EVOLUTION & DEVELOPMENT 9:5,416-431 (2007)

Brefeldin A and monensin inhibit the D quadrant organizer in the and columbiana

Eric E. Gonzales, =<''°'''''='<^ Maurijn van der Zee,^ Wim J.A.G. Dictus,^ and Jo van den Biggeiaar^ ^Department of Developmental Biology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands ''Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA '^Smithsonian Marine Station, Smithsonian Institution, 701 Seaway Drive, Fort Pierce, FL 34949, USA "^Center for Integrative Genomics, University of California at Berl

SUMMARY The D quadrant organizer is a developmental oweniid, Oweniia fusiformis. To interfere with cell signaling signaling center that is localized to the vegetal D quadrant in during the period predicted for organizer specification and different spiral-cleaving lophotrochozoan embryos and may patterning in A. vittata and S. columbiana, we use two general be homologous to axial organizing regions in other inhibitors of protein processing and secretion: Brefeldin metazoans. Patterning by this organizing center creates a A (BFA) and monensin. In A. vittata, we detail subsequent secondary developmental axis and is required for the embryonic and larval adult development and show that transition from spiral to bilateral cleavage and later treatment with either chemical results in radialization of the establishment of the adult body plan. Organizer specification embryo and subsequent body plan. Radialized larvae in equal-cleaving embryos is thought to involve inductive differentiate many larval and adult structures despite the interactions between opposing and vegetal loss of bilateral symmetry but do so in either a radially blastomeres. To date, experimental demonstration of this symmetric or four-fold radially symmetric fashion. Our results interaction has been limited to molluscs and nemerteans. suggest that the D quadrant organizer is functionally Here, we examine three families of equal-cleaving polychaete conserved in equal-cleaving , but that details of annelids for evidence of animal-vegetal contact. We find that its specification, induction, and patterning have diverged contact is present in the polynoid, Arctonoe vittata, but is relative to other spiral-cleaving phyla. absent in the serpulid, Serpula columbiana, and in the

INTRODUCTION Spemann's organizer, in the frog, the blastopore rim and adult hypostome in anemones and polyps, the cumulus, a Developmental organizers are signaling centers that pattern germ disk thickening, in spiders, and the vegetal D quadrant the surrounding cells or region. A developmental organizer in molluscs and other spiral-cleaving lophotrochozoans. Here, commonly patterns the early embryo through gastrulation for we refer to these and other similar taxon-speciñc organizers proper estabUshment of the adult body plan in diverse and collectively as axial organizers, i.e., developmental organizers distantly related animal phyla (Martindale 2005). These or- required for proper establishment of the primary body axis. ganizers were first identified through classical experiments and Although the homology of axial organizers and the body have since been shown to express similar sets of transcription plans they give rise to remain to be determined, functional factors and signaling pathway components (reviewed in Mar- and molecular genetic similarities in development suggest that tindale 2005; Lebreton and Jones 2006). Within taxa as di- the now distinct axial organizers of extant metazoans may vergent as cnidarians, molluscs, arthropods, and chordates, share a potentially ancient origin and that the ancestral or- the organizing regions are often localized, at least initially, to ganizer may have played a fundamental role in early body the vegetal pole. Although clear differences exist between plan evolution. Thus, the comparative characterization of clades, organizer loss or duplication results in a similar loss or axial organizers in a variety of metazoans offers potential new duplication of the primary body axis in each (Crampton 1896; insight as to the enigmatic origin and diversification of animal Wilson 1904; Browne 1909; Penners 1924; Spemann and body plans, particularly with respect to poorly understood, Mangold 1924; Tyler 1930; Holm 1952). Morphologically, but phylogenetically important, clades, including spiral-cleav- these organizing regions include the dorsal blastopore lip, or ing lophotrochozoans, such as anneUds.

416 e 2007 The Author(s) Journal compilation e 2007 Blackwell Publishing Ltd. Gonzales et al. Radialized development in annelids 417

Spiral-cleaving lophotrochozoans include annelids, mol- organizer-specific determinants are localized to the vegetal luscs, nemerteans, kamptozoans, and polyclad flatworms cortex (Clement 1968; Guerrier 1968). In many unequal- (Halanych 2004). Early development is characterized by spiral cleaving embryos, such as the gastropod mudsnail Ilyanassa cleavage, which, through its Stereotypie patterns of division obsoleta, the vegetal region is then repeatedly segregated to a and determinant segregation, creates a 4-fold radially sym- single-daughter blastomere through either unequal cleavage metric developmental architecture that is polarized along the or polar-lobe formation during early divisions, such that, with animal-vegetal axis, allowing individual blastomeres to be establishment of the embryonic four quadrants at the four-cell identified in a nomenclature of quadrants and quartets stage, a single lineage is already destined to give rise to the D (Wilson 1892; Sweet 1998; Lambert and Nagy 2002). Before quadrant organizer. How 3D's organizing function is later gastrulation, a signaling center localized to the vegetal D activated remains unclear in many unequal-cleaving embryos quadrant, commonly known as the D quadrant organizer, but must occur before the transition from spiral to bilateral patterns the early embryo, thereby establishing a secondary cleavage, as this transition is dependent upon patterning by developmental axis (Crampton 1896; Clement 1962). As the organizing center (Clement 1962; Lambert and Nagy shown by patterns of division (van den Biggelaar 1977), 2001). In equal-cleaving embryos, such as the gastropod lim- cell lineage contribution (Dictus and Damen 1997), and gene pet Patella vulgata and the pond snail Lymnaea stagnalis, the expression (Lartillot et al. 2002b) in gastropod molluscs, or- vegetal region is equally partitioned during cleavage and the ganizer patterning transforms the developmental architecture, vegetal lineages of all four quadrants remain equivalent, ex- such that it becomes bilaterally symmetric and is hibiting the same developmental potential to act as the D polarized along two perpendicular axes: the primary ani- quadrant organizer until midway through the sixth cleavage mal-vegetal axis and the secondary organizer-dependent axis. (van den Biggelaar and Guerrier 1979; Arnolds et al. 1983; The nature of this secondary axis is poorly understood. It is Freeman and Lundelius 1992). In this case, organizer spec- commonly referred to as either the anteroposterior or dorso- ification involves selecting one of four equivalent 3Q vegetal ventral axis, with the D quadrant or D quadrant midline as blastomeres to take on the organizing role of 3D (van den posterior or dorsal, respectively. However, cell lineage studies Biggelaar 1977). This is achieved through contact-dependent, indicate that blastomere contributions at the time of pattern- inductive interactions between cells in the animal and vegetal ing are neither axis nor structure specific and that the adult poles (van den Biggelaar and Guerrier 1979). Specifically, the axes arise later in development after gastrulation (Dictus and animal Iq" and vegetal 3Q blastomere quartets extend into Damen 1997). In molluscs, annelids, and nemerteans, exper- the cleavage cavity at the 32-cell stage and establish contact imental loss or duplication of the vegetal D quadrant results with one another (van den Biggelaar 1977). Following con- in the subsequent loss or duplication of bilateral symmetry in tact, one of four vegetal blastomeres becomes highly central- the embryo and later body plan, i.e., radialization and twin- ized within the cleavage cavity, occluding its counterparts ning (Crampton 1896; Wilson 1904; Tyler 1930; Clement from their animal contacts, and establishes contact with near- 1952; Render 1983; Henry and Martindale 1987; Henry 2002). ly all other blastomeres (van den Biggelaar 1977). Experi- Thus, the D quadrant organizer is an axial organizer. In mental inhibition of animal-vegetal extension and contact, molluscs and nemerteans, the blastomere 3D functions as including the use of monensin, a potent Na(-I-) ionophore, the organizer (Crampton 1896; Clement 1962; Wilson 1904; and Brefeldin A (BFA), an inhibitor of protein translocation Henry 2002). In contrast to axial organizers in other meta- in the Golgi, results in the radialization of subsequent devel- zoans, the D quadrant organizer acts only as a transient sig- opment (Martindale et al. 1985; Kuhtreiber et al. 1988; Go- naling center and is not maintained during gastrulation nzales et al. 2007). In particular, a single 3Q blastomere is not (Clement 1962). Still, in various molluscs and annelids, a centralized, the transition from spiral to bilateral cleavage number of genes characteristic of, although not specific to, does not occur, and the subsequent body plan is radially or axial organizers in other phyla are expressed during gas- 4-fold radially symmetric, similar to lobeless development in trulation within midline lineages whose origin is directly unequal-cleaving embryos (Martindale et al. 1985; Kuhtreiber dependent upon patterning by the D quadrant organizer et al. 1988; Gonzales et al. 2007). Despite noted differences in (Arendt et al. 2001; Nederbragt et al. 2002; Lartillot et al. organizer specification, both equal- and unequal-cleaving em- 2002a, b). Overall, classical experimental and recent molecular bryos activate Mitogen-Activated Protein Kinase (MAPK) in studies collectively suggest that the D quadrant organizer in 3D just before organizer patterning, and both are similarly spiral-cleaving lophotrochozoans is potentially homologous radialized by treatment with an inhibitor of MAPK activa- at some level to axial-organizing regions in other metazoans. tion, U0126 (Lambert and Nagy 2001; 2003; Lartillot et al. Specification of the D quadrant organizer is often distinct 2002b), suggesting that the divergent specification systems in even closely related species, particularly in equal- versus converge onto a pathway or network before patterning. In unequal-cleaving embryos (Freeman and Lundelius 1992; addition, variations and intermediate forms of the two sys- van den Biggelaar and Haszprunar 1996). In general. tems clearly exist, for instance, the slipper snail Crepdiula 418 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007 fomicata, where cleavage is unequal with a polar-lobe but To test for the D quadrant organizer in equal-cleaving animal-vegetal contact is required for organizer specification polychaetes, we used the chemicals BFA and monensin, two and where 4d, rather than 3D, may function as the organizing small organic molecules that rapidly and reversibly inhibit center (Henry 2006). protein processing and secretion in eukaryotes through dis- To determine whether 3D's role as the D quadrant orga- tinct mechanisms (Mollenhauer et al. 1990; Dinter and Berger nizer in gastropods is representative of spiral-cleaving lop- 1998; Mayer 2003). As demonstrated in P. vulgata and other hotrochozoan development in general, it is necessary to test molluscs (Kuhtreiber et al. 1988; Gonzales et al. 2007), both for the organizer in other phyla, including annelids. Annelids chemicals show great promise as simple yet effective exper- are a paraphyletic clade of segmented, or secondarily non- imental tools with which to inhibit the inductive interactions segmented, worms (Halanych 2004). The group is composed underlying organizer specification in species where more spe- of some 80 families split into three large groups: the cific molecule genetic tools have not yet been developed. We polychaetes, oligochaetes, and clitellates. Monophyly and initially looked for evidence of animal-vegetal contact in three phyletic relations within and between the different groups are families of equal-cleaving polychaetes: the polynoid, Arctonoe unresolved (Halanych 2004). However, equal-cleaving poly- vittata, the oweniid, Oweniia fusiformis and the serpulid, chaetes may retain many ancestral features (Nielsen 2004). Serpula columbiana. We then tested for the presence and Development in polychaetes is described in classical studies function of the organizing center in A. vittata and S. colum- (Wilson 1892; Mead 1897; Treadwell 1901) and is reviewed biana by using BFA or monensin to interfere with cell sig- for the Nereid, Platynereis dumerilii (Fischer and Dorresteijn naling during the interval predicted for organizer specification 2004). The presence of the D quadrant organizer in and patterning, i.e., after fifth cleavage but before the onset of polychaetes is suggested by a number of different studies. bilateral cleavage. We examined the effect of treatment on The organizer-dependent transition to bilateral cleavage was subsequent development, focusing on A. vittata in particular. first recognized in spiral development in the nereid, Neanthes Our results demonstrate that a functional homolog of the D (formerly Nereis), and is classically detailed for a number of quadrant organizer is present in equal-cleaving polychaetes, equal-cleaving families, including the polynoid, Lepidonotus, although its blastomere identity remains unknown. the hesionid, Podarke, and the echiuran, Thalassema (Wilson 1892; Mead 1897; Treadwell 1901; Torrey 1903). In contrast to molluscs, this transition in polychaetes occurs after the METHODS onset of the sixth cleavage in the animal Iq" and vegetal 3Q quartets, rather than before it (Freeman and Lundelius 1992). Collection and culturing Despite this difference, experimental work in polar lobe- A. vittata, O. fusiformis, and S. columbiana adults were collected forming species indicates that the vegetal D quadrant func- from intertidal localities on San Juan Island, Washington between 1996 and 2004. A. vittata spawned after removal from its host, the tions as an organizing center. In the polar lobe-forming keyhole limpet Diadora áspera, whereas O. fusiformis and S. co- sabellarid, Sabellaria cementarium, removal and suppression lumbiana spawned upon removal from their tubes. Successful of the polar lobe at the first cleavage, which normally locaUzes A. vittata fertilizations exhibited >90% synchrony within 5 min to the vegetal region of the D blastomere, results in the of first and second cleavage. Developmental synchrony of respective loss and twinning of bilateral symmetry in the em- O. fusiformis and S. columbiana embryos was uncharacterized, or bryo and body plan (Hatt 1932; NovikoflF 1938; Render 1983). was variable and <90% within 5 min. Embryos and larvae were In the equal-cleaving polynoid, Lepidonotus sp., animal-veg- cultured in 0.45 |iM filtered seawater (FSW) at ambient sea table etal contact occurs before the transition to bilateral cleavage temperatures (10-12°C). (Mead 1897) and is suggestive of organizer specification. However, in contrast to equal-cleaving molluscs and nemerte- Treatment with BFA and monensin ans, contact in Lepidonotus is between the animal Iq'^^ and One millimolar BFA (Sigma, St. Louis, MO, USA) and monensin 4Q sixth-cleavage quartets, rather than the Iq^^ and 3Q fifth- (Sigma) stocks were prepared in 100% ethanol and stored for up to cleavage quartets (Freeman and Lundelius 1992). In the 6 months at • 20°C. Based on previous BFA and monensin dose- equal-cleaving serpulid Hydroides, MAPK is activated in response experiments in the gastropod mollusc P. vulgata (Ku- the sixth cleavage 4d mesentoblast, a pattern that may be htreiber et al. 1988) (per obs), A. vittatae and S. columbiana em- bryos were incubated for 1-5 h in freshly prepared 1 |iM BFA or homologous to its organizer-related activation in the fifth- monensin FSW solutions. Embryos were then washed five times cleavage 3D blastomere in molluscs (Lambert and Nagy with FSW after treatment. To test for the stage-specific specificity 2003). Overall, characteristic elements of D quadrant orga- of treatment relative to organizer specification versus patterning nizer specification and patterning in molluscs are present in in A. vittata, BFA and monensin treatments were initiated either annelids (Freeman and Lundelius 1992; Lambert and Nagy before or else after the onset of bilateral cleavage. Treatments in 2003) but the presence and function of the organizing center S. columbiana were initiated only before the onset of bilateral remain untested in equal-cleaving polychaetes. cleavage. To show that observed experimental phenotypes are Gonzales et al. Radialized development in annelids 419 specific to either BFA or monensin and not to the solute EtOH, mental series for Ught microscopy, the series extending from control experiments of 0.001% EtOH treatment were made in fertilization through the onset of gastrulation, in A. vittata, O. A. vit tata. To score treatments in all species, experiments of tens of fusiformis, and S. columbiana. In A. vittata, animal Iq'" and thousands of embryos were initially surveyed on a dissecting scope. Iq"^ and vegetal 4q and 4Q quartets extended into the cleav- Subsamples of 50-100 larvae were then examined at higher mag- age cavity following their formation at the 44-cell stage and nifications on dissecting or compound microscopes and scored as established contact by the 56-cell stage, some 2 h after the 32- nornial, radialized, abnormal, or other. cell stage onset and before the transition to bilateral cleavage (Figs. lA, 2, and 4, A-E). In contrast, the same blastomeres in Embryonic and larval analysis O. fusiformis and S. columbiana exhibited only a slight exten- To assess animal-vegetal contact in A. vittata, O. fusiformis, and S. sion into the cleavage cavity. In these embryos, the cleavage columbiana, and cleavage patterns in A. vittata, embryos were fixed cavity was still present around the 128-cell stage, some 4-5 h every 20-30 min through early gastrulation and prepared for whole-mount or semithin sectioning according to Kuhtreiber et al. after onset of the 32-cell stage (Fig. 1, B and C). In O. fu- (1988). For each stage, 20-30, or more, embryos were examined siformis, a more general animal-vegetal contact was observed and sketched, and of these, a selected few were either photographed several hours later but appeared to be a feature of gastrulat- or else drawn using camera lucida and the drawings were then ion. S. columbiana was not examined at these later stages. scanned and traced using Adobe Illustrator and Photoshop. In A. vittata, all cells and patterns of cleavage (spiral vs. bilateral) were identified for each set of divisions through the 64-cell stage; how- Development in A. vittata ever, for later stages, only landmark cells and those preparing to To design experiments to test for the D quadrant organizer in divide were identified. In O. fusiformis and S. columbiana, only A. vittata, we first made a detailed description of early de- blastomeres of the more animal and vegetal quartets were fully velopment. Proceeding from fertilization, the primary features identified and the transition to bilateral cleavage was not charac- of early development were spiral cleavage, a transition to bi- terized. Overall, developmental rates (tiiTiing of cell divisions after lateral cleavage, gastrulation in the larval trochophore, and fertilization and the onset of ciliation and swimming) in all three initial estabhshment of the adult body plan in the larval species were nearly identical, i.e., within 20-30 min or less of one presetiger, i.e., estabhshment of adult axes and analagen but another. To assess larval morphology in A. vittata, larvae were limited adult structures. photographed live, or fixed and analyzed by scanning electron mi- In the embryo, we focused on the presence and timing of croscopy according to Kuhtreiber et al. (1988). To assess muscle differentiation in A. vittata, larvae were fixed and stained with developmental features characteristic of organizer specifica- Phalloidin (Molecular Probes, Carlsbad, CA, USA) for confocal tion and patterning in equal-cleaving molluscs and nemerte- analysis according to Wanninger and Haszprunar (2002). A min- ans. These features included animal-vegetal interactions imum of 20 larvae were examined per stage. (specification) and the transition from spiral to bilateral cleavage (patterning). The division chronology of early cleav- age in A. vittata is previously undescribed and is summarized RESULTS in Fig. 2. Divisions were synchronous within, but not neces- sarily between, blastomere quartets along the animal-vegetal An I ma!-vegetal contact in equal-cleaving axis (Fig. 2). Sets of divisions occurring in rapid succession polychaetes exhibited a shght variability with regard to the sequence of To survey initially for animal-vegetal contact in different division between embryos. The fifth cell-cycle was the last families of equal-cleaving polychaetes, we fixed a develop- cycle to be fully completed before the onset of the next. It

Fig. 1. Animal-vegetal contact is present in Arctonoe vittata but absent in Oweniia fusiformis and Serpula columbiana. (A) shows contact between the Iq and 4Q quartets at 9 h in A. vittatae (lateral). (B) and (C) show the absence of contact at 11 h in O. fusiformis and in S. columbiana, respectively (lateral and lateral). Scale bar • 20 |im. c, contact; cc, cleavage cavity. 420 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007

Contact Cells 16 20 28 32 40 44 56 60 68 71 75 79 84 92

Quartets

Animal 1qlll Iqiii iq 11 1121 iq 1q1121 1q112 1qll221 1q11221 iq 1122 11222 1q1 iq 1qll222 iq 1211 1ql2l 1q1211 1q1212 iq 1ql2 1q1212 1q122 1q122 1q211 1q2l 1q211 1q2l2 1q2 1q212 1q22l 1q22 1q221 1q222 1q222 2q 111 2q 11 2q111 Q 2q 112 2q1 2q112 2q 12 2q12 2q 2d 211 2q 21 2d2ll 2d 212 2q2 2d212 2a-c22i 2q 22 2a-c22l 2a-c222 IQ 2a-c222 3q 11 3q1 3q11 3q 12 3q 3q12 3c-d 21 3a-b2i 3q2 3q2l 2Q 3c-d 22 3a-b22 3q22 4q 3Q 4q 4Q 4Q Vegetal

Fig. 2. Arctonoe vittata undergoes a developmental transition from spiral to bilateral cleavage. The division chronology of early devel- opment in A. vittatae shows the developmental tiiTiing of animal-vegetal contact and the subsequent transition to bilateral cleavage. concluded with the division of the 2q quartet at the 32-cell Newly formed animal Iq'" and Iq"^ and vegetal 4q and 4Q stage (Fig. 2). A subsequent resting stage, which occurs in blastomere quartets extended into the cleavage cavity at the equal-cleaving gastropods, was absent and the sixth cell cycle 44-cell stage and established contact with one another around was initiated in the animal Iq" blastomeres and vegetal 3Q the 56-cell stage (Figs. 2 and 4, B-E). Contact occurred spe- blastomeres (Fig. 2). Cell divisions remained spiral until mid- cifically between the animal-pole Iq'" and vegetal-pole 4Q way through the sixth cell cycle, when a localized transition to quartets and between the more peripheral animal Iq"^ and bilateral cleavage occurred in blastomeres along the D quad- vegetal 4q quartets (Fig. 4, B-E). Both sets of quartets main- rant midline (Figs. 2 and 4, F and G). A cleavage cavity was tained contact through the onset of bilateral cleavage and into present in the embryo beginning at the 32-cell stage (Fig. 4A). later development (Fig. 4, B-E). Centralization of a single Gonzales et al. Radialized development in annelids 421

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Fig. 3. Brefeldin A (BFA) and monensin treatment before bilateral cleavage results in radial development in Arctonoe vittata and Serpula columbiana. (A) shows a schematic of the developmental timing of BFA and monensin treatments and shows that treatments initiated after onset of the 32-cell stage result in radial development in A. vittata and S. columbiana, whereas treatments initiated after the onset of bilateral symmetry result in nornial development in A. vittata (S. columbiana was not tested). (B) shows a bar chart showing the number of normal versus radialized embryos resulting from 32-ceII onset BFA treatments made at different concentrations and shows that concentrations of 1 \iM radialized development, whereas concentration of 0.1 |iM had no discernable effect. (C) and (D) show normal and monensin-radialized 8-day A. vittatae presetigers. (C) shows normal development with the body plan symmetrically organized about the adult anteroposterior and dorsoventral axes (posterior). (D) shows monensin-radialized development with the body plan radially organized about a novel aboral- oral axis (lateral). (E-H) show normal and monensin-radialized gallocyanin-stained 7-day S. columbiana presetigers. (E and F) show normal development with the body plan symmetrically organized about the adult anteroposterior and dorsoventral axes (lateral and posterior). (G and H) show monensin-radialized development with the body plan radially organized about a novel aboral-oral axis (lateral and oral). Scale bar • 10|im. a, anus; ab, aboral; an, anterior; d, dorsal; e, extruded material; es, eyespot; fg, foregut; g, uncharacterized gut; hg, hindgut; o, oral; po, posterior; pr, prototroch; s, stomodeum; si, stomodeum-like opening; st, stomach; v, ventral. 422 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007 blastomere did not occur before the onset of bilateral primary trochoblasts, which were arranged in a 4-fold radially cleavage, or in later stages (Fig. 4E). After contact, divisions symmetric pattern of four clusters of four cells around the became increasingly asynchronous both between and middle of the embryo, and of the nonmotile apical tuft, which within quartets (Fig. 4, F and G). Patterns of division was located at the animal pole, began at around the 68-cell asynchrony within quartets were bilaterally symmetric about stage (Fig. 4, B-D and K). and polarized along the B-D quadrant midline (Fig. 4, F We next examined larval development, focusing on mor- and G). phological features that characterized the developing body The transition to bilateral cleavage was initiated at the 56- plan of the gastrulating trochophore and subsequent prese- cell stage, shortly after animal-vegetal contact (Figs. 2 and 4). tiger. A distinct blastopore was not observed in the embryo Although not all divisions contributing to this transition were through the Ill-cell stage (data not shown); however, a gen- true bilateral cleavages, i.e., divisions in which the cleavage eral ingression of cells at the vegetal pole was later observed in spindle is oriented with respect to a plane of bilateral sym- the 18-h trochophore (Fig. 5D). The blastopore became slit- metry, division asynchronies and asymmetries within quartets like and appeared to give rise to both the stomodeum and were bilaterally symmetric with respect to the B-D quadrant anus through convergence of the lateral lips by 27 h (Fig. 5, midline and created a more global pattern of bilateral sym- A-C). However, it is possible that either the stomodeum, metry in the embryo. The transition to bilateral cleavage was anus, or both formed as secondary invaginations unrelated to first evident during the sixth division of the 2q^ quartet the blastopore during the interval between 18 and 27 h. The (Fig. 4, F and G). Blastomere 2d^ was bisected by the D stomodeum and anus formed opposing invaginations at either quadrant midline and future plane of bilateral symmetry, but end of what had been the vegetal pole and were bisected by divided spirally, producing a large 2d^' blastomere and a the B-D quadrant, or now bilateral, midline (Fig. 5, C and E). small 2d^^ blastomere (Fig. 4G). Equivalent 2q^ blastomeres The apical tuft was present at the anterior tip of the pretroc- outside the D quadrant midline also divided spirally, but with hal episphere in 12- to 27-h trochophores, whereas paired a reversal in the size of daughter blastomeres, producing small meniscotrochal cilia lay to either side of the anterior ventral 2a^'-c^' blastomeres and large 2a^^-c^^ blastomeres (Fig. 4, F midline (Fig. 5D). The four sets of primary trochoblasts and G). At the seventh division, patterns of asynchrony with- shifted from their initially opposing positions at the midpoint in the 2q^ daughter blastomeres were bilaterally symmetric of the embryo to form a ring of beating cilia, the prototroch. with respect to the B-D quadrant midline, with 2d^' dividing The prototroch gradually became the base of the larva over before 2a' -c and 2d' dividing after 2a '-c (Fig. 4, F and the course of vegetal ingression and gastrulation (Fig. 5D). G). In the third quartet, the sixth division of 3q occurred Overt morphogenetic movements appeared to be localized before animal-vegetal contact and was spiral with radial to the posterior hyposphere, i.e., posttrochal or future trunk sjonmetry (Fig. 2). The seventh division of 3q^ occurred after region. Here, the initial stomodeal invagination migrated, contact and was a mixture of bilateral and spiral cleavages perhaps through ingression of B quadrant midline lineages, with bilateral symmetry. Blastomeres 3c^ and 3d^ lay to either along the B quadrant midline to a more anteroventral posi- side of the D quadrant midline and divided in mirror image, tion just beneath the prototroch and gave rise to the whereas 3a^ and 3b^ lay to either side of the opposing B stomodeum proper and foregut in the presetiger (Fig. 5, quadrant midline and divided slightly later with spiral cleav- C-G). In the presetiger, the posterior hyposphere extended age (Fig. 4G). Presumably, the 4d mesentoblast underwent its perpendicularly from the prototroch along the forming ante- characteristic bilateral division across the plane of symmetry, roposterior axis of the trunk (Fig. 5, D and F). Owing to this but this division was not observed through the 93-cell stage elongation, the prototroch now occupied a more anterior po- (Fig. 2). The 4D blastomere was equally likely to be a cross- sition along the body axis. In addition, the stomodeum and furrow or noncross-furrow blastomere in different embryos. foregut became increasingly ciliated with short motile cilia Overall, novel asymmetries and asynchronies were observed (Fig. 5, E and G). The stomodeum was bent midway at a within quartets relative to the D quadrant midline following right angle and was largely bilaterally symmetric (Fig. 5, F animal-vegetal contact. These asymmetries and asynchronies and G), but had an asymmetrically placed oral brush of cilia suggested the underlying formation of a secondary develop- (used in food capture) just outside it (not pictured). A short, mental axis and transformed the developmental architecture narrow band of cilia, the neurotroch, formed along the ven- from 4-fold radial to bilateral symmetry (Fig. 2). In the pre- tral midline between the mouth and anus (Fig. 5, F and G). trochal region, divisions remained synchronous and spiral Paired larval lateral glands formed laterally in the episphere through the 93-cell stage; however, in contrast to the orien- or head region (Fig. 6, A and B). Larvae exhibited muscular tation predicted by spiral cleavage, seventh division of the contractions and Phalloidin labeling of f-actin revealed a bi- Iq"^ quartet, i.e., the quartet that previously established laterally symmetric, intricate series of circular muscle bands contact with the 4q blastomeres, was not dexiotropic but around the stomodeum and foregut, with muscular connec- either laeotropic or, perhaps, radial (Fig. 4K). Ciliation of the tions running up into the head region and to a circular muscle Radialized development in annelids 423

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K 1q''''2i 1^ ^ 6th cleavage

-|qii22 ^ 1^ 7th cleavage 1q121 n 1q122 •

Fig. 4. Monensin-radialized Arctonoe vittata embryos establish animal-vegetal contact but do not make the transition to bilateral cleavage patterns. (A-G) show normal development. A shows initiation of animal-vegetal extension into the cleavage cavity (lateral). (B-E) show contact between the central Iq'" and 4Q quartets and between the peripheral Iq"^ and 4q quartets (animal midsection +3, 0,-3 jim, and lateral). (F) and (G) show bilateral cleavage in sixth division asymmetry of 2q^ and seventh division asynchrony of 2q^' and 3q, respectively (animal and vegetal). (H-J) show monensin-radialized development. (H) shows contact between the central Iq'" and 4Q quartets (lateral). (I and J) show spiral cleavage with 4-fold radial symmetry in the 2q^ and 3q quartets, respectively (animal and vegetal). Brefeldin A- radialized embryos exhibited identical patterns of contact and division. (K) and (L) show the laeotropic, rather than dexiotropic, seventh division of the Iq"^ and its similar orientation to the sixth division in the Iq'^ quartet. (K) shows a 68-cell control (animal). (L) shows a 68- cell monensin-radialized embryo (animal). Semithin sections, (A-F), (H), (I), (K), (L); Whole-mounts, (G), (J); *dividing cells. 424 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007

Fig. 5. Brefeldin A (BFA)-radialized larvae undergo radial patterns of gastrulation to form a radially symmetric mouth at the oral-pole. (A- K) show scanning electron micrographs of normal and BFA-radialized development. (A-G) show the normal transformation of the blastopore into mouth and anus in the trochophore and presetiger. (A) shows the blastopore as a slightly oblong invagination at the vegetal pole in an 18-h trochophore (vegetal). (B) and (C) show the apparent convergence of the lateral lips to form the stomodeum and anus in 27- and 40-h trochophores (ventral). (D) and (E) show 2-day trochophores with fully formed mouth and anus at opposing ends of the posterior pole and bisected by the plane of bilateral symmetry (lateral and ventral). (F) and (G) show bending of the mouth by 90° and ciliary differentiation of the mouth and neurotroch in 4-day presetigers (lateral and posterior). (H-K) show the radial transformation of the blastopore into a radial mouth at the oral pole in BFA-radialized trochophores and presetigers. (H) and (I) show stomodeal-like inv- aginations formed at 90° to one another around a central disk, or, perhaps, invagination, in 2-day trochophores. (J) and (K) show a single circular ciliated oral field with a single stomodeal-like opening in 4-day presetiger. Scale bar = lOjim; a, anus; at, apical tuft; bl, blastopore; d, central disk; 11, blastopore lateral lips; in, stomodeal-like invagination; lg, lateral glands; m, meniscotrochal cilia; n, neurotroch; pr, prototroch; s, stomodeum; si, stomodeal-like opening.

band beneath the prototroch (Fig. 6E). Paired eyespots were and through the onset of bilateral cleavage (Fig. 3A). This present at 8 days (Fig. 3C). interval includes the period during which organizer specifica- tion and patterning is predicted to occur. Following highly synchronous fertilizations, a 1.5-3.5-h treatment with a min- BFA and monensin treatment in A. vittata imum concentration of 1 |íM BFA initiated at timepoints In a preliminary investigation of the effect of BFA and within 90 min after the onset of fifth cleavage resulted in monensin treatments on development in A. vittata, embryos nearly 100% radialized development in thousands of embryos were incubated in either drug beginning at the 32-cell stage (Fig. 3, A and B). Consequently, there was a loss of bilateral Gonzales et al. Radialized development in annelids 425

Fig. 6. Radial larvae differentiate larval adult structures in a radial pattern. (A-D) show scanning electron inicrographs of normal and monensin-radialized development. (A) and (B) show 2-day nornial trochophores with paired larval lateral glands at each side of the episphere and paired meniscotrochal cilia at either side of the anterior-ventral midline (anterior-lateral). (C) and (D) show 2-day Brefeldin A (BFA)-radialized trochophores with four sets of lateral glands and four sets of menisco trochal cilia and members of each set at 90° to one another and the two quartet sets offset from each other by 45° (aboral-lateral). (E-G) show confocal projects of muscle differentiation (Phalloidin staining) in 4-day normal and radialized trochophores. (E) shows a bilaterally symmetric musculature with circular bands around the stomodeum and esophagus (posterior). (F) and (G) show radial rings of muscle organized about the aboral-oral axis in monensin- and BFA-radialized trochophores, respectively (oral). Lateral glands and meniscotrochal cilia were not characterized in monen- sin-radialized larvae. Scale bar= 10|im; ar, apical ridge cilia; Ig, lateral glands; m, meniscotrochal cilia; pr, prototroch; s, stomodeum; si, stomodeal-like opening. symmetry in the embryo and subsequent differentiation of ment of monensin- and BFA-radialized embryos and larvae. larval and adult structures about a novel aboral-oral axis in a In radialized embryos, animal-vegetal extension of the Iq'", radial or 4-fold radial symmetric pattern (Fig. 3D; Table 1). Iq"^, 4q, and 4Q quartets still occurred during the period Treatments at lower concentrations (0.5-0.01 |íM) resulted in of treatment (Fig. 4H). However, in contrast to normal normal development, similar to that observed in controls (Fig. development, subsequent patterns of division within quartets 3, A and B; Table 1). Treatments made after the onset of remained synchronous and spiral, with a 4-fold radial bilateral cleavage also had no effect on development (Fig. symmetry throughout the embryo. This radiaUzation of the 3A). Identical treatments using monensin, rather than BFA, embryo represented a loss of divisions specific to the D quad- had a similar effect on embryonic and larval development. rant midline (Fig. 4, I and J). Division of the 2q^ quartet Treatments made with working concentrations of EtOH, resulted in small 2q^' blastomeres and large 2q^^ blastomeres which is used as a solvent in BFA and monensin stocks, had (Fig. 41), identical to 2a^-2c^ divisions in controls. Similarly, no effect on development (data not shown). Thus, BFA and the seventh division of the 2q^^ and 2q^' quartets remained monensin treatments speciñc to the interval predicted for or- spiral and synchronous within each quartet (data not shown). ganizer specification and patterning resulted in radialization The seventh division of the 3q' quartet was also spiral and of the embryo and body plan, suggesting the presence and synchronous, identical to 3a^-3b^ divisions in controls (Fig. experimental loss of the D quadrant organizer in A. vit tata. 4J). The seventh division of the 4q quartet did not occur before the 92-cell stage. In the pretrochal region, seventh di- Radialized development in A. vittata vision of the Iq"^ quartet was either laeotropic or possibly To further explore the apparent loss of patterning by the radial (i.e., spindles aUgned parallel to the animal-vegetal ax- organizing center in A. vittata, we characterized the develop- is), a pattern identical to controls (Fig. 4L). Formation of the 426 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007

Table 1. Scoring normal and radialized development in A. vittata, S. columhiana, and O. fusiformis

Taxon Character change(s) Sarcopterygii 200." Metapterygial segmentation: absent (0) -^ present (1). This change is optimized to the sarcopterygian stem group below the divergence of the extended 'onychodont' clade comprising Onychodus, Strunius, Achoania, and Psarolepis. 207. Entepicondyle (posterior process from most proximal mesomere): absent (0) -^ present (1). This change is optimized to the sarcopterygian stem group crownward of Onychodus, which lacks an entepicondyle (Andrews et al. 2006). Latimeria 196. Pectoral fin (A: 6): unrotated (0) -^ rotated (1). Although the state for this character cannot be assessed for Shoshonia, the pectoral fin appears unrotated in Miguashaia. If Shoshonia is placed below or as sister to Miguashaia, then pectoral fin rotation would have evolved on the branch leading to Latimeria in Fig. 3. Regardless of the condition in Shoshonia, the cladistic solution presented here indicates that fin rotation is homoplastic between lungfishes and coelacanths. Pectoral fin web: asymmetrical (0) -^ symmetrical (1). 202. Pectoral radiais: all bear fin rays or lepidotrichia (0) -^ some 'naked' (1). Tetrapodmorpha 112. Deltoid and supinator processes (F: 114): absent (0) -^ present (1). Sauripterus 201. Pectoral radiais: do not bifurcate (0) -^ bifurcate (1). 204. Ball-shaped caput humeri (JA: 15): absent (0) -^ present (1). 212. Distal fin or limb domain expanded across A-P axis (C: 1): no (0) -^ yes (1). Tiktaalik and higher 114. Pectoral fin radiais (F: 116): unjointed (0) -^ jointed (1). tetrapodomorphs 115. Pectoral fin radiais (F: 117): 'preaxial' radiais only (0) -^ 'preaxial' plus 'postaxial' radiais (1). 201. Pectoral radiais: do not bifurcate (0) -^ bifurcate (1). 205. Body of humérus (AJ: 89): cylindrical (0) -^ flattened with rectangular cross-section (1). 209. Radius of equal length or shorter than humérus (C: 17): no (0) -^ yes (1). Dipnomorpha 113. Metapterygial 'axis' of pectoral fin skeleton (jointed 'axes' only) (F: 115): short (0) -^ long (1). 198. Pectoral fin web: asymmetrical (0) -^ symmetrical (1). 208. Humeral radiais (A: 8): present (0) -^ absent (1). Glyptolepis 202. Pectoral radiais: all bear fin rays or lepidotrichia (0) -^ some 'naked' (1). Neoceratodus 196. Pectoral fin (A:6): unrotated (0) -^ rotated (1). 201. Pectoral radiais: do not bifurcate (0) -^ bifurcate (1). 203. Postaxial radiais: restricted distal to ulna (0) -^ first present on ulna (1).

blastopore and subsequent morphogenetic movements were region that centered on the vegetal pole (Fig. 5, H and I). not observed directly; however, anterior migration of the Owing to larval retention of the embryonic viteUine mem- vegetal region failed to occur, as a single stomodeal- brane and the radial symmetry of development, it was not like structure differentiated with radial, or perhaps 4-fold possible to clearly identify either the invaginations or disk radial, symmetry at what had been the vegetal pole (Fig. 5, I region as stomodeal or anal; however, the entire region gave and K). In general, the differentiation of larval adult struc- rise to a large circular stomodeal-like structure at the oral tures occurred in a radially or 4-fold radially symmetric pole, suggesting that the four invaginations and disk were pattern about a novel aboral-oral axis. The aboral pole of the stomodeal in nature (Fig. 5, J and K). The stomodeum and aboral-oral axis corresponded to the animal pole; however, foregut were ciUated similar to controls but, in contrast to despite the general similarity of spatial position, the lineage controls, the stomodeum now formed a flat radial circle that composition of the oral pole did not correspond to that of the was not bent at a right angle (Figs. 5, J and K and 6, F and vegetal pole. This difference was due to the radially symmetric G). Similarly, the gut, which normally runs anterior, turns 180 internalization of the vegetal region during gastrulation. Thus, degrees, and runs posterior, was a simple in-pocketing that the aboral-oral axis of radialized larvae was parallel to but ran parallel to the aboral-oral axis. The gut appeared to be distinct from the animal-vegetal axis of the embryo. everted in the later stages of development (data not shown). The origins of the radiaUzed stomodeum were unclear. In the pretrochal region, anterior tufts of meniscotrochal However, based on the later relative position of the pro- cilia and larval lateral glands differentiated in a 4-fold radial totroch, it appeared to be derived from second- or third- pattern oriented at right angles from one another (Fig. 6, C quartet lineages from all four quadrants. In forming the and D). Larvae exhibited muscular contractions and Phalloi- stomodeum, 48-h radialized trochophores exhibited four sites din staining of preeversion presetigers revealed radial rings of of invagination. These sites were perpendicular to one another muscle bands encircUng the stomodeum and foregut, stacked and formed a 4-fold radially symmetric ring around a disk along the aboral-oral axis and extending up into the aboral Gonzales et al. Radialized development in annelids 427 region (Fig. 6, F and G). Altliough paired eyespots are pres- of equal-cleaving molluscs and nemerteans (van den Biggelaar ent in 8-day control larvae (Fig. 3C), they were not readily 1977; Henry 2002). It is suggested that equal-cleaving apparent in radialized larvae. Diffuse pigmented patches of polychaetes are similar, but with specification occurring a eyespot-like material were observed in some radialized larvae, cell-cycle later and with 4d, rather than 3D, acting as the but could not be clearly scored as differentiated larval eye- organizer (Freeman and Lundelius 1992; Lambert and Nagy spots (data not shown). Differentiation of additional adult 2003). In contrast, we observed two types of equal-cleaving structures was not characterized, as radialized larvae died be- polychaete embryos, those exhibiting overt contact before bi- tween 2 and 3 weeks, potentially from an inability to feed due lateral cleavage {A. vittata) and those lacking contact (O.fu- to eversión of the gut (data not shown). siformis and S. columbiana), neither of which fully supports previous models. In A. vittata, contact occurs, as predicted, BFA and monensin treatment in S. columbiana between the sixth cell-cycle animal (Iq'" and Iq"^) and veg- etal (4q and 4Q) quartets. However, contact does not result in Based on A. vittata, BFA and monensin radialization treat- centralization of a single blastomere, as all eight vegetal ments were made at similar developmental stages in S. co- blastomeres are centralized and in full contact with overlying lumbiana, which, unlike A. vittata, does not establish animal- animal blastomeres, and it is morphologically unclear which, vegetal contact. Although the early embryo and trochophore if any, of the vegetal blastomeres act as the signaling center. were not characterized, later presetigers of monensin and Given that animal-vegetal contact is required for organizer BFA-treated embryos exhibited a similar radialization of de- specification in other equal-cleaving embryos (van den Big- velopment about a novel aboral-oral axis in 2- to 8-day larvae gelaar and Guerrier 1979; Arnolds et al. 1983; Martindale et (Table 1; Fig. 3, E-H). al. 1985; Henry 2002), the absence of contact in O.fusiformis and S. columbiana might suggest that the organizer is absent. However, BFA and monensin radialization of S. columbiana DISCUSSION development and known MAPK activation in 4d of Hyroides sp., a related serpulid in which contact is presumably also D quadrant organizer inhibition absent, suggest that it is present (Lambert and Nagy 2003). A developmental signaUng center homologous to D quadrant Thus, it appears that a novel, noncontact mechanism of or- organizer in molluscs, nemerteans, and unequal-cleaving an- ganizer specification is present in the serpulids S. columbiana nelids has been proposed for equal-cleaving polychaetes and Hydroides sp., and, perhaps, the oweniid O. fusiformis, (Freeman and LundeUus 1992; Lambert and Nagy 2003) but the nature of this mechanism is unclear. Animal-vegetal but has not been experimentally tested. Using BFA and contact is not required in many unequal-cleaving embryos, monensin to block cell signaling during the predicted window such as /. obsoleta, which segregate organizer-specific deter- of organizer specification and patterning in A. vittata and 5*. minates during division (Clement 1952, 1967). It is possible columbiana, our results show that either chemical radiaUzes that noncontact equal-cleaving polychaetes have convergently subsequent development, with a loss of bilateral symmetry in evolved a similar mechanism, or are secondarily equal-cleav- the embryo and body plan, and that this effect is specific to ing and have conserved noncontact specification from an un- treatments initiated before the organizer-dependent transition equal-cleaving ancestor. Alternatively, in the equal-cleaving to bilateral cleavage. Given that radialization in other spiral- hesionid P. obscura, overt contact is initially absent. However, cleaving taxa is a result of inhibiting the D quadrant organizer fine filamentous protrusions extend between animal and veg- (Wilson 1904; Clement 1952, 1962; van den Biggelaar and etal blastomeres before bilateral cleavage (Treadwell 1901) Guerrier 1979; Lambert and Nagy 2001, 2003; Henry 2002), and it is suggested, although not demonstrated, that filament- BFA- and monensin-induced radialization of ^. vittata and S. ous contact may underly organizer specification in P. obscura columbiana indicates that a functional homolog of the orga- and Hydroides sp. (Lambert and Nagy 2003). It is unclear nizing center is present in equal-cleaving polychaetes and that from our results whether filamentous contact occurs in O. it is inhibited, either directly or indirectly, by treatment with fusiformis and 5*. columbiana, or whether it might precede either chemical. overt contact in A. vittatae. If present in all three, it could provide a unifying mechanism for organizer specification in contact and noncontact equal-cleaving polychaetes, or even Organizer specification different equal- and unequal-cleaving species and phyla. Clear We found that organizer specification in equal-cleaving comparative documentation and functional studies of non-, polychaetes has diverged, both among polychaetes and rela- filamentous, and overt animal-vegetal contact will help to tive to other spiral-cleaving phyla. Specification with overt resolve the evolution and conservation of organizer specifica- animal-vegetal contact between the fifth cell-cycle Iq" and tion in equal-cleaving polychaetes and other spiral-cleaving 3Q quartets and subsequent 3D centralization is characteristic lophotrochozoans. 428 EVOLUTIONS DEVELOPMENT Vol. 9, No. 5, September-October 2007

Developmental timing of organizer patterning velopment in equal-cleaving gastropods and chitons (Ku- We found that development in A. vit tata is generally similar to htreiber et al. 1988; Gonzales et al. 2007). However, while the other equal-cleaving polychaetes (Mead 1897; Treadwell 1901; direct targets and immediate effects of either chemical are Torrey 1903) and in agreement with previous descriptions largely conserved across eukaryotes (Mollenhauer et al. 1990; (Pernet 2000). One distinction is that in Podarke and Dinter and Berger 1998; Mayer 2003), and although within a Thalassema, the Iq"^ of the cross are the first quar- species, the initially distinct effects of each chemical converge tet to exhibit bilateral symmetry in cleavage, with asymmetric to radialize development similarly, details of how radialization divisions at the B quadrant midline and symmetric divisions is achieved differ between phyla, if not between species, in a at the D quadrant midline and subsequent divisions asyn- development-specific manner. In both annelids and molluscs, chronous between midlines (Treadwell 1901; Torrey 1903). In development is radialized only if treatment is initiated before the polynoid, Lepidonotus sp., Iq"^ divisions are all equiv- the transition to bilateral cleavage. In radialized gastropods alent and spiral, but become bilateral and asynchronous in and chitons, BFA and monensin inhibit organizer specifica- subsequent patterns of divisions, similar to subsequent divi- tion by blocking, at the morphological level, Iq" and 3Q sions in Podarke and Thalassema (Mead 1897). In A. vittata, extension and contact (Kuhtreiber et al. 1988; Gonzales et al. Iq"^ divisions are identical to Lepidonotus, but we did not 2007). However, similar treatments in A. vittata do not block follow subsequent divisions in sufficient detail to determine extension and contact still occurs in radialized development, whether they take on a bilateral pattern. If bilateral, it is making it unclear whether contact is functionally required. In possible that, in the polynoids A. vittata and Lepidonotus sp., addition, the absence of clear morphological markers also the organizer-dependent secondary axis is established within makes it unclear when it is that BFA and monensin act during the Iq^^^ quartet, as it is in Podarke and Thalassema, but is organizer specification and patterning, or even if they interfere not morphologically evident. More generally, the timing of with the same or different features of either process. Still, the the developmental versus morphological onset of the orga- actions of both drugs are clearly downstream of extension and nizer-dependent secondary axis is a critical distinction that contact and, therefore, distinct from their mode of action in will be central to understanding the D quadrant organizer in molluscs. The combined absence of centralization and occur- A. vittata and other spiral-cleaving lophotrochozoans. rence of an additional division in animal and vegetal quartets make it is unclear which blastomere, or blastomeres, function BFA and monensin developmental targets in as the organizing center in equal-cleaving polychaetes. Similar annelids and molluscs to other spiral-cleaving lophotrochozoans tested, the orga- nizing center might be localized to vegetal lineages of the D The direct targets of BFA and monensin are distinct (MoU- quadrant or D quadrant midline, but we have no direct ev- enhauer et al. 1990; Dinter and Berger 1998; Mayer 2003); idence of this in A. vittata and, given clear morphological however, both chemicals similarly inhibit protein processing differences with regard to contact and centralization, the or- and secretion and treatment with either results in an identical ganizer might reside in additional, or distinct, lineages. Based radialization of development. This suggests that downstream on MAPK activation patterns, it is suggested that 4d in molecular, subcellular, or physiological effects of either treat- polychaetes acts as the functional homolog of 3D in molluscs ment converge to interfere with the same developmental pro- (Lambert and Nagy 2003). However, MAPK has numerous cess or processes and that the radialization phenotype is not roles in early development (Schohl and Fagotto 2002) and it is the result of chemical-specific toxicity. Both the radialization unclear whether its role in Hydroides embryos is related to the phenotype, with its characteristic loss of bilateral symmetry in organizer, or might be directed toward other potentially re- the embryo and body plan, and the specificity of treatment, lated or unrelated functions, such as endomesoderm specifi- i.e., radialization is limited to treatments made during the cation. Future blastomere ablation experiments within the period predicted for organizer specification and patterning, animal and vegetal quartets, functional assays of MAPK in suggest that BFA and monensin, either directly or indirectly, polychaetes, and the identification and functional testing of a inhibit patterning by the D quadrant organizer. It is also clear molecular marker for the D quadrant organizer are all possible that additional, unrecognized developmental pro- important steps toward establishing whether contact is re- cesses unrelated to the organizer also act within the same quired for organizer specification and which blastomeres take developmental window, are sensitive to treatment, and have on its organizing function in polychaetes. contributed to the observed phenotypes.

Organizer specification in molluscs versus Models of organizer specification and patterning annelids Current mollusc-based models of organizer specification and BFA- and monensin-radialized development in A. vittata is, in patterning are problematic when applied to equal-cleaving a general sense, similar to BFA- and monensin-radialized de- polychaetes. A stochastic model has been proposed for orga- Gonzales et al. Radialized development in annelids 429 nizer specification in equal-cleaving gastropods (Arnolds et al. equal-cleaving polychaetes, although the underlying molecu- 1983). In this model, all four vegetal 3Q blastomeres make lar genetic pathways of patterning may still be conserved for overt animal contact and a single blastomere, designated 3D, spiral-cleaving lophotrochozoans in general. is selected as the organizer through contact-dependent animal interactions (Arnolds et al. 1983). However, despite develop- mental equivalence within the 3Q quartet, 3D is almost al- Developmental function of the D quadrant ways a cross-furrow, and only rarely a noncross-furrow, organizer blastomere (van den Biggelaar 1976, 1977; van den Biggelaar The developmental function of the D quadrant organizer in and Guerrier 1979). Therefore, it is proposed that organizer spiral-cleaving lophotrochozoans is enigmatic. Organizer pat- specification is stochastically determined by differences in the terning is said to estabhsh anteroposterior, dorsoventral, or relative amount or duration of contact that each 3Q blasto- structure-specific developmental fates (Clement 1962; van den mere makes with overlying animal quartets and that, due to Biggelaar and Guerrier 1979; Martindale et al. 1985; Lambert the geometry of spiral cleavage, the more central and elevated and Nagy 2001; Lartillot et al. 2002b). However, lineage cross-furrow blastomeres have an inherent advantage. This studies indicate that axis- or structure-specific fates are estab- model has two problems in equal-cleaving polychaetes. The lished later in development (Serras and van den Biggelaar first is that both S. columbiana and O. fusiformis do not es- 1990; Dictus and Damen 1997). RadiaUzed development pro- tablish animal-vegetal contact and so organizer specification vides a functional test of the D quadrant organizer because does not involve overt contact-dependent interactions with patterning by the organizer is absent. Previous studies recog- the animal pole. The second is that A. vittata establishes overt nized a limited ability of radialized larvae to differentiate dis- contact, but animal contacts remain equivalent between veg- organized masses of adult tissue (Wilson 1904; Clement 1952, etal blastomeres and the plane of bilateral symmetry, which is 1962; Atkinson 1971; van Dongen 1976; Kuhtreiber et al. a proxy for the location of the organizing center, is as Ukely to 1988; Lambert and Nagy 2001, 2003) and it has been recently bisect cross-furrow as noncross-furrow blastomeres. These shown in BFA- and monensin-radialized molluscs that these features both suggest that spatially and stochastically deter- structures are organized about a novel aboral-oral axis (Go- mined relative differences in animal contacts by vegetal nzales et al. 2007). Similarly, we show here that radialized A. blastomeres do not play a role in D quadrant organizer spec- vittata larvae differentiate various larval and adult structures ification in A. vittata. An understanding of what, if any, el- in a radial or 4-fold radial fashion that is organized both ements of organizer specification are conserved between around, i.e., larval glands, menisotrochal cilia and stomo- molluscs and annelids will require the characterization of un- deum, and along, i.e., gut and muscles, a novel aboral-oral derlying molecular mechanisms in each clade. axis. This radiaUzed body plan is reminiscent of the 4-fold Mechanistic models of organizer patterning in molluscs radially symmetric developmental architecture that is estab- commonly suggest that the D quadrant organizer acts as a lished by spiral cleavage prior patterning by the organizer in signaling point-source within the radially symmetric develop- normal development. However, due to morphogenetic move- mental architecture estabhshed by spiral cleavage (Raven ments in gastrulation, the aboral-oral axis is parallel, but not 1976; van den Biggelaar and Guerrier 1979; Sweet 1998; identical, to the animal-vegetal axis. Similar to radialized Lambert and Nagy 2003). In general, these models argue that molluscs (Gonzales et al. 2007), the ability of radialized A. 3D centralization places 3D in contact with nearly all other vittata larvae to gastrulate demonstrates that patterning by blastomeres in the embryo but that the onset or duration of the D quadrant organizer is not required for gastrulation to contact and the position of blastomeres relative to the nucle- proceed. It also argues against, although does not disprove, us, vegetal cortex, or some unknown subcellular feature of 3D the idea that the radiaUzed development is a passive result of differs between blastomeres in a bilaterally symmetric fashion. default developmental fates or autonomous differentiation. It is this combination of centralized global contact and spatial Instead, we propose that the organizer-dependent develop- asymmetry that then allows 3D to pattern the embryo, cre- mental architecture at the onset of gastrulation, whether ra- ating the distinct, symmetrical developmental identities ob- dial or bilateral, dictates subsequent patterns of gastrulation served within quartets relative to the D quadrant midline. In and it is active patterning during gastrulation that regionally contrast, our results show that while the functional homolog establishes lineage contributions to and architecture of the of the D quadrant organizer is present in equal-cleaving adult body plan. In this way, organizer-dependent patterning polychaetes, global centralized contact by a vegetal blasto- of the embryo is transformed into the body plan of the adult mere is absent in both 5*. columbiana (no centralized contact) without directly patterning adult anteroposterior and dorso- and A. vittata (centralized contact is equivalent and localized ventral axes, or directly specifying specific larval and adult to immediate neighbors for each 4q and 4Q blastomere). structures. Even if this is true, the nature of the organizer- Thus, novel noncontact noncentralized and contact noncen- dependent secondary axis remains unknown, as is the tralized mechanisms of organizer patterning are required for relationship of the organizing center in spiral-cleaving 430 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September-October 2007 lophotrochozoans with axial organizers in other metazoans. proliferation and a mixed benthic/pelagic life cycle. Bioes.mys 26: 314• Future research focusing on genome-enabled spiral-cleaving 325. Freeman, G., and Lundelius, J. W. 1992. Evolutionary implications of the lophotrochozoans, including the polychaete annelid Capitella mode of D quadrant specification in coelomates with spiral cleavage. /. spl, the clitellate annelid Helobdella robusta, and the gastro- Evol. Biol. 5: 205-247. pod mollusc Lottia gigantea, will be central to understanding Gonzales, E. E., van der Zee, M., Dictus, W. J., and van den Biggelaar, J. 2007. Brefeldin A or monensin inhibits the 3D organizer in gastropod, both the developmental function of the D quadrant organizer polyplacophoran, and scaphopod molluscs. Dev. Gene Evol. 217: 105- and the potential role that an ancestral axial organizer may 118. have played in early animal evolution. Guerrier, P. 1968. Origine et stabilité de la polarité animale-vegetative chez quelques Spiralia. Ann. Embryol. Morphogen. 1: 119-139. Halanych, K.M. 2004. The new view of animal phylogeny. Annu. Rev. Ecol. Acknowledgments Evol. Syst. 35: 229-256. We wish to thank B. Pemet, C. Hennans, and R. Strathmann for Hatt, P. 1932. Essais expérimentaux sur les localisations germinales dans guidance and advice on local polychaetes, R. Rubicz for field and lab l'oeuf d'un annélide {Sabelllaria alveolata L.). Arch. dAnat Mie. Morph. assistance, G. Zwaan for preparing and drawing semithin sections of Exp. XXVIII: 81-98. Henry, J. 2002. Conserved mechanism of dorsoventral axis determination in A. vittata, L. Nederbragt for contributions on the O.fusiformis anal- equal-cleaving spiralians. Dev. Biol. 248: 343-355. ysis, J. Priano and M. Rice for their generosity of lab space and Henry, J. J., and Martindale, M. Q. 1987. The organizing role of the D assistance with SEM, V. Foe, S. Santagata, G. V. Dassow, and A. quadrant as revealed through the phenomenon of twinning in the Wanninger for advice on confocal microscopy, and the FHL NIH polychaete Chaetopterus variopedatus. Roux's Arch. Dev. Biol. 196: Center for Cell Dynamics for the generous use of their confocal. We 499-510. also thank E. Begovic, D. Rokhsar, K. O'Day, and two anonymous Henry, J. Q., Perry, K. J., and Martindale, M. Q. 2006. Cell specification reviewers for their helpful comments and suggestions regarding the and the role of the polar lobe in the gastropod mollusc Crepidula for- manuscript. We greatly appreciate the generosity, kindness, and sup- nicata. Dev. Biol. 297 (2): 295-307. port of staff and scientists at the University of Washington Friday Holm, A. 1952. Experimentelle Untersuchungen über die entwicklung und entwicklungsphysiologie des spinnenembryos. Zool. Bidrag. 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