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Cytoplasmic transport of the BBSome Primer PCM-1 Rab8GDP asymmetry

Rabin8 A. Richard Palmer Rab8GTP For decades morphological BBS8 BBS4 asymmetries have evoked curiosity BBIP10 Docking and fusion of vesicles and wonder (Figure 1). Although BBS1 with the base of the cilium BBS2 largely studied by natural history connoisseurs, many wonderful stories emerged: for instance, lopsided BBS7 BBS9 that lie on one side of their body and have both eyes on the BBS5 other; the narwhal’s spectacular, sinistrally- coiled and left-sided tusk; Leptin receptor Velella velella, the by-the-wind sailor that drifts on the ocean surface and Sensing of fat stores has right- and left-sailing forms; the and weight-lowering response Current Biology ability of oppositely coiled snails to mate — sometimes it’s easy and sometimes it’s not; male theridiid Figure 1. Molecular interactions of the BBSome. See text for details. spiders that rip off one palp and eat it, leaving only one for mating; male problems and defects in mucus What remains to be explored? fiddler crabs with a massive claw (up clearance that are characteristic of Nearly everything! What are the to 40% of body weight) that is used primary cilliary dyskinesia, a disorder membrane proteins that require for signaling and fighting. of motile cilia. the BBSome for their trafficking? Morphological asymmetry is Does the BBSome function only in one of those exceedingly rare Anything related to signaling? This trafficking to cilia or is it also involved characteristics of (and is one of the most exciting aspects of in IFT or trafficking out of cilia? protists and plants) that has evolved primary cilium biology and BBSome What is the molecular activity of the independently many times (Table 1). function. In the ear of bbs knockout BBSome? Does it have any enzymatic In a 1932 compilation not since animals, hair cells frequently fail to activity? What is the function of the equaled, Wilhelm Ludwig tallied align with one another, a characteristic BBS proteins that do not belong to all known examples and kinds of of defective planar cell polarity (PCP). the BBSome? In particular, does animal asymmetries: large, small, Although cilia are now known to be BBS3/Arl6 function in vesicular bilateral, helical, morphological essential for PCP in vertebrates, no trafficking? How and where is the and behavioral. But little general one knows the identity of the relevant BBSome assembled? Do the type II insight emerged from this Herculean signals sensed and transduced by chaperonin-like BBS proteins BBS6, exercise other than an attempt cilia. Do cilia sense a morphogen BBS10 and BBS12 play a role in to standardize terminology, some gradient that instructs polarity within the folding or assembly of BBSome speculations on common causes, the plane of the epithelium? Or are subunits? and a nearly 100-page screed on cilia facilitating planar cell polarization handed behavior in humans and established at cell–cell contacts by Where can I find out more? other primates, a subject that, Fliegauf, M., Benzing, T., and Omran, H. (2007). relaying a permissive signal? On the When cilia go bad: cilia defects and ciliopathies. astonishingly, remains poorly obesity front, bbs mutant mice are Nat. Rev. Mol. Cell. Biol. 8, 880–893. understood even today. Nachury, M.V., Loktev, A.V., Zhang, Q., Westlake, unable to transduce leptin signals in C.J., Peränen, J., Merdes, A., Slusarski, D.C., specialized hypothalamic neurons that Scheller, R.H., Bazan, J.F., et al. (2007). A core A simplified perspective control feeding behavior. Since the complex of BBS proteins cooperates with the on morphological asymmetry GTPase Rab8 to promote ciliary membrane leptin receptor was found to interact biogenesis. Cell 129, 1201–1213. Despite the great diversity of with a BBSome subunit, it has been Pazour, G.J., and Bloodgood, R.A. (2008). Targeting asymmetrical forms, a focus proteins to the ciliary membrane. Curr. Top. Dev. hypothesized that the leptin receptor Biol. 85, 115–149. solely on direction of asymmetry may get trafficked to cilia by the Seo, S., Guo, D.F., Bugge, K., Morgan, D.A., renders broad-scale comparative BBSome. However, to this date, no one Rahmouni, K., and Sheffield, V.C. (2009). studies of asymmetry variation Requirement of Bardet-Biedl syndrome proteins has succeeded in visualizing the leptin for leptin receptor signaling. Hum. Mol. Genet. tractable. This is because the receptor in cilia of the relevant neuronal 18, 1323–1331. development and evolution of a cell types. Nonetheless, the discovery simple and well- defined qualitative that IFT dysfunction also causes Department of Molecular and Cellular trait — direction of asymmetry — can Physiology, Stanford University School unregulated weight gains in mice of Medicine, 279 Campus Drive, Stanford, be easily compared among makes the ciliary hypothesis of leptin CA 94305-5345, USA. organisms with very different body signaling extremely appealing. *E-mail: [email protected] plans. If only direction is considered, Current Biology Vol 19 No 12 R474

two-allele polymorphism, a result made somewhat puzzling by the fact that either dextral or sinistral may be dominant. In flatfish, eye-side inheritance has only been studied in starry flounder, a rare polymorphic species whose eye-side frequencies depart significantly from random. Eye side is clearly heritable, but, curiously, only about 70%. Some odd observations in cultivated flatfish raise eyebrows even further. Despite the rarity of reversed individuals in nature, flatfish in cultivation exhibit up to 20% reversal, suggesting that eye- side determination remains sensitive to environmental effects. Another peculiar mode of inheritance involves an internal, anatomical asymmetry. Like all vertebrates, mice have an asymmetrical, left-sided heart. Curiously, heart side is random in iv mutant mice, regardless of the parents’ direction of asymmetry. Figure 1. Examples of morphological asymmetries in different animal groups. Therefore, unlike snails, the two (A) Bothus lunatus, a flatfish whose eyes lie on the left side of the head (photo by Arthur Anker). alleles are not for left and right, but (B) Amphidromus heerianus, a polymorphic snail having both dextral (left image) and sinistral (right image) forms (photo by Bill Frank). (C) Chama lazarus, a bivalve that cements one valve to for left and random. the substratum, in this case, the left one (photo by George Sangiouloglou). (D) Pandora inaequiv- alvis, a bivalve that lies horizontally near the sediment-water interface on either the right or left Random asymmetry: inheritance valve (photo with permission from www.femorale.com). (E) Torquirhynchia, a brachiopod where A more consistent, albeit surprising, the dorsal (upper) and ventral (lower) valves are raised on one side (right side of valve in this pattern of inheritance emerges from image) at random (reproduced with permission from Fürsich, F. T. and T. Palmer (1984). Commis- studies of random asymmetries sural asymmetry in brachiopods. Lethaia 17, 251–265. With permission from Wiley-Blackwell). (F) Verruca sp., a peculiar group of barnacles with only two movable opercular plates, the re- (Table 1, Figure 1). Because maining two (right or left at random) are fused into the rigid ring of lateral wall plates. (G) Circeis dextral and sinistral forms are amoricana, a coiled tube-building polychaete (Spirorbinae); tubes attach to the substratum along equally common in such species, the dorsal or ventral side so the worm’s body bends to the right or left (photo by Tara Macdon- tests for inheritance are easier to ald). (H) Spirographis, a feather-duster type polychaete worm with a coiled tentacle fan (photo conduct. Remarkably, among 13 by Cristophe Quintin). (I) Loxia leucoptera, a crossbill finch where the upper mandible crosses animal studies only one suggested randomly to the left or right of the lower one (to the left here) (photo by Frode Falkenberg, www. cyberbirding.no). (J) Neotrypaea californiensis, a large male thalassinid mud shrimp with a greatly that direction of asymmetry was enlarged right claw (photo by Greg Jensen). inherited, and doubts remain about that exception. Add to this even three types of conspicuous What role do genes and environment more results from plants — in 15 of asymmetry typically occur within play in the evolutionary origin of 16 cases direction is not inherited — species: dextral (all individuals morphological asymmetry (i.e., in and a broad generalization emerges. right- sided or dextrally coiled), the breaking of symmetry during With only one exception — albeit sinistral (all individuals left-sided development when morphological a highly informative one (see or sinistrally coiled) or random (half asymmetries first appear below) — direction of asymmetry of the individuals are right/dextral evolutionarily)? is not inherited in cases of random and half are left/sinistral; sometimes asymmetry. called antisymmetry). An even Fixed asymmetry: inheritance In cases of random asymmetry, simpler grouping is: fixed asymmetry Fixed asymmetries occur in many therefore, right-sided and left-sided (all individuals asymmetrical in groups (Figure 1, Table 1), and may are conspicuous phenotypic variants the same direction, regardless of be either right-sided (dextral) or that almost always lack a genetic direction) and random asymmetry. left-sided (sinistral). In most such basis. This lack of a heritable basis One or more of these kinds of species, reversed individuals occur to direction of asymmetry raises asymmetry occur in the external form occasionally. These variants permit some fascinating questions about of many animal groups (Table 1), as breeding studies to test whether how right and left forms develop. they do in internal organs. direction of asymmetry is controlled Perhaps direction is entirely Two questions emerge: of what by a few or many genes. The answer stochastic, or random influences significance is the observation that is mixed. In all snails studied so from the environment induce direction of asymmetry is fixed in far, coiling direction is inherited asymmetry in a particular direction some species and random in others? predominantly as a single-locus, in an individual. Magazine R475

Ontogeny of asymmetry Table 1. Selected examples of conspicuous external morphological asymmetries in animals.* Two examples of how morphological asymmetries develop, one fixed and Taxon (example) Asymmetric trait Asymmetry type** R L R+L one random, show how symmetry- breaking is coupled developmentally Mammals to other asymmetries, either in Cetacea (whales, dolphins) Dorsal skull midline deviation X the intracellular environment or Monodontidae (narwhal) Side of elongate tusk in male X in influences from the external Birds environment. Carduelinae (crossbill finch) Side of upper mandible tip X In some gastropods, the Charadriidae (wry-bill plover) Direction of bill twist X orientation of shell coiling may Strigiformes (owls) Larger/higher ear opening X X be traced back ontogenetically to Reptiles the orientation of cleavage planes Serpentes (snakes) Side of larger hemipene X in early spiral cleavage. In 1895, Fish Henry Crampton first noted that Pleuronectiformes (flatfish) Eye side XXX Phallostethidae Side of male clasper (priapium) X X X spiral cleavage orientation was Scale eating species of Cichlidae, Side of mouth deflection reversed in a sinistral gastropod, Triacanthodidae and Characidae X Physa heterostropha, compared Cephalochordates (lancelets) Side of larval mouth X to the more typical orientation in Hemichordates (acorn worms) Side of proboscis pore (if single) X the vastly more numerous dextral gastropods — and, indeed, in Echinodermata most other spirally cleaving Larvae of all classes (exc. crinoids) Side of juvenile rudiment X animals. In a classical study with Brachiopods † † the polymorphic freshwater snail Orthida and Rhynchonelloida Dorsalmost side of gape X Lymnaea peregra, Gary Freeman Bryozoa (moss animals) and Judith Lundelius confirmed Fenestrata† (Archimedes) Colony coiling direction X that coiling direction was inherited Gymnolaemata and Stenolaemata Colony coiling direction X predominantly as a single- locus Crustaceans two- allele polymorphism, with Cirripedia (verrucomorph barnacles) Side of lost lateral plates X dextral being dominant. They Copepoda (looking glass copepod) Side of ‘black organ’ + others X Ostracoda (seed shrimp) Side of larger valve XX also showed that embryos from Malacostraca- Decapoda genetically sinistral mothers Astacidea (clawed lobsters) Side of larger first claw X exhibited a reversed orientation Caridea (snapping and river shrimp) Side of larger first claw X of spiral cleavage compared to Thalassinidea (mud shrimp) Side of larger first claw XXX embryos from genetically dextral Brachyura (true crabs) Side of larger first claw XXX mothers (in snails, the asymmetry Anomura (hermit crabs) Side of larger first claw XX phenotype of the offspring reflects Insects the genotype of the mother). Orthoptera (katydids, crickets) Side of wing cover with file X X Therefore Crampton’s original Thysanoptera (thrips) Side of mandibular stylet X observations of differences among Coleoptera (carabid beetles) Side of notched mandible X X X species also apply to shell coiling Many insect orders Male genital asymmetry XXX direction within species. Most Chelicerates remarkable of all, motivated by a Arachnida (theridiid, pholcid spiders) Side of palp in male X hunch that the recessive sinistral Acari (feather mites) Side of elongated legs X allele was a loss-of-function Polychaete Annelids allele, Freeman and Lundelius Spirorbinae (coiled tube worms) Coiling direction of tube XXX transplanted egg cytoplasm from Serpulidae (calcerous tube worms) Side of opercular plug X fertilized eggs of dextral mothers Sabellidae (feather duster worms) Coiling direction of fan X into those of sinistral mothers and Cephalopods managed to reverse spiral cleavage Coleoidea (squids, octopus) Side of hectocotylus in male XX orientation. Some cytoplasmic (likely Ammonoidea† and Nautiloidea† Shell coiling direction XX cytoskeletal and chiral) component Bivalves in the egg is clearly responsible Anomiacea, Pectinacea Attached/smaller side X for orienting spiral cleavage and, Ostreacea, Chamacea, Hippuritacea Attached/larger side XXX ultimately, body asymmetry and shell Gastropods coiling. Regrettably, the identity of Prosobranchia (marine snails) Coiling direction of shell XXX this factor remains elusive. Pulmonata (land snails) Coiling direction of shell XXX American lobsters (Figure 2), much Hydrozoan Cnidarians appreciated as a culinary delicacy, Hydroida (by-the-wind-sailor) Direction of sail twist X are a textbook example of random Siphonophora (Port. man-o-war) Direction of sail twist X asymmetry. In large samples, half *For each taxon, an X indicates at least one example is known; this does not mean this form of asym- have the large crusher claw on the metry is representative of all species in that taxon. **R, right, dextral or clockwise; L, left, sinistral or left side and half on the right. Simple counterclockwise; R+L, random asymmetry (both R and L forms found commonly within species, yet elegant laboratory experiments typically in equal frequencies). †Extinct species only. Current Biology Vol 19 No 12 R476

phenotype already existed, a pattern seen in many taxa that include both random and fixed asymmetries. If direction of asymmetry is inherited in cases of fixed asymmetry but not inherited in cases of random asymmetry, two evolutionary scenarios are possible. If a species with fixed asymmetry evolved from a randomly asymmetrical ancestor then mutations that induce right- sidedness or left-sidedness most likely arose evolutionarily after the conspicuous morphological phenotypes right-sided (dextral) and left-sided (sinistral) already existed as a polymorphism. Alternatively, if a species with fixed asymmetry Figure 2. An anomalous two-toned American lobster. evolved directly from a symmetrical This Homarus americanus possesses asymmetrical claws (left side: crusher type, right side: ancestor, then mutations that induce cutter type), and a stunningly crisp midplane (background digitally removed). right-sidedness or left-sidedness likely initiated the morphological asymmetry. by the late C.K. Govind showed how how developmental mechanisms Take flatfish (Pleuronectiformes), differential claw use by juveniles, evolve. They address a question for instance: as adults, they lie which initially have symmetrical that might, at first glance, seem horizontally on one side of their claws, induces one to transform into a innocuous: Did animals with body, with both eyes facing upwards crusher claw. Young juveniles reared fixed morphological asymmetries on the other. Yet they begin life like through five molts with hard objects evolve directly from symmetrical any other planktonic fish larvae, to manipulate or with a second small ancestors or from ancestors that swimming upright with two eyes lobster for interaction develop the exhibited random asymmetry? symmetrically placed on opposite crusher claw at random on the right But this question actually sides of the head. Then, as they or left. Autotomy or denervation of the represents a fundamental one in approach the time to settle they right claw induces a crusher claw on evolutionary biology: which comes pass through an extraordinary the left. Preferential exercise of the first evolutionarily, mutations transformation. One eye actually left claw induces it to transform into that yield novel phenotypes, or migrates across the midline of the a crusher claw. Most amazing of all, novel phenotypes, followed later skull to lie wholly on the other side of without sufficient stimulation during by mutations that facilitate their the head. Only then, with both eyes the critical developmental window development? In other words, firmly ensconced on one side, do for symmetry breaking, neither claw from the perspective of left-right they settle into life on the bottom. becomes a crusher claw. Clearly, asymmetry, are mutations for The evolutionary history of the developmental program that rightness or leftness what generates (Figure 3) is at least as yields a crusher claw still requires an new right- and left-sided phenotypes, remarkable as their ontogeny. Two appropriate environmental trigger to or do new right- and left-sided of the three most ancient extant initiate it. phenotypes arise first, followed by lineages exhibit random eye-side This example raises an interesting mutations that stabilize development asymmetry. Add to this the recent question: does handed behavior of rightness and leftness? report of random asymmetry in induce or orient morphological A simple comparative test of early flatfishes, and it seems asymmetry during ontogeny? If these alternative modes of evolution very likely that random eye-side lateralized behaviors, such as is possible by way of a single asymmetry was the ancestral state. preferred use of the right or left assumption. In species that exhibit From random-eyed ancestors, both limb for feeding, are learned, random asymmetry, we assume right-eyed and left-eyed species and if differential use induces that direction of asymmetry is not arose independently at least twice, differential development of one inherited, at least in the absence of with occasional evolutionary side in the same way it enhances direct evidence for inheritance. This reversals of direction or reversions development of many structures, assumption seems safe because to polymorphism in both crown such learned behaviors might it has been verified in 28 of the clades (I and II). Eye-side direction is greatly facilitate both the ontogeny 29 cases examined (see above). clearly an evolutionarily labile trait. and evolution of morphological Moreover, the sole exception — style One startling conclusion emerges asymmetry. bending in enantiostylous flowers from this evolutionary history: of some monocot plants — actually genes directing larval flatfish to Evolution of asymmetry confirms that genetic control become right-eyed or left-eyed Phylogenetic studies of asymmetry of bending direction appeared likely arose evolutionarily after variation offer surprising insights into evolutionarily after the bent-style conspicuously right-eyed and Magazine R477

I II Of course, these puzzles apply to all cases where fixed asymmetries evolved from randomly asymmetrical

ancestors, or where direction of asymmetry changes evolutionarily. But that’s one reason the study of right-left asymmetry remains so Amphistium,PsettodidaeCitharidae HeteronectesTephrinectes (2) Scophthalmidae(7) †ParalichthyidaeBothidae (9) (145)(95)ParalichthodidaePoicilopsettidae (60)Rhombosoleidae (1)Achiropsettidae (30)Samaridae Achiridae(19) (4) (28) (31)Cynoglossidae (139) (145) fascinating.

Emerging generalities The flatfish example illustrates nicely how morphological asymmetries offer a rich buffet of puzzles about development, Eye side functional morphology, ecology and evolution. By studying the Random simple characteristic — direction + Polymorphic* of asymmetry — solutions to these Right ONLY puzzles can be compared among (2 spp.) groups with highly divergent body -10–40% reversal to R * Left ONLY plans, including protists, plants and Pleuronectidae (2 spp.) animals. Broad generalities about the -25–100% reversal to L Equivocal Current Biology interplay between development and evolution, and between genes and Figure 3. Evolutionary relations among living and fossil flatfish. environment, seem possible — if only Numbers of living species are given in parentheses. Two basal clades of living flatfish (Psetto- we take the time to look. didae, Tephrinectes), and both genera of extinct fossil flatfish (†), all exhibit random asymmetry (green), so random asymmetry was the ancestral state in the Pleuronectiformes. From this Acknowledgments ancestral state, right-eyed (red) and left-eyed (blue) descents evolved independently in both I gratefully acknowledge NSERC Canada the Citharidae and in the crown flatfish groups (I and II). Therefore, genes for direction of asym- for sustained and generous research metry (red and blue lineages, where eye-side is fixed) arose evolutionarily after right- and left- funding of my long-term research program eyed flatfish already existed but where eye-side was random and not inherited (green lineages). on biological asymmetries. Thanks to Eye-side reversals have also occurred evolutionarily at least three times (Pleuronectidae, Achi- ropsettidae, Cynoglossidae). ‘Polymorphic’ species differ from randomly asymmetrical species L. Hammond for valuable editorial advice, because their eye-side frequencies depart significantly from random. Inset: anterior views of and to those who gave permission to right-eyed (left) and left-eyed (right) individuals of the polymorphic flatfishPlatichthys stellatus; reproduce their pictures, as noted in the the anatomical dorsal side is up, but in life both individuals would lie horizontally with their legends to Figures 1 and 3. light-colored side on the substratum (photo by Carolyn Bergstrom).

Further reading Asami, T., Cowie, R.H., and Ohbayashi, K. (1998). Evolution of mirror images by left-eyed flatfish already existed. typically not inherited). So, fixed sexually asymmetric mating behavior Random eye side in the earliest asymmetries evolved almost as often in hermaphroditic snails. Am. Nat. 152, 225–236. flatfish very strongly suggests that via a phenotype-precedes-genotype Freeman, G., and Lundelius, J.W. (1982). The eye-side was determined either mode of evolution as via the more developmental genetics of dextrality and purely stochastically or by randomly conventional genotype-precedes- sinistrality in the gastropod Lymnaea peregra. Roux’s Arch. Dev. Biol. 191, 69–83. lateralized environmental cues. Only phenotype mode. Friedman, M. (2008). The evolutionary origin of later did genes arise that biased Despite their evident success flatfish asymmetry. Nature 454, 209–212. Govind, C.K. (1989). Asymmetry in lobster claws. eye-migration predictably towards (over 700 living species), and despite Am. Sci. 77, 468–474. a particular side of the head. In our progress in understanding their Huber, B.A., Sinclair, B.J., and Schmitt, M. (2007). other words, in flatfishes — a wholly evolutionary history (Figure 3), The evolution of asymmetric genitalia in spiders and insects. Biol. Rev. 82, 647–698. novel form of fish if there ever was one big question remains: what Ludwig, W. (1932). Das Rechts-Links Problem one — eye-side asymmetry appears possible advantage is there to im Tierreich und beim Menschen (Berlin: Springer). to exhibit a phenotype-precedes- having eyes on the right side of the Neville, A.C. (1976). Animal Asymmetry (London: genotype mode of evolution during head versus the left, or vice versa? Edward Arnold). the early radiation of the group. Eye side became genetically fixed Palmer, A.R. (2004). Symmetry breaking and the evolution of development. Science 306, When this logic is applied to presumably because individuals 828–833. many clades of animals, an even carrying genes for right-eyedness in Palmer, A.R. (2005). Antisymmetry. In Variation, B. Hallgrímsson and B.K. Hall, eds. (New more surprising result emerges: some lineages and left-eyedness in York: Elsevier), pp. 359–397. between one-third and one-half of other lineages somehow had higher Palmer, A.R. (2006). Caught right-handed. Nature the cases of fixed asymmetry (where fitness. But any invoked advantages 444, 689–691. genes play a role orienting the must account for how eye-side asymmetry in a particular direction) became fixed to one side from some Systematics and Evolution Group, Department of Biological Sciences, arose evolutionarily from ancestors random- eyed ancestor, and how University of Alberta, Edmonton, that exhibited random asymmetry right-eyed descendents evolved from Alberta T6G 2E9, Canada. (where direction of asymmetry is left- eyed ancestors, and vice versa. E-mail: [email protected]