract asymmetry (Arun, 2004). snail shell coiling and chirality in sidedness of asymmetrically posi- There are also links to more subtle some plants) or even the asymme- tioned organs and asymmetric paired features of human physiology; as- try (lack of quantum parity conser- organs), heterotaxia (a loss of con- ymmetric histamine skin responses vation) in weak nuclear decay (Wu cordance where each organ makes in L versus R arms are altered in et al., 1957)? At what developmen- an independent decision as to its si- patients with left cerebral epileptic tal stages is asymmetry initiated in tus), and right or left isomerism (in focus (Meador et al., 2004), and in which the organism is completely mice, interleukin level difference in L symmetrical, for example, polysple- and R cortex corresponds to their how can the LR axis nia or asplenia). Heterotaxia and iso- paw preference (Shen et al., 2005a, merismoftenresultinserioushealth 2005b). Synergy between clinical be consistently problems for the patient (Burn, data and model systems has signifi- oriented with respect 1991). The LR asymmetry of the heart cantly contributed to the under- to the anterior- is intimately connected to its function, standing of LR patterning (Bisgrove and errors in cardiac situs represent and Yost, 2001; Zhu et al., 2006), posterior and a significant source of human heart and clinical data have suggested im- dorsal-ventral axes in disease (Kathiriya and Srivastava, portant components of the pathway. 2000). Laterality defects can arise in a One example is PA26 (sestrin), dis- the absence of any single individual (Winer-Muram, covered through the analysis of macroscopic feature 1995; Kosaki and Casey, 1998) but human laterality patients (Peeters are especially associated with mono- et al., 2003) but not yet explored in of the world that zygotic twinning (see below). model systems. Another is the inver- distinguishes left Interestingly, complete (and rare) sion of various organs long observed situs inversus totalis is not associ- in the context of human conjoined from right? ated with severe difficulties in most twins (Torgersen, 1950; Aird, 1959; patients, although it does appear to Cuniff et al., 1988; Burn, 1991; be accompanied by an estimated Winer-Muram, 1995). The discovery higher incidence of congenital heart of the spatial signals propagated by vertebrate embryos? Are the disease on the population level asymmetric gene expression in chick establishment of bilaterality, impo- (Ramsdell, 2005). This is at first embryos has allowed a partial under- sition of asymmetry, and bias of puzzling: if everything is exactly standing of laterality defects in that asymmetry with respect to the mirror-image, why should there be human conjoined twins (Kapur et al., other two axes separate events? any problem at all? All connections 1994), which appear to be induced How conserved are the molecular and structures should be preserved by crossover of LR morphogen mole- mechanisms establishing correct by this reflection. However, it was cules from one twin to the adjacent asymmetry in animals with drasti- recently found that in situs inversus one (Levin et al., 1996). cally different modes of cleavage patients, where the large-scale as- and gastrulation? And, how can ymmetry of the heart is reversed, The LR Pathway in Three theLRaxisbeconsistentlyori- asymmetric myofiber orientation ented with respect to the anterior- (small scale structure) in the apex of Easy Phases posterior and dorsal-ventral axes left ventricle is not, making it oppo- Conceptually, LR patterning is di- in the absence of any macroscopic site to that of the large-scale mor- vided into three phases (Levin and

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 193

Figure 1. Overview of LR patterning pathway. The LR pathway proceeds from an initial microscopic orientation event (either extracellular, as in the cilia model, or intracellular, as in the cytoskeleton model; A), through the imposi- tion of LR positional information on cell fields (B), to downstream asymmetric transcriptional cascades (C).

Mercola, 1998b); the flow of infor- ber of studies have shown that the to shape organogenesis include the mation is schematized in Figure 1. individual organs’ lateralities are set, extracellular matrix (Tsuda et al., In the final phase, individual organs and can be experimentally random- 1996; Yue et al., 2004) and actin utilize cell migration, differential ized, independently of each other bundles (Itasaki et al., 1989, 1991; proliferation, cytoskeletal organiza- (Levin et al., 1997; Chin et al., Latacha et al., 2005) in the heart tion, and other mechanisms to 2000). The topological deformations tube, as well as differential rates of achieve asymmetries in their loca- undergone by asymmetric tissues elongation in the frog gut tube tion or morphogenesis (Stalsberg, are more complex than usually as- (Muller et al., 2003). A number of in- 1969a, 1969b; Manasek, 1981; sumed (Manner, 2004), and com- formative recent studies have Horne-Badovinac et al., 2003;). plete understanding is likely to addressed the mechanics of asym- Consistent with their downstream require mathematical or physical metric organogenesis (Ramsdell position, and counter to earlier pro- models in addition to molecular biol- et al., 2005, 2006), including com- posals (Waddington, 1937), a num- ogy. Biophysical mechanisms used putational models and direct meas-

Birth Defects Research (Part C) 78:191–223, (2006) 194 LEVIN urements of stiffness in tissues of axis is probably specified after the (Levin et al., 1995; St Amand et al., heart tube (Zamir et al., 2003). Me- anterior-posterior (AP) and dorsal- 1998; Garcia-Castro et al., 2000; chanical forces in looping have now ventral (DV) axes, and is likely to be Ohuchi et al., 2000; Wang et al., been analyzed (Voronov and Taber, 2004) suggested an immediate 2002; Alford and Taber, 2003; Zamir question: what mechanisms are up- et al., 2003; Voronov et al., 2004; stream of the very first asymmetri- Zamir and Taber, 2004; Latacha a unique and cally-expressed gene? Contrary to et al., 2005; Nerurkar et al., 2006; fascinating aspect of the paradigm of genetically sepa- Taber, 2006), though myosin does rate L and R compartments that not appear to be involved (Remond LR patterning: the applies after mid-gastrulation, it et al., 2006). Genetic control over physiological compo- was observed that events occurring these pathways is mediated proxi- nents, which reveal on the far R side were required for mately by genes such as flectin, the establishment of L identity on the L bHLH family members EHAND and surprising aspects of side at the beginning of streak initia- DHAND, and the transcription factor evolutionary tion (Levin and Mercola, 1999). Tbx5 (Srivastava, 1995; Tsuda et al., Thus, gap junctional communication 1996; Sparrow et al., 1998; Bruneau conservation, (GJC) was examined as a candidate et al., 1999; Angelo et al., 2000; Fer- feedback loops, and nandez-Teran et al., 2000; Hatcher et al., 2000; Takeuchi et al., 2003). biophysical events occurring Upstream of these processes lies mechanisms. a pathway of asymmetric genes on the far R side that are expressed in cell fields only were required for on one side of the embryo’s midline. By inducing or repressing transcrip- establishment of L determined with respect to them tion of downstream asymmetric tar- (McCain and McClay, 1994; Danos identity on the L side gets, they propagate signals among and Yost, 1995). The timing of the subpopulations of cells (such as at the beginning initiation of LR asymmetry in the node and lateral plate mesoderm) various species is particularly con- of streak that eventually dictate sidedness troversial, but the mechanisms initiation for the organs undergoing asym- underlying different aspects of LR metric morphogenesis (Lowe et al., patterning in various species are 1996; Levin et al., 1997; Lohr et al., beginning to be uncovered in signif- 1998; Piedra et al., 1998). These icant detail. cascades of asymmetric gene ex- for a mechanism that would enable A number of excellent recent repression form the middle phase of cells to communicate across large views have summarized the pro- LR patterning (Levin, 1998; Whit- distances along the LR axis and gress in various areas of asymme- man and Mercola, 2001; Mercola, assign LR identities to cell fields. try (Burdine and Schier, 2000; 2003), and include gene families Gap junctions are channels con- Hamada et al., 2002; Hobert et al., such as Hedgehogs, bone morpho- necting adjacent cells that allow the 2002; McGrath and Brueckner, genetic proteins (BMPs), and Pitx direct transfer of small molecule 2003; Palmer, 2004; Ramsdell, (see Levin (2005) for an exhaustive signals. The cell biology of gap junc- 2005; Tabin, 2005; Vallortigara and list of asymmetric genes and their tions has been described exten- Rogers, 2005; Raya and Belmonte, respective lateralities). sively (Goodenough et al., 1996; 2006). In this review, moving among However, for whichever asym- Falk, 2000), and gap junctional flow model species as needed, I focus on metric gene is at the top of the is involved in a number of important whatIbelievetobeauniqueandfas- pathway, it is necessary to ask what patterning events in embryonic de- cinating aspect of LR patterning: the determined its asymmetry. Thus, in velopment and tumor progression physiological components, which re- the first phase of LR patterning, an (Guthrie and Gilula, 1989; Lo, veal surprising aspects of evolution- as-yet unknown mechanism orients 1996; Levin, 2001). Based on a ary conservation, feedback loops, the LR axis with respect to the other report that several unrelated and biophysical mechanisms. two axes. While theoretical candi- patients with visceroatrial hetero- date mechanisms have been pro- taxia contain potential mutations posed (Brown and Wolpert, 1990), PHYSIOLOGICAL within Connexin43 (Britz-Cunning- no mechanism has been conclu- COMPONENTS OF THE ham et al., 1995), and data from sively shown to initiate asymmetry LR PATHWAY frog embryos that indicated asym- de novo. The developmental timing Gap Junctional metric patterns of GJC (Guthrie, of each phase differs among spe- 1984; Guthrie et al., 1988) in early cies, though asymmetric gene Communication blastomeres, Levin and Mercola expression almost always begins at The fairly dense pathway of LR cas- (1998a) tested the hypothesis that or shortly after gastrulation. The LR cade members in chick embryos gap junctional paths were a mecha-

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 195 nism by which LR information was epiblast but not across an insulating cated four target genes involved in communicated across large cell zone at the streak. In support of this Hþ and Kþ flux (Adams and Levin, fields. Xenopus embryos at early model, surgical incisions made along 2003; Chen and Levin, 2004). Two of cleavage stages were shown to con- various radii emanating from the these, the Hþ/Kþ-ATPase and V-Hþ- tain a junctional path across the developing node abolish node asym- ATPase, are already known to func- dorsal blastomeres, and a zone of metry. While a topological transfor- tion during early cleavage stages junctional isolation on the ventral mation is required to map the GJC (Levin et al., 2002 Adams et al., midline (confirming with a double- system onto the different embryonic 2006); the Kþ channel components dye system in previous observation architectures of the chick and Xeno- have not yet been extensively char- using a single small-molecule probe pus, the basic schematic is the same acterized. Maternal Hþ/Kþ-ATPase (Guthrie, 1984; Olson et al., 1991; in both systems: correct laterality mRNA and protein subunits of both Brizuela et al., 2001); but see Land- determination upstream of asym- pumps are asymmetrically localized esman et al. (2000)). Injection of metric gene expression depends on during the first two cell divisions, mRNA encoding a dominant nega- an uninterrupted contiguous region demonstrating that asymmetry in tive connexin protein into dorsal of GJC around a small zone of junc- Xenopus is generated by about 2 hr blastomeres or wild-type connexins tional isolation. postfertilization. Analysis of the situs into ventral blastomeres both re- of asymmetric genes (xNR-1, xLefty, sulted in heterotaxia and random- and xPitx-2) following pharmacologi- ization of XNR-1 expression in the Ion Flows: Asymmetrical cal and genetic inhibition of these absence of other developmental de- Bioelectricity pumps (as well as gain-of-function fects (Levin and Mercola, 1998a). experiments) showed that, consis- These results indicate that an en- One of the key aspects of the GJC tently with the early asymmetrical dogenous path of GJC between dor- model is that the junctional flow localization, the ion flux mechanism sal and lateral blastomeres, as well must be net unidirectional, in order is upstream of asymmetric gene as the isolation across the ventral to derive a LR asymmetry from the expression. Equalization of mem- midline, is necessary for normal LR existing dorsoventral difference in brane voltage and cytoplasmic pH by asymmetry in Xenopus.Pharmaco- GJC (although no individual small methods independent of V-ATPase logical blocker experiments sug- molecule needs to make it all the and Hþ/Kþ-ATPase also randomize gested that the gap-junctional sys- way around the path). In some con- asymmetry, demonstrating that it is tem begins to function in LR asym- texts, chemically-rectifying or one- indeed the ion transport that is cru- metry during cleavage stages. These way junctions have been observed cial for laterality, not some other data led to the hypothesis that a cir- (Flagg-Newton and Loewenstein, cryptic function of these protein cumferential path of GJC, around a 1980; Robinson et al., 1993; Xin and complexes, which has been reported zone of isolation, could be the mech- Bloomfield, 1997; Zahs and New- for some other channels and pumps anism that bridges asymmetry at man, 1997; Zahs, 1998), and it is (Paul et al., 2003; Baumgartner the level of a cell (step 1) to the tempting to visualize unidirectional et al., 2004). Voltage-sensitive dyes embryo-wide cascades of asymmet- paths of heterotypic gap junctions and self-referencing ion-selective ric gene expression (step 2). It was arranged appropriately to provide a probe measurements during early proposed (Levin and Nascone, 1997; ratchet mechanism for accumulation cleavage stages in vivo revealed the Levin and Mercola, 1998a) that small of LR morphogen on one side of the predicted consistent asymmetries in molecule determinants are initially midline. However, because thermo- Hþ efflux from L versus R ventral cells randomly distributed, but traverse dynamics forbids the generation of a and the resultant membrane voltage the circumferential GJC path net uni- gradient without an expenditure of differences across the midline. directionally, accumulate on one side energy, GJC models require an ener- Because the GJC system is con- of the midline, and then induce getic process to drive the chiral dis- served to both chick and Xenopus, asymmetric gene expression in contribution of the small molecules Levin et al. (2002) and Adams et al. ventional ways. through the circumferential path. (2006) tested whether embryonic Similarly to the results in Xeno- Hypothesizing that a voltage differ- laterality was dependent upon ion pus, it was discovered that differen- ence might provide an electromotive flux in the chick and zebrafish. An tial GJC is required upstream of force that can be used to electro- asymmetryincurrentflowsinthe asymmetric Shh expression in the phorese charged molecules in pre- chick streak was already observed chick node and one Connexin, Cx43, ferred directions through GJC paths, (but not discussed with respect to LR was implicated by treatment with Levin et al. (2002) tested the hy- patterning) in Jaffe and Stern specific antisense oligonucleotides pothesis that ion fluxes (needed to (1979). Analysis of the chick embryo or blocking antibodies (Levin and generate the standing voltage gra- usinganinvivoreporterofmem- Mercola, 1999). Interestingly, Cx43 dients) were an obligatory aspect of brane voltage indicated that cells on mRNA is broadly expressed in the early LR patterning in Xenopus. theleftsideoftheprimitivestreak epiblast of streak stage embryos, A pharmacological screen of hun- were consistently depolarized with but not in the streak itself. Thus, GJC dredsofvarioustypesofionchan- respect to those on the contralateral required for LR asymmetry may nels, pumps, and cotransporters side. This indicates that the chick propagate signals throughout the (Levin et al., 2002) specifically impli- embryo has assigned L and R identi-

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Figure 2. Integrative model of early physiological events based on Xenopus system (time-sequence moving left to right). Initially randomly-distributed maternal mRNAs and proteins encoding Hþ and Kþ transporters become asymmetrically distributed due to the action of motor proteins (cued in turn by an asymmetric cytoskeleton deriving chirality from a basal body or other oriented ‘‘F-molecule’’). The function of asymmetrically-distributed ion channels and pumps results in a LR gradient in membrane voltage. When the gap junction system provides an open circuit around the zone of isolation (battery), small molecule determinants (e.g., serotonin) become asymmetrically localized, inducing downstream gene expression (such as Shh). ties by stage 3-prior to the earliest (Levin, 2003a) and the gap junctions tion of movement via ion pump-de- known asymmetric gene expression. that determine the distribution of pendent voltage gradients) (Levin þ þ Specific inhibition of the H /K - isopotential and iso-pH cell fields in and Nascone, 1997; Levin, 2003c). ATPase prior to gastrulation equal- embryos (Levin, 2001), another pas- Moreover, it should be water-solu- ized the depolarization of cells across sive component shapes current ble: signaling molecules such as the midline, and randomized the paths (leaks between cells) and thus retinoic acid (which has been impli- asymmetric expression of Shh, controls resulting voltage gradients: cated in later LR patterning steps cWnt-8C, and other markers (in- tight junctions. Consistently with the (Chazaud et al., 1999; Adam et al., cluding Cerberus-a marker of head role of bioelectric signals in asymme- 2003; Kawakami et al., 2005; asymmetry). Subsequent work in try, protein components of tight Tanaka et al., 2005; Vermot and another laboratory revealed that in junctions have been implicated in LR Pourquie, 2005; Sirbu and Duester, the chick, the asymmetric function of patterning (Brizuela et al., 2001; 2006)) do not need gap junctions to þ þ the H /K -ATPase functions through Simard et al., 2006). move between cells. The neuro- þþ downstream Ca and Notch signals transmitter serotonin (5HT) fits (Raya et al., 2004). Although no these criteria, has been demon- direct measurements of early volt- Serotonin: A Small Molecule strated to go through gap junctions age or pH have yet been reported With a New Role in some contexts (Wolszon et al., for zebrafish, functional data indi- 1994), has been suggested to have þ þ Another main line of inquiry raised cate that both the H /K -ATPase by the implication of gap junction nonneural roles in early embryo- and the V-ATPase are utilized at genesis (Buznikov and Shmukler, paths in asymmetry was, what is early stages (before Kupffer’s vesi- 1981; Buznikov et al., 2001), and the molecular nature of the small- cle formation) of zebrafish develop- offers the benefits of a well-devel- ment to direct the normal asymme- molecule LR signals that are oped pharmacological tool set try of gene expression and organ si- exchanged between cells on the L (Gaster and King, 1997). Serotonin tus (Kawakami et al., 2005; Adams and R sides? The ideal candidate is thus an ideal candidate for an et al., 2006). would be smaller than the size cut- early LR signal (Levin et al., 2006). Alongside the ion transporters that off of gap junctions (< 1 kDa) and Fukumoto et al. (2005a, 2005b) produce asymmetric ion flows would be charged (to enable regula- took advantage of the large number

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Figure 3. Feedback loops involving early physiological systems. The physiological module consists of several components: tight junctions and gap junctions that control current paths and thus voltage gradients, cytoskeletal elements that control localization of ion transporters, the ion flow module consisting of pH and membrane voltage (regulated by several transporters), and the serotonergic module (consisting of 5HT itself, receptors, serotonin binding protein, MAO degradation enzyme, and the SERT plasma membrane transporter). Arrows indicate functional relationships. Some connections are bidirectional; for example, gap junction paths dictate membrane voltage in cells but are themselves gated by transjunc- tional potential.

of well-characterized reagents avail- Analysis of endogenous localiza- able to test and characterize sero- serotonin signaling tion of serotonin revealed a striking tonin’s role in LR asymmetry in shift from homogenous distribution chick and frog embryos. Using was shown to be to a gradient, ultimately culminat- analysis of endogenous localiza- upstream of early ing in coalescence of serotonin in a tion of 5HT and related proteins, single blastomere-precisely as pre- and functional experiments de- asymmetric gene dicted by the GJC path model (sche- signed to probe the properties of expression in both matized in Fig. 2). This serotonin the 5HT pathway, serotonin sig- gradient was dependent on open naling was shown to be upstream chick and frog, gap junctions and on the function of of early asymmetric gene expres- the previously-implicated ion pumps. sion in both chick and frog, and MAO appears to function by destroy- revealed new developmental as- ing the maternal serotonin by about pects of this versatile signaling mol- uncharacterized) intracellular 5HT the 64-cell stage, presumably after ecule. An inverse drug screen and receptor in the right ventral cells. the relevant signaling has taken subsequent molecular loss- and Manipulation of any of these com- place. In the chick, MAO and SERT gain-of function specifically impli- ponents resulted in randomization of proved to be asymmetrically local- cated receptor subtypes R3 and asymmetric gene expression and ized in Hensen’s node, adding to the R4, the cell-membrane serotonin organ situs. Interestingly, manipula- available markers of asymmetry transporter SERT, and the degradation of R3 activity led mostly to com- (Levin, 2005). Serotonin itself was tion enzyme monoamine oxidase plete situs inversus, a very rare phe- localized throughout the streak, (MAO). Moreover, evidence indicat- notype in the LR field (Hyatt and consistent with older radiographic ed the importance of a novel (as yet Yost, 1998). (Emanuelsson et al., 1988) and fluo-

Birth Defects Research (Part C) 78:191–223, (2006) 198 LEVIN rescence immunocytochemical data mechanisms; for example, it is and loss-of-function approaches, to- (Wallace et al., 1982). The asym- known that embryonic pH gra- gether with real-time in vivo imag- metric catalytic activity of MAO is dients regulate GJC paths (Turin ing of Caþþ content, produced a likely to provide differential 5HT lev- and Warner, 1980), and some of mathematical model of gene activa- els on the R versus L sides of Hen- the components of the serotonin tion by ion fluxes transduced by sen’s node, but this has not been Notch signaling (Raya et al., 2004). demonstrated quantitatively. This important contribution used A number of important questions The ultimate goal of data from the chick system to ana- remain. First, the chick data do not lyze a complex and robust genetic directly support a GJC morphogen the field is to network that locally activates asym- model for 5HT function, because synthesize the metric Notch signaling in Hensen’s asymmetric localization of 5HT itself node. This in turn is driven by a tran- has not been demonstrated in this molecular sient extracellular calcium spike, species, and no evidence has been data back into a itself dependent on asymmetric Hþ/ þ presented to support the circumfer- coherent model of the K -ATPase activity. Although it has ential gradient of serotonin in chick not yet been explored, given the se- that was observed in Xenopus integrated rotonin data it is tempting to hy- þþ (Fukumoto et al., 2005b). Moreover, system. pothesize that the Ca flux may be the available chick data concern due to asymmetric 5HT-R3 activity. macroscopic (embryo-wide localiza- Asymmetric signaling by Caþþ is tion). Subcellular investigation of also suggested to function in the 5HT movement as well as sensitive mouse node (McGrath et al., 2003), pathway both contribute to and are comparisons between the two sides leading to the hypotheses of sen- modulated by membrane voltage. of Hensen’s node will be required to sory cilia transducing physiological Also, it should be noted that cilia understand the role of SERT-medi- signals to downstream cascades can contain ion channels and sero- ated transport and 5HT signaling in (McGrath and Brueckner, 2003; tonin receptors (Brailov et al., detail. Moreover, in the frog, direct Tabin and Vogan, 2003) similar to 2000; Shin et al., 2005), and read- movement of 5HT in vivo has not the way ion flow is upstream of the ers are invited to produce models been demonstrated. Unfortunately, direction of ciliary beating in ciliates involving the known components. given the small size of serotonin, (Machemer and Eckert, 1973). Of necessity, this effort must be current technology does not permit Modeling has also been applied to quantitative, and a true under- labeling it in such a way as to allow in the biomechanics of cilia movement standing of asymmetry will be dem- vivo analysis of its movement: all in mammalian asymmetry. While onstrated by a quantitative model available tags that are compatible the role of cilia is controversial (see that is predictive with respect to with detection in living cells alter its below), modeling of ciliary hydrody- symmetry outcomes under a range molecular weight by an order of namics (Cartwright et al., 2004) of stochastic and targeted perturba- magnitude, likely significantly alter- have illuminated necessary proper- tions in system size, physiological ing its interaction with endogenous ties, such as tilting (Nonaka et al., parameter level, gene expression, binding partners, as well as the rate 2005) and a significant degree of and geometric (surgical) rearrange- and extent of its movement. New robustness (Brokaw, 2005). It ment (Cooke, 1972a, 1972b, techniques will need to be developed should also be noted that like the 1972c, 1973, 1979; Cooke and so that serotonin movement can be model described below, ciliary Webber, 1985a, 1985b; Meinhardt, tracked in living embryos. movement is a potential class of 2001, 2004; Oviedo et al., 2003; mechanisms that can transduce a Ravasz and Barabasi, 2003). biochemical (subcellular) chirality Robustness in particular is a key into an embryo-wide asymmetry. You Haven’t Understood It trait that must be explained (Eldar Until You Can Model It on a et al., 2002, 2003, 2004; England Computer and Cardy, 2005; Houchmandzadeh et al., 2005). For example, Xenopus The Embryo is an The ultimate goal of the field is to embryos reliably orient the LR axis Electrophoresis Chamber: synthesize the molecular data back under a pH range of 6–11 and under One Model Synthesizing the into a coherent model of the inte- the influence of numerous drugs, grated system. Available Data with a base heterotaxia rate of 1% (Mimura and Nishiura, 1979). The The considerable amount of quanti- (Levin et al., 2002; Adams et al., complexity of many physiological tative data now available on early 2006). pathways requires a computer sim- LR-relevant physiology in Xenopus ulation because the effects of per- One of the key questions is how can be synthesized into a unified turbations can be difficult or impos- asymmetries in epigenetic signals model (Fig. 2) explaining the de- sible to predict intuitively. Figure 3 become stabilized in asymmetric pendence of early asymmetric gene reveals the functional relationships gene cascades. A recent elegant expression (and subsequent organ between the elements of the early study that used a variety of gain- situs) on ion flows, serotonergic sig-

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 199 naling, and gap junctional paths model is a bias in asymmetric local- One key feature of the serotonin (Levin, 2004; Levin et al., 2006). ization of ion transporters driven by dataset is that all of the important This hypothesis suggests that net asymmetric subcellular motor pro- parameters can be quantitatively asymmetric serotonin movement tein transport, which was suggested estimated. This model was tested occurs through gap junctions, is previously in the context of LR by construction of a computer simu- driven by electrophoretic forces asymmetry (Levin and Nascone, lation of electrophoresis of sero- generated by ion pump-dependent 1997) and other systems (Brawley tonin in a GJC-coupled system with membrane voltage differences across and Robinson, 1985; Goida et al., the geometry of an early Xenopus the ventral midline, and ultimately 1992; Bregestowsky et al., 1993; embryo (Esser et al., 2006). The induces asymmetric downstream Brezhestovsky et al., 1993). Indeed, simulation revealed that electro- genes (Levin, 2004). This class of it is known that the motor protein phoresis under the force generated models has been previously pro- KIF3A, and the ion channal Polycys- by realistic ion pump-generated posed for a number of morphogen tin-2, both of which are important gradients can indeed result in a sig- systems (Rose, 1966; Novak and for normal LR asymmetry in mouse nificant gradient of serotonin in the Bentrup, 1973; Lange and Steele, embryos, are now known to interact two hours in which the serotonin 1978; Larter and Ortoleva, 1981) directly, as is predicted by this gradient is known to form. More- and is compatible with many other model (Li et al., 2006). This scheme over, it allowed investigation of the small molecule signals. Inositol poly- shows how subcellular asymmetries dependence of the kinetics of this phosphates are also likely candi- can be transduced into asymmetric gradient on relevant parameters dates, as they have been implicated gene expression through physio- such as GJC density, voltage gradi- in asymmetry (Albrieux and Villaz, logical mechanisms and suggests ent, and morphogen size/charge. 2000; Sarmah et al., 2005), as well that the origin is to be sought Since it is possible to individually as being downstream of a voltage inside early blastomeres within the manipulate all of the relevant pa- sensor (Murata et al., 2005). first hour after fertilization (see rameters in this system, the specific In this scheme, the early embryo discussion below on cytoskeletal quantitative predictions of this is viewed as an open circuit (GJC subcellular chirality), but it should model must now be tested in exper- zone) around a region of differential be noted that the originating event imental work. voltage potential (zone of junctional (cytoskeletal chirality) has not An essential feature of this class isolation). In the frog, the circuit is yet been experimentally demon- of models is circumferential GJC the set of animal pole cells during strated. around a zone of junctional insula- early cleavages. In the chick, this Roles for electrophoretic move- tion (the primitive streak in chick, occurs through the blastoderm ment of morphogens (Cooper, the ventral midline in Xenopus). around the primitive steak. Sero- 1984; Cooper et al., 1989; Fear and While consistent with the idea that tonin accumulates on one side of Stuchly, 1998a, 1998b, 1998c) have the epiblast influences node asym- the midline and thus induces differ- been observed in the context of fol- metry, this set of findings in chick ent genes on the R side through a licle-egg systems (Woodruff and Tel- also indicates that the information cytoplasmic receptor. In Xenopus, fer, 1980; Telfer et al., 1981; Bohr- does not originate from a single the initial asymmetry of the battery mann and Gutzeit, 1987; Woodruff source, but that contiguity of the et al., 1988; Woodruff and Cole, blastodisc on both sides of the mid- 1997; Adler and Woodruff, 2000), line is necessary (Levin and Mer- self-electrophoresis in Fucus sym- cola, 1999). The GJC model pre- metry-breaking (Jaffe et al., 1974; dicts that the midline cells receive subcellular Jaffe and Nuccitelli, 1977), and re- LR information from lateral tissue asymmetries can be generation in both vertebrate and in- during gastrulation. In the chick, vertebrate systems (Rose, 1966, current data strongly indicate that transduced into 1970; Smith, 1967; Lange and Steele, indeed Hensen’s node is instructed asymmetric gene 1978). Theoretical analysis indicates with respect to the LR axis by adja- expression through that gap junctional coupling increases cent lateral cell groups (Psychoyos the sensitivity of cells to electric fields and Stern, 1996; Pagan-Westphal physiological produced in their milieu (Cooper, and Tabin, 1998; Yuan and Schoen- mechanisms 1984; Cooper et al., 1989). However, wolf, 1998; Levin and Mercola, having proposed a role for this mech- 1999), rather than generating LR anism in chick and frog, it was impor- information intrinsically (which would tant to answer the question: are be required by the cilia-based mod- physiological-strength endogenous els). Another essential feature that (source of electrophoretic force electric fields actually sufficient to must be investigated is selectivity, through gap junctions) is estab- produce a meaningful gradient in se- since clearly it cannot be the case lished by the asymmetric localiza- rotonin in the time provided, given that every free charged small mole- tion of channel and pump subunits the known properties of embryonic cule in the embryo is driven into a LR to the L or R side. Thus, the earliest cells and the geometry of the early gradient. Gap junctions are known to known step of asymmetry in this frog embryo? be selective for permeability to differ-

Birth Defects Research (Part C) 78:191–223, (2006) 200 LEVIN ent small molecule signals (Weber old. Then, each side would be et al., 2004; Ek-Vitorin and Burt, A mechanistic model equally likely to drive themselves to 2005; Ek-Vitorin et al., 2006), and a yes or no for Shh expression, giv- the basis for endogenous selectivity, for this process would ing the four possible different states and perhaps even chemical rectifica- have to explain not (left, right, bilateral, or no expres- tion (Robinson et al., 1993; Zhang simply consistent sion). Even more puzzling for sim- et al., 2003), must be addressed ple gene cascade models is the ob- experimentally. induction (or servation that in several vertebrate repression) of genes and invertebrate systems, symmetric expression of an upstream nor- such as Shh by GJC or MAJOR PUZZLES: THE mally asymmetric gene does not cell depolarization, lead to uniformly bilateral or missing FUTURE OF ASYMMETRY expression of downstream genes (in AND SOME HYPOTHESES but a process by the case of positive and negative re- Everyone in the Field Scores which cells in both gulation, respectively) but rather re- this Phenotype, But sides of the node can sults in a randomization of down- What Exactly Is stream gene expression (Morokuma be driven to randomly et al., 2002), or does not affect ‘‘Randomization’’? downstream LR pathway targets at One of the key remaining questions express Shh or not. all (Kelly et al., 2002)! is the molecular meaning of ‘‘ran- Regardless of the mechanism, it is domization,’’ which is the pheno- clear that heterotaxia can present type most often observed when significant problems for the embryo organ situs or asymmetric gene is not that single cells make this de- (Casey, 1998; Ramsdell et al., 2006; expression is scored after experi- cision (resulting in a speckled pat- Zhu et al., 2006). Thus, a candidate mental perturbation. Upon the ini- tern on both sides of the node), but mechanism explaining randomiza- tial discovery of the LR pathway, it that L and R sides of the node each tion may also answer the long- was observed that embryos with makes its decision as a group. One standing puzzle of why populations double-sided Nodal expression or candidate for such a bistable mech- are biased and not split 50-50 lack of Nodal expression (produced anism would be a short-range acti- among situs inversus and situs soli- by Shh or Activin implants, respec- vation/long-range inhibition system tus-perhaps the amplification and tively), show a randomization of such as that which establishes cell synchronization mechanisms do not visceral situs (Levin et al., 1995, polarity via the Notch-Delta path- allow such fluctuating asymmetry to 1997)-not a symmetrical heart and way (Hermann et al., 2000; occur without a significant back- gut, but heterotaxia. This was inter- Delattre and Felix, 2001; Krebs ground of heterotaxia. Indeed, this preted as suggesting that this path- et al., 2003; Przemeck et al., 2003; is observed in mouse mutants such way of genes imparts LR informa- Raya et al., 2003, 2004; Vincent, as inv, which are not purely reversed tion to the organs but does not con- 2003). It is possible that such a (Morgan et al., 1998, 2002;Eley trol their morphogenesis per se, mechanism works in the node to et al., 2004). This in turn suggests a leading the organs to independently integrate a number of epigenetic search for models that do not sepa- and randomly choose their situs biasing factors into stable domains rate clearly between the symmetry when presented with identical mo- of downstream gene expression. breaking phase and the orientation lecular signals from the L and R Future work is necessary to under- of the LR axis with respect to the sides. However, it is now known stand how this works in the node other two axes. that global equalization of signal- and streak; recent mathematical ing in a number of LR pathways models are beginning to tackle this also induce randomization of asym- issue (Meinhardt and Gierer, 2000; Evolutionary Conservation metric genes such as Shh and Rasskin-Gutman and Izpisua-Bel- 1: What Is Known More Nodal. A mechanistic model for monte, 2004). this process would have to explain Some sort of feedback amplifica- Broadly Through Phyla? not simply consistent induction (or tion system in cell fields on each How many different asymmetry repression) of genes such as Shh side is likely, to turn a morphogen mechanisms have been invented by GJC or cell depolarization, but a concentration into a sharp yes/no throughout evolutionary history? processbywhichcellsinbothsides for (for example) Shh expression. If Table 1 summarizes the physiologi- of the node can be driven to ran- this mechanism’s threshold is simi- cal mechanisms and their functional domly express Shh or not. More- lar to the initial ubiquitous concen- implication in various model sys- over, existing in situ hybridization tration of morphogen (before the tems. Consistent LR asymmetry data require that the cells making electrophoresis acts), then loss-of- has been described in plants (Luo this decision be synchronized. function of the gap junction or the et al., 1999; Andalo et al., 2000; For example, Shh expression on a ion pump systems will cause the Waites and Hudson, 2001; Galego given side is always homogenous-it morphogen to stay near this thresh- and Almeida, 2002; Rao et al.,

Birth Defects Research (Part C) 78:191–223, (2006) TABLE 1. Conservation of Physiological Mechanisms among Phyla (in rough Evolutionary Order)

Embryonic time: c Cytoskeleton/ Model system motor proteins Ion flux GJC 5HT Cilia

X Plants

Ciliates X

C. elegans X

Snails X

Drosophila X og vltoaytm (complexity) time Evolutionary Rough

Sea urchin X

Ciona X

Zebrafish XX

Xenopus XXXX

Chick XXX

Rabbit X *

Mouse X

Human X !

X ¼ functionally implicated; * ¼ described but not yet functionally tested. 202 LEVIN

2002; Rolland-Lagan et al., 2003; hibit invariant LR asymmetries in mollusks (chirality is mathemati- Corley et al., 2005; Costa et al., the nervous system and gut. It is cally equivalent to asymmetry but 2005; Korn, 2006), and chiral forms now known that induction at the does not require a linear midline). exist among the protozoa (Nelsen 12-cell stage by the mesomere However, recent morphometric anal- et al., 1989; Frankel, 1991). The (MS) blastomere is necessary to es- ysis has revealed a subtle but real heart is asymmetrically located in tablish the differences between left directed asymmetry in wing size the mollusks (McMurrich, 1894) and right pairs of blastomeres in the (Klingenberg et al., 1998), suggest- and in sea urchins, the asymmetric anterior part of the embryo (Hutter ing that mechanisms orienting the LR position of the adult rudiment in the and Schnabel, 1995). The micro- axis in fruit flies remain to be discov- larva has been studied (McCain and RNA lsy-6 controls the neuronal LR ered. In another genus of flies, a McClay, 1994; Aihara and Ame- asymmetry of chemosensory re- large-scale (not subtle, requiring miya, 2000, 2001; Kitazawa et al., ceptor expression (Johnston and statistical analysis) asymmetry in 2004; Duboc et al., 2005). Hobert, 2003). Two lateral blast wings has been reported (Runyon Several kinds of mollusks cells P(11/12)L and P(11/12)R are and Hurley, 2004). The fly system undergo spiral cleavage and secrete symmetric at hatching but migrate has the potential to shed light on the an exoskeleton shaped like a coni- subsequently in opposite AP direc- linkage of the LR axis with the AP cal spiral (Crampton, 1894; Sturte- tions during the first larval stage axes (since inversion of AP polarity vant, 1923; Meshcheryakov and and adopt different fates; this is does not alter LR polarity of proven- Beloussov, 1975). In three-dimen- downstream of the Notch pathway triculus (Hayashi et al., 2005); sional space, such spirals can have (Delattre and Felix, 2001; Hermann though see Ligoxygakis et al. two possible variants: a left-handed et al., 2000), as are the consistent (2001)), as well as on the potential and a right-handed helix (which are cell movements leading to a twist of role of motor proteins in invertebrate otherwise identical). Each particular the intestinal primordium. asymmetry (Hozumi et al., 2006; species of snail has invariant (con- Amphioxus exhibits many LR asym- Speder et al., 2006) that may parallel sistent) chirality, but there are spe- metries (Jefferies et al., 1996); one the known roles of motor proteins in cies that utilize each type of coiling. of the most striking is the asymmetry rodent embryos (Supp et al., 1997; Murray and Clarke (1966) found of somitogenesis (Minguillon and Marszalek et al., 1999; Takeda et al., that the direction of coiling of P. Garcia-Fernandez, 2002), which has 1999). suturalis is maternally inherited and recently been shown to be linked to sinistrality is dominant to dextrality. the LR axis in vertebrates through Evolutionary Conservation Freeman and Lundelius (1982), retinoic acid signaling (Saude et al., 2: Which of the Known studying a different species, found 2005; Vermot and Pourquie, 2005; that dextrality is dominant; inter- Sirbu and Duester, 2006). Down- Mechanisms Are Conserved? estingly, the dextral gene appa- stream asymmetric gene cassettes A number of mechanisms appear to rently functions via a cytoplasmic appear to be conserved (Terazawa be conserved. For example, chick component since it is possible to and Satoh, 1995, 1997; Araki et al., Syndecan-2 is asymmetrically ex- rescue the sinistral phenotype by 1996; Shimeld, 1999; Boorman and pressed (Fukumoto and Levin, direct transfer of cytoplasm from Shimeld, 2002a, 2002b; Minguillon 2005), and syndecans are known to the dextral variant of the snail into and Garcia-Fernandez, 2002; Moro- be crucial for embryonic asymmetry the sinistral variety. The biochemi- kuma et al., 2002). in Xenopus (Kramer et al., 2002; cal nature of this activity has not Drosophila, which has provided Kramer and Yost, 2002). Even more yet been identified, and although inroads to so many other develop- strikingly, intracellular microtubules molecular work is ongoing (Hosoiri mental questions, has not played are important for asymmetry in et al., 2003; Harada et al., 2004; an early role in uncovering mecha- plants (Hashimoto, 2002; Thitama- Shibazaki et al., 2004; Wandelt and nisms of LR asymmetry but has re- deeetal.,2002;Abeetal.,2004), Nagy, 2004), it is not yet known cently provided some interesting similar to their likely role in verte- whether the mechanisms of chiral data. While selection for LR asym- brate asymmetry (Yost, 1991; Qiu blastomere cleavage are related to metries in Drosophila has not been et al., 2005). While the details of ion those regulating asymmetric motor successful (Tuinstra et al., 1990), it flux usage in the LR pathway of vari- protein movement and cytoskeletal is now known from mutant analysis ous species differ, Hþ and Kþ flux components in vertebrates (Yost, that Drosophila possesses genes roles are conserved to sea urchin 1991; Qiu et al., 2005). that govern the helical torsion of and Ciona (Duboc et al., 2005; In C. elegans, the embryonic cell the body (Martin-Blanco and Gar- Hibino et al., 2006; Shimeld and lineage is asymmetrical: although cia-Bellido, 1996) and the rotation Levin, 2006). In the zebrafish, the the animal is generally bilaterally of the embryonic gut proventriculus Hþ/Kþ-ATPase (Kawakami et al., symmetrical with only a few LR (Hayashi and Murakami, 2001; 2005; Adams et al., 2006) and Naþ/ asymmetries, many of its contralat- Ligoxygakis et al., 2001; Adam Kþ-ATPase (Ellertsdottir et al., 2006) erally analogous cells arise via dif- et al., 2003). Both of these asym- are also known to be important for ferent lineages on the two sides of metries are instances of chirality, normal asymmetry, but no asym- the embryo (Wood and Kershaw, which appears to dominate in other metric localization has yet been re- 1991). Larvae and adults also ex- invertebrates such as ciliates and ported. Rhythmic Caþþ waves have

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 203 been described during fish gastrula- vertebrates and even invertebrates ric architecture than taxonomical tion (Gilland et al., 1999), consistent is considerable; the situation with relationships (Fischer et al., 2002). with the calcium fluxes discovered in mammalian embryos is more com- mice and chick (McGrath et al., plex. For example, no mouse mu- 2003; Raya et al., 2004), although tants in gap junction genes have as What Happens When There the connection to downstream LR yet reported a true LR phenotype; Are Many Small Cells Instead markers is not yet characterized. thus, knockins of dominant negative of a Few Large Ones? The GJC, ion flux, and serotonin constructs will be required to deter- pathways have been extensively mine whether GJC plays a role in LR Consideration of early mechanisms compared in chick and frog em- asymmetry of rodents (since many in chick or rabbit raises an important bryos (Levin, 2005) and, while all of different connexin genes exist in issue about what occurs in organ- the basic components appear to be embryos and are thus potentially isms that do not have the benefit of utilized by both systems despite able to exhibit compensation during large cell cleavages. What happens their radically-different gastrulation single gene deletion experiments). when early asymmetry steps occur architecture, details differ in inter- Likewise,althoughtheionchannel in a cell field of thousands of small esting ways. For example, while the polycystin is known to be important cells as opposed to a few large Hþ/Kþ-ATPase and V-ATPase ion in mice (Pennekamp et al., 2002; blastomeres whose cleavage planes pumps are expressed, as predicted McGrath et al., 2003) as well as are oriented with respect to the by the GJC model (which requires zebrafish (Bisgrove et al., 2005), final embryonic body-plan? (as in the motive force ‘‘battery’’ to be existing knockouts for many mem- Xenopus)? The alignment of axes located in the zone of isolation) in bers of the serotonin pathway and in mammalian cleavage is some- the primitive streak during early various channels and pumps deleted what controversial (Gardner, 1996, gastrulation of the chick, no asym- as part of neurobiology efforts have 1997; Zernicka-Goetz et al., 1996; metry in pump localization has not resulted in any reported asym- Piotrowska and Zernicka-Goetz, been reported in the chick at the metry phenotypes. Thus, it is possi- 2001; Plusa et al., 2002), but it is 0 level of mRNA or protein. Thus, ble that the relevant physiological known that the human 3 untrans- while asymmetric ion flux is pro- components remain to be identified, lated region (UTR) for squint mRNA vided by asymmetric localization of since experiments with early mouse mRNA in early frog embryos, it embryos are much more difficult appears to be established in the than in other model systems and chick embryo by a posttranslational many of the elements have not been What happens when mechanism (such as gating of elec- directly examined. Especially in the trogenic activity of mature pump case of ion transport, considerable early asymmetry complexes). Likewise, the differ- molecular divergence may occur since steps occur in a cell ence in GJC in frog embryos takes any number of channels or pumps field of thousands of place posttranslationally, by gating can produce the same physiological control of existing gap junctions effect and thus potentially substitute small cells as opposed (Levin and Mercola, 1998a). In con- for one another through evolutionary to a few large trast, the chick embryo establishes change. Another possibility is that the zone of isolation at the level of while the downstream effectors such blastomeres whose mRNA, by not transcribing Cx43 as the Nodal-Lefty-Pitx cascade are cleavage planes are mRNA in the primitive streak (Levin conserved throughout phyla, early and Mercola, 1999). With respect to components diverge significantly. oriented with respect serotonin, both species use 5HT as Important insight into the evolu- to the final embryonic part of an early LR signaling step; tionary conservation of GJC mecha- both rely on MAO, R3, R4, and SERT nisms is expected from analysis of body-plan? (Fukumoto et al., 2005a, 2005b). GJC in rabbits; the rabbit embryo However, frog embryos operate exhibits circumferential patterns of with a maternal pool of serotonin connexin expression (Liptau and that is meant to be degraded by the Viebahn, 1999), and GJC has been is able to drive asymmetric localiza- blastula stage, while chick embryos functionally implicated in rabbit tion in cleaving zebrafish embryos utilize zygotically-synthesized sero- asymmetry (Muders et al., 2006). (Gore et al., 2005), so future work tonin during gastrulation. The asym- Thus functional analysis of GJC in a must determine the extent to which metric serotonin gradient in Xeno- mammal with the flat architecture cleavage-based localization mecha- pus is generated by progressive of the chick is likely to shed signifi- nisms are also utilized in mammals. relocalization of maternal 5HT through cant light on the evolutionary con- One of the most exciting new devel- paths, while it appears to be generated servation and origin of the GJC sys- opments has been the discovery that in chick by differential degradation on tem as it participates in LR patterning. this cytoplasmic asymmetry may in the L versus R sides. Data on downstream components fact be a rotational (East-West) chir- The conservation of subcellular and suggests that asymmetry mecha- ality in Xenopus (Danilchik et al., physiological mechanisms throughout nisms track more closely to geomet- 2006); this is conceptually very

Birth Defects Research (Part C) 78:191–223, (2006) 204 LEVIN attractive because the sperm entry motion driving extracellular move- sally involved in organisms other point (which defines the DV axis) can ment of signaling molecules (see than the mouse and fish. Ciliary be anywhere on the circumference of below) and embryo-wide gap junc- data have given rise to two main the egg, making a rotational model tion paths (through which unidirec- physiological hypotheses: a sen- more likely. Rotational chirality pro- tional transport is guided by events sory model (McGrath and Brueck- vides information on LR direction oriented within single cells at the ner, 2003; McGrath et al., 2003; (not absolute position), and is a good zone of isolation) are natural candi- Tabin and Vogan, 2003) and one candidate for asymmetry generation dates for such a signal exchange. in which cilia are wafting morpho- in small cells because it is an ideal Does asymmetric gene expression gens such as Sonic hedgehog match for a subcellular ‘‘F-molecule’’ exist prior to gastrulation? It has (Okada et al., 2005; Tanaka et al., function. It is likely that subcellular been suggested (Levin and Mercola, 2005; Hirokawa et al., 2006). events related to the cytoskeleton 1998b) that the computation which (perhaps organizing centers and ba- aligns the LR axis with the DV and AP salbodies)arewheretheoriginof axes in the chick takes place at the asymmetry is to be found, and this initiation of gastrulation, at the base theme is discussed in the context of of the primitive streak (which reli- while cilia are likely polarized cells below. ably progresses from the periphery to be an The next phase of asymmetry is to the center of the blastoderm). also very important (Fig. 1B and C), However, no detailed model of this important aspect of and cell size and number are crucial process in the chick has been pro- mouse asymmetry, it to formulating models of how it posed, and may have to wait for a is not at all clear that might happen. Because no macro- good understanding of how (and scopic force distinguishes R from L, whether) individual cells in the chick they initiate a powerful paradigm has been pro- blastoderm determine their AP po- asymmetry in posed to leverage large-scale asym- larity (Wei and Mikawa, 2000). metry from the chirality of subcellu- Existing data suggest that the sim- mammals, or that lar components (Brown and Wolpert, ple early cleavage and ion trans- they are causally 1990; Brown et al., 1991). In this porter partitioning model cannot be class of models, some molecule or the whole story. First, it cannot be involved in organelle with a fixed chirality is ori- mapped directly onto the chick or organisms other than ented with respect to the AP and DV rabbit body-plan. Second, even in axes, and its chiral nature is thus Xenopus, ectopic organizers can be the mouse and fish. able to nucleate asymmetric proc- induced at later stages (when there esses such as transport of ion chan- are approximately 1000 cells) that nel and pump proteins to the proper correctly orient their own LR asym- side of the early embryo (Levin and metry (Nascone and Mercola, 1997), Mercola, 1998b). Thus, the first de- showing that even frog embryos do While the results of a true ‘‘no flow’’ velopmental event that distinguishes not have to rely on large early blasto- condition (such as in viscous me- LfromRwouldtakeplaceonasub- meres. The model shown in Figure 2 dium) have not yet been demon- cellular scale. However, a mechanism is a beginning, toward a framework strated, and culture of rodent must then exist to transduce subcel- that can be used to think about the embryos randomizes situs in and of lular signals to cell fields (Brown and components known to be important itself (Fujinaga et al., 1990; Fuji- Wolpert, 1990; Levin and Nascone, for asymmetry, but it clearly must be naga and Baden, 1991), in rodent 1997; Levin and Mercola, 1998b). modified in important ways to be embryos, cilia at the node are likely Asymmetric gene expression in em- more widely applicable. to play a functional role in the LR bryos requires that fairly large fields pathway, although it is not known of cells already know on which side of whether they generate LR informa- the midline they are located (such as tion de novo or function in transmis- the expression of the left-sided gene Cilia sion of as yet unknown upstream LR Nodal). In contrast, proposed mech- An important physiological mecha- signals. Consistent with the theo- anisms of step 1 of asymmetry (such nism that has been implicated in LR retically pleasing hypothesis that as the F-molecule model) rely on asymmetry is ciliary motion. This cilia initiate LR orientation, no ear- subcellular mechanisms for deter- subject has been extensively cov- lier LR mechanisms have been mining which direction is L and which ered in recent reviews (Hirokawa described in rodents. However, is R. Thus, one of the key questions et al., 2006), and we have pre- many types of cilia can reverse the concerns how orientation informa- sented the minority view (Levin, direction of their beat (Bone, 1958), tion can be turned into information 2003c, 2004) that while cilia are and it is not clear whether the bio- on a cell’s location, relative to the likely to be an important aspect of chemical structure of cilia uniquely midline, within the context of the mouse asymmetry, it is not at all determines their function (allowing whole embryo. This information flow clear that they initiate asymmetry asymmetry to be generated from must take place between cells; ciliary in mammals, or that they are cau- molecular chirality).

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 205

TABLE 2. Parallels between the Serotonin and Auxin Systems

Component Plant Vertebrate Kþ channel (Bandurski et al., 1990; Fuchs et al., (Adams and Levin, 2004; 2003; Philippar et al., 1999; Chen and Levin, 2004; Philippar et al., 2004; Levin, 2003b) Vicente-Agullo et al., 2004) Hþ pump Hþ-ATPase Hþ-ATPase, Hþ,Kþ-ATPase (Coenen et al., 2002; Hager, 2003; (Adams et al., 2006; Palmgren, 1998; Hibino et al., 2006; Rober-Kleber et al., 2003) Levin et al., 2002b) Small molecule auxin serotonin signal

Influx cell membrane AUX1 SERT transporter (Reinhardt et al., 2003; Yang et al., 2006) (Fukumoto et al., 2005a) Soluble receptor TIR1, ABP57 SBP (Dharmasiri et al., 2005; (Del Rio, 1995; Kim et al., 1998; Kim et al., 2001; Fukumoto et al., 2005b; Woodward and Bartel, 2005a; Jimenez Del Rio, 1993; Woodward and Bartel, 2005b) Tamir et al., 1994) Long-range gradient 10-fold 5-fold (estimated) (Edlund et al., 1995; (Esser et al., 2006; Uggla et al., 1996) Fukumoto et al., 2005b) Electrophoretic (Bandurski et al., 1990; (Esser et al., 2006; mechanism Goldsworthy and Rathore, 1985; Fukumoto et al., 2005a; proposed Rathore and Goldsworthy, 1985; Fukumoto et al., 2005b) Schrank, 1951; Webster and Schrank, 1953)

Because rodent embryos in which et al., 2006). Interestingly, OFD1 is a The rodent embryo is somewhat molecular motors have been centrosomal/basal body protein, and unusual in its large scale and node mutated are also likely to have is an ideal candidate for intracellular architecture, compared to more typi- impaired cytoplasmic function of F-molecule orientation-based mod- cal mammals such as rabbit and motor transport (which may be very els. In contrast to extracellular ciliary human. Consistent with the possibil- important for asymmetry (Qiu et al., movement (Romio et al., 2004). The ity that the functional involvement of 2005)), it has not been possible to resolution to this question will come cilia in asymmetry is not specific to cleanly separate the ciliary functions through a more sophisticated mu- rodents, recent studies in Zebrafish of the LR-relevant motors from their tant: if a mouse can be produced in demonstrated that knockdown of the cytoplasmic transport roles in mam- which loss-of-function of LR dynein ntl gene specifically in the dorsal malian embryos. Most ciliary proteins or other ciliary components can be forerunner cells (ciliated cells in have subcellular roles, such as motor restrictedtothenodeormadeto Kupffer’s vesicle) results in random- protein transport, cell polarity deter- begin only at day 7, and if this mu- ization of situs (Amack and Yost, mination, and transcription control tant has a LR phenotype, it will be 2004; Bisgrove et al., 2005; Essner (Wang et al., 2006); it is also possible convincingly demonstrated that it is et al., 2005; Kramer-Zucker et al., that ciliary motion itself has physical indeed the nodal ciliary roles of the 2005). These data argue for a more (traction) effects on cytoplasmic relevant proteins that are important widely-conserved role of ciliary com- components through the ciliary root- for mouse asymmetry. Regardless of ponents. Since an Naþ/Kþ-ATPase let (Yang et al., 2002, 2005; Yang the outcome in rodent embryos, mutation in fish resulted in laterality and Li, 2005). Ciliary protein deletion human primary ciliary dyskinesia defects but normal cilia (Ellertsdottir mutants tend to give laterality patients still have normal cerebral et al., 2006), it is possible that the defects, but are also often implicate lateralization, demonstrating that fish uses parallel pathways. Taken other roles, like the OFD1 knockout cilia are not a unique symmetry- together, the data in vertebrates and that also has altered Hoxa and Hoxd breaking event in humans (McManus invertebrates suggest the possibility expression in the limb (Ferrante et al., 2004). that subcellular motor protein- and

Birth Defects Research (Part C) 78:191–223, (2006) 206 LEVIN cytoskeleton-driven localization LR by upstream physiological events. In ment, etc.) are now known to be pathways and ion flows were evolu- Xenopus, the first known asymmetric crucial components of auxin signal- tionarily older and were the way that gene is Nodal, which is expressed ing (Arend et al., 2002; Coenen evolution first oriented asymmetry long after neurulation begins. Thus, et al., 2002; Pasternak et al., 2002; (now having been implicated in a number of interesting mechanisms Hager, 2003; Rober-Kleber et al., plants, snails, C. elegans, Drosoph- must yet remain to be identified 2003; Wind et al., 2004). ila,seaurchins,ciona,fish,frog,and functioning between early serotonin In addition to the similarity of the chick). At some point, ciliary move- signaling and Nodal regulation. One molecular components involved, a ment became involved in the middle possibility is syndecan signaling, functional loop mechanism appears of the pathway (perhaps to strengthen which functions in frog embryos dur- to be conserved as well. The ability or transmit earlier asymmetries), ing the correct timeframe (Kramer and rodent embryos may indeed et al., 2002; Kramer and Yost, have dispensed with the earlier 2002). Future experimental work activation of mechanisms once the ciliary compo- must uncover the receptors for sero- nents were fully in place. Consistent tonin and other GJC-permeable LR asymmetric ion flux with this imaginary timeline (Table morphogens, and perhaps array by serotonin and the 1), organisms with fully-fledged LR approaches can identify proximal im- asymmetry, such as sea urchins and mediate-early response genes to unidirectional þþ þ þ Drosophila, do not seem to have any- Ca ,H,andK asymmetries. þ movement of 5HT due thing resembling a node or an inter- Screens for pH- and [K ]-sensitive nal ciliated pocket (Amemiya, 1971, promoter elements may reveal to an electrophoretic 1986, 1989; Martinez-Campos et al., genes sensitive to intracellular ionic force may thus be a 2004). The cilia is likely to have en- conditions. Likewise, the identifica- tered the evolutionary stream around tion and characterization of a morph- positive-feedback the time of fish, since recent data in ogen that depends on ciliary move- loop that could zebrafish implicate cilia (Amack and ment remains a high priority. magnify small Yost, 2004; Bisgrove et al., 2005; Essner et al., 2005; Kramer-Zucker The Minds of Plants: asymmetries on the et al., 2005), though a direct extrac- Parallels Between Serotonin cellular level into ellular viscosity test has not been done in this system. and Auxin Asymmetry asymmetry on the The question of timing and possible The patterning of the LR axis scale of cell fields; ciliary roles in asymmetry has been has some fascinating similarities to addressed in another mammal. Rab- mechanisms of other patterning bit embryos (like most mammals systems. One of these concerns the including humans) develop as a flat parallels between serotonin and the blastodisc similar to chick embryos, plant hormone auxin (Table 2), in of 5HT to localize to a specific blas- and their study has contributed sig- the determination of morphogene- tomere depends on gap junctional nificant advances which complement sis and LR asymmetry in plants, communication and Hþ,Kþ-ATPase and contrast the mouse system (Vie- which is now being addressed function, which (in concert with a bahn et al., 1995; Viebahn, 2001; Fi- molecularly (Endress, 1999, 2001; potassium channel) may provide an scheretal.,2002).Inrabbit,Cer1 Theissen, 2000). Auxin, a plant hor- electromotive force for moving expression is asymmetric at stage 3 mone that bears a striking struc- charged small molecules (such as (Idkowiak et al., 2004), prior to cili- tural similarity to the neurotrans- serotonin) between cells (Levin, ary movement, although it is not mitter serotonin, is a positional sig- 2004). Conversely, a number of ion clear whether this is consistently bi- nal in a number of plant patterning transporters are controlled by 5HT, ased. Mice may not recapitulate all events (Sabatini et al., 1999; Vroe- most notably R3 and SERT (Maricq phenomena important for LR pat- men et al., 1999; Baluska et al., et al., 1991; Quick, 2003). As this terning (see discussion of midline 2003; Friml, 2003; Barlow, 2005) field matures, it will be necessary to determination below). This becomes and is involved in establishment of develop quantitative models of the of particular importance when con- bilateral symmetry (Lee and Evans, bidirectional relationship between sidering the mechanistic implications 1985; Liu et al., 1993; Zgurski 5HT movement in an electric field of laterality phenotypes observed in et al., 2005) and even LR asymme- and the regulation of ion transport- human monozygotic twins. try (Pekker et al., 2005) in plant ers by 5HT. The activation of asym- systems. Indeed, many of the same metric ion flux by serotonin and the players that have been implicated unidirectional movement of 5HT How Do Physiological Events þ in LR asymmetry (K channels, due to an electrophoretic force Control Gene Expression? plasma membrane Hþ flux, cell may thus be a positive-feedback One of the challenges facing this field membrane transporters and gra- loop that could magnify small is to understand precisely how asym- dients of serotonin/auxin, regulatory asymmetries on the cellular level metric gene expression is controlled roles of pH, electrophoretic move- into asymmetry on the scale of cell

Birth Defects Research (Part C) 78:191–223, (2006) INTEGRATION OF PHYSIOLOGICAL SIGNALS 207 fields; such feedback loops are 3-3 protein (PAR-5) is required for vertebrates, myosin in Drosophila) likely to be absolutely central to cellular asymmetry in the early both result in randomization! This establishment of asymmetry. embryo (Morton et al., 2002). PAR-5 spatial distribution is paramount for The movement may be intracellu- likewise functions in axial patterning these physiological mechanisms and lar or intercellular in various pat- in Drosophila (Benton et al., 2002). is not like many pathways, in which terning systems. Indeed, plant root Interestingly, it was shown that the loss-of-function and gain-of-func- tips set up a sink-driven gradient of PAR homolog 14-3-3E protein is LR- tion have opposite effects. The ne- auxin (Friml et al., 2002); the sero- asymmetrically localized during the cessity for high GJC/5HT/membrane tonin gradient in frog embryos is first two cleavages in frog embryos voltage in one area and simultane- likewise dependent on the degrada- and functions in the LR pathway ously low GJC/5HT/membrane volt- tion machinery to function after the upstream of Nodal; moreover, this age elsewhere, precludes simple ‘‘one relocalization steps have taken signaling can be perturbed by fusi- knockout-one phenotype’’ models place. A model has been proposed coccin-a fungal compound previ- that are common in other aspects of (Paponov et al., 2005) in which ously thought to only interact with morphogenesis. On the other hand, auxin flow, starting by diffusion, plant cells (Bunney et al., 2003). The recent efforts to mathematically induces formation of the polar involvement of 14-3-3 proteins in model morphogen gradients in Dro- transport system. This in turn pro- cellular asymmetry in early clea- sophila have also revealed that such motes auxin transport, leading to vages of both C. elegans and Xeno- robust patterning mechanisms must canalization of auxin flow along a pus is further evidence of a deep and keep the system between uniformly narrow column of cells and ulti- fundamental underlying conserva- low or uniformly high morphogen mately controlling differentiation. A tion of mechanisms by which asym- levels across the embryo (Gregor similar amplification loop between metry and polarity, whether on the et al., 2005; Goentoro et al., 2006)- 5HT and Kþ flow through R3 is pre- cellular level, or on the scale of the as in the case of LR asymmetry, driv- cisely the kind of mechanism that organism, is established. The finding ing the system in either direction has been proposed to account for that elements of fusicoccin/14-3-3 (through a very high or very low the function of 5HT movement dur- signaling are conserved from fungi- Thiele modulus) destabilizes the ing LR patterning (Fukumoto et al., plant interaction to animal embryo- axial-patterning event. 2005b). The involvement of elec- genesis presents a new perspective Another interesting parallel of the trophoretic mechanisms relevant to from which to investigate novel as- LR axis exists in the way neurons serotonin-like molecules in plant pects of large-scale morphogenetic regulate their membrane voltage. and animal patterning (Goldsworthy control in vertebrates and highlights Normal neurons build up a voltage and Rathore, 1985; Fischer et al., the possible connection between potential using the Naþ/Kþ-ATPase 1997; Palmgren, 1998; Rober- fundamental subcellular polarity main combination with a Kþ channel to Kleber et al., 2003) suggest that fas- chinery (cytoskeleton and motor provide the negative membrane cinating aspects of evolutionary con- proteins) and morphological asym- voltage as the excess Kþ escapes servation of such patterning mecha- metry. (Hille, 2001). The early Xenopus nisms await investigation. Such Another example of this polarity is embryo utilizes exactly the same wide conservation of mechanism seen in the development of the system, involving an Hþ/Kþ-ATPase between the common ancestor of nervous system. Neuroblasts are exchanger in combination with a Kþ animals and vascular plants requires able to polarize without external channel to set up its LR voltage dif- that protists had access to sero- cues, but such cues are needed to ferences (Levin et al., 2002; Levin, tonin-like molecules. Though we do orient their polarization to a consist- 2003b; Chen and Levin, 2004), al- not have access to the protist basal ent angle (Pearson and Doe, 2003; though there is an additional hyper- to both plants and animals, we know Rolls and Doe, 2003). The LR axis polarizing pump involved in Xeno- that protists such as plasmodium may also involve a similar distinction pus-the V-ATPase (Adams et al., and tetrahymena have both sero- between generating asymmetry and 2006). Gastric cells appear to do tonin and its receptors (Brizzi and establishing its orientation with the same (Fujita et al., 2002), using Blum, 1970; Janakidevi et al., 1966; respect to the other two axes. Caþþ- the Hþ/Kþ-ATPase pump and the Pan et al., 1994). dependent polarization in pollen same KCNQ1 channel that is impli- tubes illustrates another unique as- cated in frog asymmetry. The LR Axis Is Like a Kidney: pect of the LR axis: excess and ubiq- In addition to the control of ion uitous Caþþ stops directional growth, current paths by subcellular local- Insights from Epithelia and þþ even though increased Ca in the ization, the reverse relationship Other Polarized Cells tip dictates the polarity of extension exists as well: ion transport is im- The mechanisms characterized in (Rathore et al., 1991; Messerli and portant for polarity on the level of the LR pathway revealed parallels to Robinson, 1997, 2003; Messerli single cells (Patel and Barber, other systems in addition to the et al., 1999, 2000; Robinson and 2005). This is yet another feedback plant connection described above. Messerli, 2002). Similarly, in the LR loop that must be incorporated into Subcellular polarity and cytoskeleton field, the global up- or downregula- the complex models of early embry- are the common theme. For exam- tion of almost every component (se- onic physiology; discoveries from ple, in Caenorhabditis elegans, a 14- rotonin, ion flow, GJC, and Shh in the kidney and gut epithelial fields

Birth Defects Research (Part C) 78:191–223, (2006) 208 LEVIN will continue to provide insights for asymmetry (Nelson et al., 1991; observed in the polarized cells of the the LR field that will contribute to Brown et al., 1992; Nelson, 1992; fly gut (Hozumi et al., 2006; Speder the development of quantitative Suzuki et al., 1995; Al-Awqati et al., et al., 2006). Moreover, b-catenin models encompassing all of the rel- 1999; Fujita et al., 2002; Yao et al., binds both inversin (Nurnberger evant physiological and genetic 2002; Beyenbach, 2001). Other et al., 2002; Eley et al., 2004; events involved (Martin and Har- components implicated in LR asym- Simons and Walz, 2006) and the fly vey, 1994; Pribyl et al., 2003; metry, such as claudins are also in- myosin I (Speder et al., 2006), sug- Fischbarg and Diecke, 2005). The volved in kidney function (Yu et al., gesting deep conservation of the future development of such models 2003; Abuazza et al., 2006; Balko- symmetry biasing mechanism and and the resulting understanding of vetz, 2006; Gonzalez-Mariscal its linkage to pathways that pattern robustness, dynamic cycles, and et al., 2006; Lee et al., 2006), as polarity along the AP and DV axes. feedback are essential to perfect they shape the transepithelial volt- Cytoskeletal elements direct the functional techniques for manipu- age gradients resulting from the tar- large-scale polarity of other axes lating these pathways rationally in geted ion flows. Thus, it may be that from the DV axis in Xenopus (Elinson biomedical settings. the reason kidney defects and asym- and Rowning, 1988; Gerhart et al., However, perhaps the most im- metry randomization often appear 1989) to the AP axis in planaria portant analogy is between kidney together is that mutations in compo- (Nentwig, 1978). nents of subcellular targeting (motor proteins, cytoskeletal components, the salient etc.) result in abnormal function Brain Asymmetry in both kidney epithelia and early While nervous system lateralization commonality asymmetry-breaking events. is spread throughout phylogeny between the kidney The intestinal epithelium, in a (Andrew, 2000) because of a num- very wide range of organisms, is ber of ecological advantages and the LR axis is another highly-polarized tissue that (Rogers et al., 2004; Vallortigara their tight linkage of expresses and utilizes key ion and Rogers, 2005), there exist a transporters implicated in LR asym- number of fascinating human be- cell polarization to ion metry (Morley et al., 1992; Kraut havioral and brain asymmetry phe- transport et al., 1995; Numata et al., 1995; nomena that have bearing on the localization. Cheng et al., 1996; Huang et al., question of exactly when different 2006; Jespersen et al., 2005; Jons aspects of the LR axis become fixed et al., 2006). during embryogenesis. While some I argue that the origin of asym- animals (e.g., mice) often show paw metry takes place intracellularly, is preference, the consistent prefer- function and the LR axis. A number conserved from invertebrates (is ence among all individuals only ap- of mouse knockout studies have related to invertebrate chirality), proaches high levels in man (ap- identified targets whose abrogation and is intimately connected to proximately 90% for right-handed- causes both a kidney and a LR motor proteins and the cytoskeletal ness), and its genetic basis is still defect (Mochizuki et al., 1998, tracks that guide their localization. controversial (McManus and Bryden, 2002; Murcia et al., 2000; Haycraft This is clear in snails (Freeman and 1992; McManus, 1995). et al., 2001; Otto et al., 2003; Lundelius, 1982; Shibazaki et al., Interestingly, brain asymmetry Kramer-Zucker et al., 2005). This is 2004) and plants (Thitamadee et al., (Toga and Thompson, 2003) does most often interpreted with respect 2002), and the data discussed not correlate with visceral asymme- to a sensory cilium being necessary above implicate it in vertebrates as try (Kennedy et al., 1999; Tanaka for kidney function and asymmetry well (Qiu et al., 2005). Alongside the et al., 1999). For example, situs in- (Pazour and Witman, 2003; Pazour, kinesins and dyneins that have been versus totalis individuals still have 2004; Barr, 2005; Pan et al., 2005). implicated in rodent asymmetry language lateralization seen in 95% In contrast, I propose that the sa- (Supp et al., 1997; Marszalek et al., of right-handed normal situs individ- lient commonality between the kid- 1999; Takeda et al., 1999), recent uals (Kennedy et al., 1999). The inci- ney and the LR axis is their tight data in Drosophila have also impli- dence of left-handedness is exactly linkage of cell polarization to ion cated the myosin motor (Hozumi the same in situs inversus individu- transport localization. Kidney cells et al., 2006; Speder et al., 2006). als as in the rest of the population (and epithelial cells in general) are Surprisingly, loss of the symmetri- (Cockayne, 1938; Torgersen, 1950). highly polarized, and exert strong cally-expressed myosin I results in This suggests that mechanisms control over ion fluxes by using mirror-image flies and not random- establishing the laterality of the brain cytoskeletal and motor protein ization, like the inversin deletion in are, at some early point in the LR transport elements to regulate the mice (Morgan et al., 1998). As in pathway, different from those that intracellular membrane transport of vertebrates, in which LR compo- determine the sidedness of visceral ion channels and pumps, specifi- nents are present in kidney and di- organs. Moreover, recent evidence cally including the Hþ and Kþ trans- gestive system epithelia, oriented suggests that human patients with porters known to be important in actin filaments and myosin I are classical primary ciliary dyskinesia

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(and the attendant heterotaxia) 1980; Newman et al., 1937; Potter ternal organs clearly is subject to do not exhibit reversals in the nor- and Nance, 1976; Gedda et al., negative evolutionary pressure. An mal prevalence of right-handedness 1981; Mensing, 1983; Yager, 1984; alternative model is that some as (McManus et al., 2004), suggesting Schneider, 1985; West, 1985; Carton yet unknown pathological mecha- that at least some aspects of lateral- and Rees, 1987; Beere et al., 1990; nism is responsible for both the ity in humans are indeed upstream Townsend and Richards, 1990; Morison process of twinning itself and the of, or exist in parallel to, mutations et al., 1994; Sperber et al., 1994; destabilization of the LR axis (Bok- affecting ciliary function. Bizarrely, Satoh et al., 1995; Cidis et al., 1997; lage, 1981, 1987). In support of this the asymmetry of wristwatch-wear- Sommer et al., 1999, 2002; Okamoto view, it has been found that in- ing behavior does indeed correlate et al., 2001; Morini et al., 2002). The creased incidence of left-handedness withsitusinversus(McManusetal., mirror/bookending phenomenon is intwinsisnotdependentonzygosity 2004). Recent work in zebrafish not just structural but also pertains or time of splitting (McManus and (Concha et al., 2003; Gamse et al., to functional parameters such as Bryden, 1992; Derom et al., 1996). 2003; Halpern et al., 2003; Barth sleep deviations, hearing, and cere- The molecular basis of these phe- et al., 2005; Lin and Burdine, 2005; bral functional localization (Springer nomena is not understood, although McManus, 2005) has shed light on and Searleman, 1978a, 1978b; Gol- analysis of laterality in twins pro- the regulation of brain asymmetry bin et al., 1993; Sommer et al., duced by splitting of embryos during by known pathways, but it is unclear 1999, 2002). Though monozygotic in vitro fertilization procedures may whether the fish or another available twins affected by genetic lesions of- eventually provide important clues. model system will provide a tracta- ten show opposite sidedness of limb Interestingly, monozygotic twins are ble entry point to the study of the abnormalities (Richieri-Costa and often discordant for the imprinting of fascinating brain asymmetry in man Opitz, 1986; Opitz and Utkus, 2001), KCNQ1 (Weksberg et al., 2002), a as distinct from the cardiac-visceral almost all bookending phenomena potassium channel which has been pathways. in healthy twins involve features of implicated in LR asymmetry in frog the head. Hair whorls originate from embryos (Levin, 2003b). the same tissue layer as the nervous Increased understanding of epi- Two Small Black Clouds in the system, and are linked to handed- genetic factors in laterality will Human Data: Cryptic ness and language dominance (Klar, come from analysis of monozygotic Asymmetry, Subtle Chirality, 2003; Weber et al., 2006), although twins (Steinmetz et al., 1995), but the proximate mechanisms deter- may also be gained by examination and Sex mining hair-whorl sidedness is not of the experimentally-tractable arti- Analogous to the seemingly small, well understood. Thus, consistently ficial twins of bovine embryos that incongruous observations in ther- with the discordance between brain later exhibit hair whorls (Lanier modynamics that turned out to rev- and body situs discussed above, et al., 2001; Meola et al., 2004; olutionize the tidy picture of classi- there may be two separate organiz- Evans et al., 2005). Why might cal physics at the beginning of the ers for the head and body (Mein- early splitting have consequences last century, a number of clinical hardt, 2002), which use different for embryonic chirality? Mirror and experimental data have been mechanisms of determining lateral- image cytoskeleton patterns and obtained that do not fit neatly into ity (Harland and Gerhart, 1997). cell migration tracks have been the existing paradigms and have The bookending phenomenon may observed following normal cell divi- the potential to reveal untapped also speak to the timing of the ear- sion in culture (Albrecht-Buehler, areas of the asymmetry field. These liest steps of asymmetry in mam- 1977a, 1977b, 1977c, 1978), once relate to subtle and cryptic asym- mals. Most healthy, nonconjoined again underscoring the importance metry, in contrast to the more ob- twins presumably result from sepa- of cytoskeleton and subcellular vious morphological and behavioral ration of cleavage, morula, or early structures for large-scale asymme- differences. blastocyst stage embryos (James, try and cellular behavior. Nonconjoined monozygotic twins, 1983). Thus, some chiral informa- Another fascinating phenomenon not only exhibiting a higher-than- tion may be present in the very early that has only begun to be explored normal incidence in laterality defects mammalian embryo, later manifest- is that of cryptic asymmetry. This (Kuehl and Loffredo, 2002), also ing as hair whorls, etc., if the cells refers to consistent asymmetries manifest many subtler kinds of mir- are separated at an early stage. In that exist in morphologically sym- ror-image asymmetry (‘‘bookend’’ contrast, the asymmetry of the metrical structures and are some- or enantiomer twin pairs). Pairs of major body organs seems to be times only revealed following ex- such twins have been noted to pres- unspecified (or plastic enough to be perimental perturbations (Cohen, ent mirror asymmetries in hand respecified) at those stages, and is 2001). One example is the consis- preference, hair-whorl direction, developed correctly for both mono- tently asymmetric defects in pla- tooth patterns, unilateral eye and zygotic twins. This may be related to narian eye regeneration only ob- ear defects, cleft lip, cleft palate, su- the fact that heterotaxic reversals in served following Hþ/Kþ-ATPase in- pernumerary teeth, and even tumor hair whorls and tooth patterns would hibition (Nogi et al., 2005) and locations and undescended testicles not be expected to be disadvanta- unilateral limb defects produced in (Lauterbach, 1925; Rife, 1933, 1940, geous, while discordant situs for in- rodents by some compounds (Barr,

Birth Defects Research (Part C) 78:191–223, (2006) 210 LEVIN

Figure 4. Two systems of asymmetry in human embryos and midline determination. A: The biomedical data suggest that human embryos have two different pathways of asymmetry, which may or may not both be downstream of a common originating mechanism. The first pathway could be cilia-related, and feeds into the known asymmetric gene cascade upstream of the situs of the heart and viscera. The other pathway is likely to be active at early cleavage stages, controls unknown downstream mechanisms, and is ultimately revealed by ectodermal structures: cryptic asymmetry in hair whorls and tooth patterns, and behavioral/anatomical asymmetries in the brain. Midline determination: asymmetric cutaneous pigmentation pattern with a sharp midline demarcation in the X-linked CHILD syndrome (B) suggests early establishment of midline in human embryos. Similar patterns are observed in gynandromorphs in butterflies (C), chickens (D), and lobsters (E). Alignment of early cleavages with the midline of the embryo is revealed in model species such as Xenopus when a marker mRNA such as b-galactosidase is injected into a blastomere at first or second cleavage and results in unilateral distribution one week later (F).

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1973; Layton and Layton, 1979; (Fraumeni et al., 1967; Clericuzio, portant implications for biomedicine Milaire, 1985). Other examples 1993; Sarkar et al., 1992; Stalens and basic developmental biology. include the consistently chiral rota- et al., 1993; Kloeppel et al., 2002; The human data on asymmetry tion observed in Xenopus following Leung et al., 2002), suggesting that suggests that there are two differ- pharmacological disruption of the a wide range of tissues within mor- ent pathways operating (Fig. 4A). cytoskeleton (Danilchik et al., 2006) phologically symmetrical organs The primary control of visceral and and asymmetries in craniofacial may not only possess positional in- cardiac asymmetry takes place structures in zebrafish mutants formation with respect to the LR axis through activity of the well-under- (Albertson and Yelick, 2005). All of but may maintain this identity for stood asymmetric cascade genes these cases demonstrate asymme- decades after completion of em- (Casey, 1998; Kosaki et al., 1999; tries that are not observed in wild- bryogenesis. Molecular insight into Bamford et al., 2000) and can be type individuals but are exposed by this phenomenon is likely to open associated with ciliary dysfunction experimental manipulation. Other important advances in developmen- (Afzelius, 1976, 1999). Interestingly, related data in vertebrates has re- tal patterning; taken together with thereappearstobeanother,largely vealed asymmetries in normal somites the bookending phenomena ob- mysterious, pathway revealed by (Saude et al., 2005) and limbs (Sien- served in monozygotic twins, these clinical data (Cohen, 2001). As knecht, 2006), structures thought to data suggest the presence of two dis- discussed above, chirality of hair be morphologically symmetrical. tinct LR pathways in human embryos. whorls, hand use preference, and Such observations hint that Most of the work in the LR field brain hemisphere language local- subtle molecular differences may has naturally addressed the mecha- ization appear to be controlled by exist between organs that are as- nisms controlling the situs of mor- a separate pathway not down- sumed to be symmetrical (such as phologically asymmetric organs. stream of the mechanisms dis- left and right limbs, eyes, etc.), However, the human and rodent rupted in situs inversus and heter- establishing a true LR axis rather data discussed above (unilateral otaxia patients. The conservation than a mediolateral one (although drug effects and global hemihyper- of asymmetry of unilateral defects the data do not require graded trophy syndromes) indicate that in monozygotic twins suggests positional information along this seemingly identical paired struc- that mirror-image LR information LR axis, but merely L vs. R identity). tures may in fact harbor subtle mo- was already present at the time of This counterintuitive and as yet lecular differences conferring posi- splitting: while we do not know the unexplored idea is also sugges- tional information along the LR axis, intermediate steps mediating con- ted by several human syndromes. and that this information may per- trol of the final phenotype, the Holt-Oram syndrome (Tbx5-related) sist well into adulthood. Recent original symmetry-breaking event studies have indicated that rodent must take place quite early (and embryo somites exhibit a striking certainly prior to the late gastrula- subtle molecular asymmetric expression of genes tion stage at which cilia act to trans- such as HB-EGF and MLC3F (Golding mit or initiate the asymmetry cascade). An important open question differences may exist et al., 2004a, 2004b); differential is whether these two pathways are gene expression in precursors of between organs that completely parallel or simply diverge paired organs and skeletal elements early from a common starting point. are assumed to be could potentially provide a hereto- To define true LR asymmetry, the symmetrical fore unsuspected mechanism for as- body-plan first must establish a signing L and R identity to seemingly midplane of symmetry. While mice identical structures. Human fetuses seem to have indeterminate early at 10 weeks of gestation are 85% development, or at least can respe- presents upper limb malformations right-handed (very close to the final cify the LR axis when disturbed which are much more common on 90% figure); this is not likely to be (Brown et al., 1990; Brown and the left-side (Smith et al., 1979; under brain control but is probably Wolpert, 1990), striking unilateral Newbury-Ecob et al., 1996; Bru- of muscular or spinal determination, pigmentation patterns (Fig. 4B) can neau et al., 1999; Hatcher et al., suggesting asymmetries in those occur in human patients with X- 2000), while fibular a/hypoplasia tissues (Hepper et al., 1998). Future linked diseases such as CHILD syn- affects the right side more often work must characterize novel molec- drome (Happle et al., 1995; Happle, (Lewin and Opitz, 1986). Indeed, a ular differences between paired 2002). The required LR-asymmetric variety of human syndromes affect- structures and address the functional X-inactivation in such patients sug- ing paired organs have a significant significance of this asymmetric gene gests that the midline decision is bias for one side (Paulozzi and Lary, expression. Identification of LR sig- made quite early in human em- 1999). Moreover, genetic conditions nals in locales other than overtly bryos. Consistent with the previous (such as hemihypertrophy syn- asymmetric organs, and an under- proposal that rodent embryos are drome) exist in which a large num- standing of the temporal extent of LR derived and atypical with respect to ber of tissues, all on one side of the information after completion of LR mechanisms, the mouse models body, resume growth in adulthood embryogenesis, is sure to have im- of CHILD syndrome recapitulate all

Birth Defects Research (Part C) 78:191–223, (2006) 212 LEVIN of the important features except Could One Properly Shake ronment. Likewise, the energy of unilateral pigmentation (Konig et al., Hands with An Alien? the right-handed a-helix of poly-L- 2000). alanine is a few tenths of a kilocalo- In orthoptera, unilateral gynan- Would the chirality of organisms with ries per mole per residue lower than dromorphs are thought to result a fundamentally different biochemis- that of the left-handed helix, imply- when one of the X chromosomes in try be the same as is observed on ing that over some length, right- an XX zygote is eliminated at the first Earth? The issue of original chirality handed forms will be more stable cleavage division (Barranco et al., (i.e., why living organisms contain (Morgan, 1977). Both asymmetries 1995). While the rodents do not only L-amino acids and D-sugars) is are presumably consequences of the appear to set midline early enough bound up fundamentally with the ori- non-conservation of parity in subato- for X-linked mechanisms to produce gin of life. In vitro synthesis almost mic weak nuclear interactions (Wu large-scale asymmetries, the human invariably results in equal mixtures et al., 1957). Testing such models cases revealed by genetic pigmenta- of enantiomer pairs of compounds, represents a formidable challenge, tion syndromes resemble examples while biosynthetic processes were but the identification and evolution- of gynandromorphs found through- able to clearly separate such racemic ary characterization of the biochemi- out phyla (Fig. 4C-E), including but- mixtures (Pasteur, 1860). Several cal mechanism that initiates asym- terflies, ants, crabs, and chickens, theories for this have been proposed. metry will ultimately reveal whether can present midline asymmetries of Perhaps, whatever type of isomer a direct link exists between the sym- sex and pigmentation (Farmer, 1972; happened to have formed first biased metry breaking events at the origin Dang and Peterson, 1979; Sivarad- the rest of evolution toward that type oftheuniverseandthehandedness jam and Bierne, 1981; Homsher and by competition (Frank, 1953). The of biological forms on Earth today. Yunker, 1981; Mey, 1982; Taber and chirality of the first one could have Francke, 1986; Taylor, 1986; Micheli, been determined by chance, or by 1991; Stevens and Munk, 1991; Bar- exogenous factors such as light CONCLUSION ranco et al., 1995; Moriyasu et al., polarization (Noyes and Bonner, 1998; Zou and Fingerman, 2000; 1975) or even the geomagnetic field The consistent macroscopic chirality Sagi et al., 1996, 2002). All of these (GMF). Interestingly, the GMF seems of embryonic structures is a crucial argue for a conserved early demarca- to have a relationship with LR chiral- part of developmental biology. This tion of the midline, and indeed in the ity (Anderson, 1988): the geological subject touches on issues ranging case of crustaceans, it is actually fossil record shows a correlation from evolutionary mechanisms of known that this takes place, as in between flipping of the GMF polarity body-plan dynamics to the subtle- Xenopus, at early cleavage stages and reversals of the chirality of sev- ties of parity conservation in quan- (Extavour, 2005). The human clinical eral types of mollusks such as Glo- tum mechanics. While a number of data also suggest a striking conser- borotalia menardi (Harrison and Fun- important advances have been vation of mechanisms establishing nel, 1964; Dubrov, 1978). Thus, the made in several model systems, the midline, at least as concerns ectoder- determination of chirality may be most interesting questions remain mal derivatives. one of the several roles the GMF open, and the field is in need of reso- Even if the midline is established probably plays in embryogenesis lution with respect to conceptual early, as is suggested by the gynan- (Cole and Graf, 1974; Shibib et al., unity (or proving the impossibility dromorph cases, is asymmetry or its 1987; Asashima et al., 1991; Leal thereof). I argue that the LR axis is biasing linked to this process? It is et al., 1992; Sandoze et al., 1995). patterned by mechanisms that are likely; in human hermaphrodites, Alternatively, there may be a fun- fundamentally conserved through- ovaries tend to develop on the left, damental reason for why biological out phyla and used in establishing while testes appear on the right forms prefer one type of molecule cell polarity in a number of tissues. (Mittwoch, 2000). This time, rodents over its enantiomer. For example, It is likely that future experiments are not the odd man out, although when racemic mixtures of the amino addressing the subcellular roles of while mice do exhibit the strong link- acids alanine, tryptophan, and tyro- motor proteins, small molecule age between sidedness of hermaph- sine in alkaline solution are sub- movement (through gap junctions roditic organs observed in human jected to decomposition by radioac- and driven by cilia in the extracellu- cases (Eicher and Washburn, 1983; tive decay of strontium-90, the D- lar space), and the mechanisms that Ward et al., 1987; Biddle et al., isomers are destroyed more quickly generate organ shape from laterality 1994), the consistent laterality of than the L-isomer (Garay, 1968). signals, will open up new areas of placement of testes versus ovaries There are also arguments based on cell, developmental, and evolution- is opposite that observed in humans weak neutral currents which show ary biology. Indeed, the increasing (van Niekerk and Retief, 1981; Krob that L-amino acids will predominate efforts to mathematically model and et al., 1994). Sex determination pro- in biochemical reactions in a period synthesize data from genetics, vides a largely-untapped entry-point of on the order of 15,000 years physiology, and biophysics to truly into understanding and gauging the (Mason and Tranter, 1984; Konde- understand the amplification and conservation of mechanisms estab- pudi,1987).Thus,radioactivedecay feedback loops will give rise to a sat- lishing midline and its coordination could plausibly have biased enan- isfying understanding of laterality in with the LR and other axes. tiomer choice in the prebiotic envi- the animal kingdom.

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ACKNOWLEDGMENTS Afzelius BA. 1999. Asymmetry of cilia Cambridge: Cambridge University Press. and of mice and men. Int J Dev Biol pp 70–93. Many thanks to the members of the 43:283–286. Angelo S, Lohr J, Lee KH, et al. 2000. Levin laboratory and the LR asym- Aihara M, Amemiya S. 2000. Inversion of Conservation of sequence and expres- metry, physiology, and neuroscience left-right asymmetry in the formation sion of Xenopus and zebrafish dHAND communities for useful discussions, of the adult rudiment in sea urchin lar- during cardiac, branchial arch and lat- vae: removal of a part of embryos at eral mesoderm development. Mech and to Dany S. Adams for her the gastrula stage. Zygote 8(Suppl 1): Dev 95:231–237. detailed comments on a draft of this S82–S83. Araki I, Terazawa K, Satoh N. 1996. review. Permissions and attributions Aihara M, Amemiya S. 2001. Left-right Duplication of an amphioxus myogenic for Fig. 4: The image of a Papilio positioning of the adult rudiment in sea Bhlh gene is independent of vertebrate urchin larvae is directed by the right glaucas in panel C was obtained with myogenic Bhlh gene duplication. Gene side. Development 128:4935–4948. 171:231–236. permission from James Adams. The Aird I. 1959. Conjoined twins. Br Med J Arend M, Weisenseel MH, Brummer M, image of the bilateral gynandromor- 1:1313–1315. et al. 2002. Seasonal changes of phy chicken in panel D is used by Al-Awqati Q, Vijayakumar S, Takito J, plasma membrane H(þ)-ATPase and et al. 1999. Terminal differentiation in permission of Michael Clinton. The endogenous ion current during cambial epithelia: the Hensin pathway in inter- growth in poplar plants. Plant Physiol image of a patient with CHILD syn- calated cells. Semin Nephrol 19:415– 129:1651–1663. drome and harlequin pigmentation 420. Arun CP. 2004. 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