The Cell Lineage of the Polyplacophoran, Chaetopleura Apiculata: Variation in the Spiralian Program and Implications for Molluscan Evolution

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Developmental Biology 272 (2004) 145–160 www.elsevier.com/locate/ydbio

The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution

Jonathan Q. Henry,a,* Akiko Okusu,b and Mark Q. Martindalec

a Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA b Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA c Kewalo Marine Lab, Pacific Biomedical Research Center, University of Hawaii, Honolulu, HI 96813, USA Received for publication 27 January 2004, revised 9 April 2004, accepted 12 April 2004 Available online 7 June 2004

Abstract

Polyplacophorans, or chitons, are an important group of molluscs, which are argued to have retained many plesiomorphic features of the molluscan body plan. Polyplacophoran trochophore larvae posses several features that are distinctly different from those of their sister trochozoan taxa, including modifications of the ciliated prototrochal cells, the postrochal position of the larval eyes or ocelli, epidermal calcareous spicules, and a collection of serially reiterated epidermal shell plates. Despite these differences, chitons demonstrate a canonical pattern of equal spiral cleavage shared by other spiralian phyla that permits the identification of homologous cells across this animal clade. Cell lineage analysis using intracellular labeling on one chiton species, Chaetopleura apiculata, shows that the ocelli are generated from different lineal precursors (second-quartet micromeres: 2a, 2c) compared to those in all other spiralians studied to date (first-quartet micromeres: 1a, 1c). This situation implies that significant changes have also occurred in terms of the inductive interactions that control eye development in the spiralians. Although radical departures from the spiralian developmental program are seen in some molluscs (i.e., cephalopods), the findings presented here indicate that important changes can occur even within the highly constrained framework of the spiral cleavage program. Among spiralians, variation has been reported for the origin of the anterior, sensory, apical organ, which arises from the 1c and 1d micromeres in C. apiculata. The prototroch of C. apiculata consists of two to three irregular rows of ciliated cells but arise from 1q and 2q daughters, similar to that of Ischnochiton rissoi, as well as the gastropod, Patella vulgata. Despite certain early claims, there is no supporting evidence that any of the shell plates arise pretrochally in C. apiculata. The first seven of eight definitive shell plates that arise in the larva originate from shell secreting grooves in the postrochal region (derived from 2c, 2d, 3d). Earlier descriptions indicate that the eighth plate arises later at metamorphosis, and as this is formed posteriorly, it too forms in the postrochal region. On the other hand, epidermal spicules originate from both pretrochal and postrochal cells (1a,1d, 2a, 2c, 3c, 3d). The significance of these observations is discussed in light of various hypotheses concerning the origin of the conchiferan shell. This study reveals conservation, as well as evolutionary novelty, in the assignment of specific cell fates in the spiralians. D 2004 Elsevier Inc. All rights reserved.

Keywords: Mollusca; Chiton; Development; Trochophore; Prototroch; Larval ocelli; Spicule; Shell

Introduction 1998). The classical trochophore possesses a sensory apical tuft and a pair of larval ocelli (eye spots) in the pretrochal Molluscs are bilaterally symmetrical metazoans that be- region (episphere), a ventral mouth just posterior to the long to the spiralian group currently called the Trochozoa prototroch, a metatroch just posterior to the mouth, and a (including: Annelida, Echiura, Entoprocta, Nemertea, and telotroch at the caudal end (Fig. 2A). Although the basic Sipuncula; Giribet et al., 2000; Peterson and Eernisse, 2001; organization underlying the trochophore larva seems to be Fig. 1A). These trochozoan animals exhibit trochophore- conserved among trochozoans, each larva has slightly dif- type larvae, which include forms with a basic ciliated ferent features that probably evolved as adaptations to their locomotory structure called the prototroch (Fig. 2A; Rouse, differing ecological settings (Rouse, 1998). Animals represented by the Trochozoa represent some- * Corresponding author. Department of Cell & Structural Biology, thing of an evolutionary paradox. Despite the fact that they University of Illinois, Urbana, IL 61801. Fax: +1-512-244-1648. demonstrate some of the most diverse and evolutionary E-mail address: [email protected] (J.Q. Henry). successful adult body plans (e.g., compare an adult oyster

1994; Render, 1997). The details that intracellular fate mapping provide have important implications for both mechanistic and evolutionary understanding of spiralian development, making it possible to identify conserved features and study evolutionary changes in the fates of individual identified embryonic cells and their descendents, between members of different spiralian assemblages. The Mollusca is a particularly interesting group for studying innovation of morphological novelty in the con- fines of the spiralian developmental program. The molluscs display highly modified adult body plans, including vermi- form aplacophorans, bilaterally shelled bivalves, torted gas- tropods, and the highly specialized cephalopods. Although the phylogenetic relationship of major molluscan taxa is still a contentious issue (Haszprunar, 2000; Salvini-Plawen, 1985; Scheltema, 1993, 1996; Fig. 1B), relatively simple features of aplacophorans and polyplacophorans have led some authors to suggest that they are the basal groups of molluscs (Haszprunar, 2000; Salvini-Plawen, 1985). The plesiomorphic molluscan features are thought to include a broad ciliated ventral foot, posterior mantle cavity housing paired gills, and epidermal calcareous spicules. Polyplaco- phorans (chitons) are a monophyletic group of mulluscs (Okusu et al., 2003) and the adults have eight calcareous dorsal shell plates, leading some workers (Ghiselin, 1988; Lemche and Wingstrand, 1959; Nielsen, 1985, 2001; Wing- Fig. 1. (A) Current phylogenetic hypothesis of Bilateria (modified from strand, 1985) to suggest that molluscs were primitively Giribet et al., 2000). Mollusca (arrow) is a member of the Trochozoa. (B) segmented like their fellow spiralians, the annelids; however, Phylogenetic hypotheses of the Mollusca. The two trees exemplify many this contention has been strongly refuted (Salvini-Plawen, controversial views on molluscan phylogeny (i.e., Scheltema, 1996 vs. 1985; Wanninger and Haszprunar, 2002). Calcareous spi- Haszprunar, 2000). cules are widely distributed within the larval (as well as adult) epidermis of chiton and aplacophoran trochophores, with a segmented bloodworm), members of this group all suggesting that this may be the plesiomorphic condition that share a highly stereotyped pattern of early embryonic gave rise to the conchiferan shell (Haas, 1981). development based on spiral cleavage. Spiral cleavage Molluscan larvae show great variations in the number of involves a regular pattern of cell divisions in which indi- trochoblasts (one, three, four, or six rows of cells) involved vidual cells can be identified simply by their position and in generating the prototroch (Figs. 2B–F). Chiton trocho- cleavage history (Heath, 1899; Fig. 3). The pattern is so phore larvae such as Chiton polii (Kowalevsky, 1883), I. highly conserved that one can draw direct homologies rissoi (Heath, 1899), Lepidopleurus asellus (Christiansen, between individual cells of different embryos from different 1954), and Chaetopleura apiculata (herein) posses a unique spiralian phyla. Tracing the fates of these identified cells prototroch that is composed of two to three irregular rows of reveals that each cell gives rise to a predictable domain of differentially ciliated trochoblasts (Figs. 2B–F). The sub- descendents and that identified structures have a common epidermal larval ocelli (eyes) of chitons are also truly unique embryological origin (Freeman and Lundelius, 1992; Henry compared to the other Trochozoa, as they are situated and Martindale, 1998, 1999; van den Biggelaar, 1996; posterior to the prototroch rather than in the pretrochal, Verdonk and van den Biggelaar, 1983; Wilson, 1898). For episphere (compare Fig. 2A with B). These larval situations example, in all spiralians thus studied, the left and right differ from the trochophore larvae typically found in other larval ocelli are generated from two cells of the first quartet spiralians. of micromeres, called 1a and 1c, respectively (Henry and We have used intracellular lineage tracers to determine Martindale, 1999; Sweet, 1998; Verdonk and van den the fates of the first three quartets of micromeres in the Biggelaar, 1983). Classic cell lineage analyses in mollusc chiton C. apiculata. We show that there are definitive embryos showed that the adult shell was generated primarily changes in the fates of cells in this spiral-cleaving animal. from the 2d micromere (Conklin, 1897; Lillie, 1895; Wil- The eyes, rather than migrating posteriorly from pretrochal, son, 1892); however, more accurate intracellular lineage first-quartet micromere derivatives (1a and 1c), arise from analyses have shown that other second-quartet micromeres second-quartet micromeres (2a and 2c). This finding has make contributions to the shell gland (Damen and Dictus, significant implications in terms of the inductive interac- J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 147

Fig. 2. (A) A typical trochophore larvae with an apical tuft, prototroch, and metatroch ciliary bands, a through gut with a mouth and an anus, and pretrochal larval ocelli. (B) Polyplacophoran trochophore larva (e.g., C. apiculata) with two to three irregular rows of prototroch cells. The epidermal spicules extend pre- and postrochally. Seven rows of shell field are present on the dorsal side of the postrochal region, while the larval postrochal ocelli and the foot are present on the ventral side. (C) Gastropod trochophore larvae (e.g., Patella modified from Patten, 1886) have two rows of prototrochal cells that can proliferate to form the third row called the accessory prototroch. (D) Scaphopod larva can have numerous ciliated bands that later diminish to three rows of prototroch (e.g., Dentale modified from Lacaze-Duthiers, 1856). (E) Solenogaster larva (E. babai modified from Okusu, 2002) has a single row of prototroch. (F) Protobranch bivalve larva (e.g., Yoldia limatula modified from Drew, 1899b) showing three rows of prototroch-like ciliary cells. an, anus; at, apical tuft; ft, foot; lo, larval ocellus; ls, larval shell; mo, mouth; mt, metatroch; pt, prototroch; sg, shell gland; sp, spicules; tt, telotroch. tions that control eye development in the spiralians. The spicules arise from different, though overlapping, popula- shell plates appear to arise from postrochal derivatives, but tions of embryonic cells has some interesting implications in the spicules arise from postrochal, as well as pretrochal terms of the perceived origins of the conchiferan shell, cells. The fact that the calcareous shell plates and epidermal which is discussed further below.

Fig. 3. Cleavage pattern in C. apiculata through the 24-cell stage. All views are from the animal pole. Stages are as indicated. 148 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160

Methods four equal-sized blastomeres. Two blastomeres meet at the animal cross-furrow, and the other two blastomeres meet at Collection of adults and embryos the vegetal cross-furrow. The first four cells are called the basic embryonic quadrants, A, B, C, and D, although they C. apiculata was collected in Woods Hole during the cannot be definitively identified in these embryos until summer months (June–August) of 2000 and 2002. The after the fifth round of embryonic cleavages (Heath, 1899). animals were kept at ambient temperature (22 jC) in sea The four quadrants generate four successive tiers of micro- tables at the Marine Biological Laboratory. They have meres at the animal pole that alternate in dexiotropic separate sexes and were spawned in separate bowls during (clockwise) and leiotropic (counter-clockwise) orientations the night several days before and after the full moon each with respect to the animal–vegetal axis. The approximate month (Grave, 1932). The eggs were artificially inseminated times of these divisions are shown in Table 1. First-quartet in a culture dish (200–400 eggs/female) by the addition of micromeres are generically called 1q micromeres or, more sperm containing water from the male cultures. specifically, 1a, 1b, 1c, and 1d depending on their quadrant of origin, while their sister cell macromeres are called 1Q, Cell lineage analysis via microinjected fluorescent tracer or more specifically, 1A, 1B, 1C, and 1D. Second-quartet (2q, or 2a, 2b, 2c, and 2d), third-quartet (3q, or 3a, 3b, 3c, The sculptured outer egg hull of the large embryos (190 and 3d), and fourth-quartet (4q, or 4a, 4b, 4c, and 4d) Am) were removed manually with forceps. Individual micromeres are named similarly as are their macromere blastomeres were pressure injected with DiIC18 (Molecular sister cells (e.g., 2A, 2B, 2C, 2D, etc.). Although we were Probes, Inc., Eugene, OR) dissolved in soybean oil using not able to identify the quadrant of origin at the time of glass microelectrodes according to Martindale and Henry injection, we were able to identify all injected cells by (1995). Injections were performed at the 8-, 16-, and 24- their consistent labeling patterns at the trochophore stage. cell stages directly through the vitelline membranes. Chiton Our efforts to induce spawning of adult animals from embryos are equal-cleaving spiralians; thus, it is not pos- Woods Hole were unsuccessful. Attempts were made to sible to identify the embryonic quadrant of origin of spawn the animals by following general procedures widely individual blastomeres at the time of the injection. On the used for other marine invertebrate animals (e.g., increasing other hand, the animal–vegetal axis is discernable, and water temperature, adjusting light–dark conditions, flip- individual tiers of micromeres and macromeres can be ping animals upside-down, regulating sea water flow, identified. Blastomeres of each tier were, therefore, injected immersing animals with spawning males, e.g., Strathmann, randomly, and labeling patterns sorted out at larval stages, 1987). Instead, animals spawned spontaneously from 11:00 based on the occurrence of their regular, clonally derived pm to 1:00 am on nights around the time of the full moon. patterns. Due to the limited amount of embryonic material, we injected first-quartet micromeres (1q) and macromeres Culture and preparation of embryonic and larval specimens (1Q) at the eight cell-stage, second-quartet micromeres (2q) and the animal descendants of the first-quartet micro- Injected larvae were transferred to culture dishes con- meres (1q1) at the 16-cell stage, and third-quartet micro- taining filtered seawater (0.45 Am) with streptomycin (50 meres (3q) and macromeres (3Q) at the 24-cell stage mg/ml) and penicillin (35 mg/ml) to reduce bacterial growth (Table 2). (Strathmann, 1987). Labeled larvae were examined live or fixed in 4% formalin in filtered seawater with 100 mM Fate of first-quartet micromere derivatives EDTA (Henry et al., 2001), with a Zeiss epifluorescence microscope and digital images taken on a Zeiss Axiocam. First-quartet micromeres in C. apiculata are given off with a clockwise or dexiotropic orientation with respect to underlying macromeres. Out of 44 injections into first- Results

Cleavage program Table 1 Approximate timing of cleavage divisions generating specific cell stages at 22C The cleavage program for equal-cleaving spiralian em- Cell stage Time (h) bryos has been described in detail elsewhere (Lymnaea stagnalis, van den Biggelaar, 1971a,b; Verdonk, 1965; Insemination 0 1st polar body 1:30 Patella vulgata, Damen and Dictus, 1994; Cerebratulus 2-cell 2:00 lacteus, Henry and Martindale, 1998; Hoploplana inqui- 4-cell 3:00 lina, Boyer et al., 1998), as well as for three species of 8-cell (first quartet) 4:00 chitons (Heath, 1899; Grave, 1932; van den Biggelaar, 16-cell (second quartet) 5:10 1996) (see Fig. 3). The first two cell divisions give rise to 24-cell (third quartet) 5:55 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 149

Table 2 The 1c blastomere gives rise to a near mirror image Distribution of injected blastomeres (8- through 24-cell stages) region of the episphere as the 1a cell, albeit on the right side Stage Micromeres of the head. Labeled clones include the apical organ and first 8-cell stage 1a 1b 1c 1d two rows of the prototroch, but label is not located posterior Number of cases 9 13 8 14 to the prototroch or include the right eye (Figs. 7C–D).It

1 1 1 1 also appears that this cell, unlike 1a, does not give rise to 16-cell stage 1a 1b 1c 1d 1 Number of cases 4 5 2 1 spicule-containing cells (Figs. 4C and 5). The 1c blastomere gives rise to ectoderm and a few prototroch cells 16-cell stage 2a 2b 2c 2d (principally the top row) on the right side of the head, as Number of cases 6 7 8 3 well as the apical organ. The 1d cell gives rise to most of the large dorsal domain 24-cell stage 3a 3b 3c 3d Number of cases 6 2 3 6 of the head. It includes the apical organ and the first two rows of the prototroch, as well as a large group of spicule- Stage Macromeres containing cells in the dorsal epidermis. There is a large 8-cell stage 1A 1B 1C 1D field of epidermis that is labeled between the spicule band Number of cases 4 4 3 3 and the prototroch (Figs. 4D and 5); however, this field seems to be uncalcified and all adult shell plates seem to 24-cell stage 3A 3B 3C 3D arise from the postrochal region, as discussed further below. Number of cases 1 2 1 2 The 1d1 micromere gives rise to the apical tuft and most of the dorsal spicules (Fig. 6B). It also gives rise to the top two quartet micromeres, four distinct and reproducible patterns rows of the prototroch on the left dorsal side of the head and were observed (Table 2). These patterns can easily be the top row toward the right dorsal side. homologized with first-quartet micromere descendents from other spiralian embryos and we thus assigned names to each Fate of first-quartet macromere derivatives cell post facto. Representative labeling patterns for the first quartet are shown in Figs. 4A–D, and the summary of these Fourteen first-quartet macromeres were injected at the four patterns is illustrated in Fig. 5. eight-cell stage (Table 2). First-quartet macromere patterns The 1a cell gives rise to the left side of the trochophore include the derivatives of the second-, third-, and fourth- epishere and includes the first two rows of the prototroch quartet micromere, as well as fourth-quartet macromere posteriorly, but does not contribute to the formation of the derivatives. Due to the larger domain of cells that are apical sensory tuft (Figs. 4A and 5). Labeled clones include labeled, which included internal structures such as the gut, spicule-containing epidermal cells on the left-dorsal side of these cases are somewhat more difficult to interpret. These the head. Injecting one of the two daughter cells of 1a at the cases, however, serve as a valuable guideline for confirming 16-cell stage, the apical descendent 1a1, reveals that this cell and organizing the subsequent micromere patterns, de- gives rise to the apical ectodermal component of the 1a scribed below. These four macromere patterns are shown staining, including the spicule-containing cells and the top in Figs. 4M–P, and summarized in Fig. 8. row of the prototroch (Fig. 6A). By subtracting the 1a1 contribution from the 1a staining pattern, it can be inferred Fate of second-quartet micromere derivatives that the 1a2 blastomere gives rise primarily to a small patch of pretrochal ectoderm and to cells contained in the second, The fate of second-quartet micromeres was determined or intermediate row of the prototroch. Significantly, high- by injecting 24 second-quartet micromeres (Table 2). Photo- magnification views confirm the fact that the 1a cell does micrographs of representative second-quartet micromere not give rise to either of the eyes, or any structures posterior injections are shown in Figs. 4E–H and diagrammatic to the prototroch (Figs. 4A, 5, and 7A). summaries shown in Fig. 5. Although it might have been The 1b cell forms a wedge of epidermis in the episphere difficult to detect technically, all derivatives of second- on the ventral surface, which does not include the apical tuft. quartet micromeres appeared to give rise to ectodermal Prototrochal staining is restricted to the apical two rows of derivatives, with no ectomesenchyme observed. This is the prototroch above the upper right lip of the stomodeum. consistent with previous descriptions of chiton development No spicule-containing cells reside on the ventral surface of (Heath, 1899; Hyman, 1967), but a closer analyses is the head. Injection of the cell 1b1 indicates that this cell warranted. gives rise to the majority of apical ectoderm on the ventral The micromere 2a gives rise to a small ectodermal surface of the head, including a portion of the anterior domain on the left lateral and left dorsal region of the prototroch (principally the top, animal row of cells) while postrochal region of the hyposphere with the most medial its sister cell gives rise to cells around the mouth, including staining seen at the posterior-most limit of clones (Figs. 4E the prototroch (second and more posterior, third rows) (Figs. and 5). Spicule-containing cells located predominantly on 4B and 5). the dorsal and lateral surfaces are also labeled. The 150 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 151 posterior-most row of the prototroch is labeled, as well as 3b micromere (Figs. 4K and 5). The dorsal shell plates on the left eye (Figs. 7E–F). There is additional deep staining the right side of the animal are also formed by this cell. seen in the region dorsal to the eye that could be the left The 3d micromere gives rise to a continuous patch of protonephridia; however, this structure is too deep to be ectoderm in the left postrochal dorsal region that extends identified unambiguously with fluorescence in whole around to the ventral surface (Figs. 4L and 5). This includes mounts. the left portion of the segmental shell plates. As shown in The cell 2b gives rise to a nearly symmetrical patch of (Fig. 4L), it appears that a small number of prototrochal staining on the ventral surface. Staining on the external cells are also included in the 3d clone on the left dorsal side surface, including a small patch of prototroch is shifted in the posterior-most row of the prototroch. This is a rather slightly to the left side, but staining is located internally and unique condition compared to other spiralians. apically inside the stomodeum in a more symmetrical fashion (Figs. 4F and 5). No spicule-containing cells are Fate of third-quartet macromere derivatives labeled. The cell 2c gives rise to ectoderm on the right side of the A limited number of macromere injections were per- animal that wraps around the right side toward the ventral formed at the 24-cell stage (Table 2). All of the derivatives midline (Figs. 4G and 5). This cell gives rise to descendents of the third-quartet macromeres give rise to internal deriv- in the most posterior row of the prototroch as well as the atives in reproducible regions (Figs. 4Q–T and 8). The 3A right eye that is situated just deep and posterior to the macromere gives rise to endodermal derivatives along the prototroch (Figs. 7G–H). left wall of the gut tube. The 3B macromere gives rise to a The cell 2d gives rise to a strip of ectoderm on the dorsal bilaterally symmetrical domain of anterior endoderm that surface. The clone of labeled cells spans the dorsal midline, extends up into the episphere, while the 3C macromere interdigitating with neighboring cells, as well as spreading gives rise to the right side of the gut. The 3D macromere to the right side of the back and extending backward to the gives rise to a large domain of dorsal posterior endoderm. posterior tip of the larvae (Figs. 4H and 5). A large number Although additional studies are needed to accurately map of spicule-forming cells, and cells that will give rise to the the full compliment of macromere descendents, injections definitive adult shell plates, are labeled. reveal that the 3D macromere also generates individual muscles in the postrochal region (Figs. 6C–D). A summary Fate of third-quartet micromere derivatives cell lineage diagram for all cells examined here is shown in Fig. 9. Seventeen third-quartet micromeres (Table 2) were injected at the 24-cell stage. (Divisions of the 1q2 and 2q quartets of micromeres are delayed relative to third quartet Discussion formation.) The 3a micromere gives rise to a patch of ectoderm on the left ventral side of the postrochal region Cell lineage and descriptive fate maps of spiralian (Figs. 4I and 5). The labeling is located up to the left side of embryos generated around the turn of the 20th century the stomodeum and includes spicule-containing cells in the revealed that the early cleavage programs and blastomere postrochal region. The staining is shifted more toward the fates were remarkably similar in certain groups as diverse ventral midline than 2a derivatives and a small wedge of the as molluscs, annelids, and polyclad flatworms (Wilson, 2a domain encroaches into the 3a clone. 1898). In more recent years, intracellular cell lineage The 3b micromeres gives rise to a small patch of techniques have enhanced our ability to follow blastomere ectoderm in the ventral-right domain posterior to the mouth descendents, and this increased resolution has allowed the (Figs. 4J and 5). This clone of labeled cells extends deep detection of even more subtle differences in fates between inside the stomodeum and passes anteriorly around the different embryos. These differences are rooted in mecha- inside lip. nistic and phylogenetic changes that have occurred during The 3c micromere generates a clone of cells in the right the evolution of these groups. A dramatic example of the dorsal posterior region that wraps around the posterior end significance of these differences is found in the comparison and toward the mouth where it abuts the staining from the of equal- vs. unequal-cleaving spiralians (Freeman and

Fig. 4. Superimposed differential interference contrast (DIC) and fluorescence micrographs showing the distribution of DiI lineage tracer in larvae following injections of first, second, and third quartet micromeres and macromeres at the 8- through 24-cell stages. All trochophore larvae are shown with the apical, anterior ends oriented toward the top of the figure. (A) Dorsal view of 1a clone. (B) Ventral view of the 1b clone. (C) Ventral view of the 1c clone. The pigmented right ocellus is visible in this image. (D) Dorsal view of the 1d clone. (E) Ventral view of 2a clone. Pigmented left and right ocelli are visible in this image. (F) Ventral view of the 2b clone. (G) Ventral view of the 2c clone. Pigmented left and right ocelli are visible in this image. (H) Dorsal view of the 2d clone. (I) Ventral view of 3a clone. (J) Ventral view of the 3b clone. (K) Dorsal view of the 3c clone. The pigmented left and right ocellus are visible in this image. (L) Dorsal view of the 3d clone. (M) Ventral view of 1A clone. (N) Ventral view of the 1B clone. (O) Ventral view of the 1C clone. (P) Dorsal view of the 1D clone. (Q) Dorsal view of 3A clone. (R) Ventral view of the 3B clone. (S) Dorsal view of the 3C clone. (T) Dorsal view of the 3D clone. clone. at, apical tuft; gt, gut; oc, ocellus; pt, prototroch; sb, spicule band; sf, shell field; st, stomodeum. Scale bar equals 100 Am. 152 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 153

Fig. 6. Superimposed differential interference contrast (DIC) and fluorescence micrographs showing the distribution of DiI lineage tracer in larvae following injections of first-quartet micromeres 1a1 and 1d1, and third-quartet macromere 3D at the 12-, 12-, and 24-cell stages, respectively. All trochophore larvae are shown with the apical, anterior end located toward the top of the figure. (A) Left lateral view showing contributions of the animal pole daughter (1a1) of the fist quartet micromere 1a. Note presence of DiI fluorescence in the cells giving rise to the spicule band in the animal episphere, and some of the prototroch cells. (B) Higher magnification dorsal view showing contributions made by the 1d1 micromere. Note that much of the spicule band is labeled in the animal episphere, as well as many of the dorsal, ciliated prototroch cells. The three bands of prototroch cells are clearly visible in this image. (C, D) Corresponding dorsal DIC and fluorescence micrographs showing the contributions of the 3D macromere to endodermal and mesodermal fates. Note the presence of DiI-labeled muscle fibers. The shaded regions in C delineate the regions compiled to make the fluorescence montage shown in D. mf, muscle fibers. All other labels are the same as those used in Fig. 4. Scale bar equals 100 Am for A, C, D, and 45 Am for B.

Lundelius, 1992; Henry and Martindale, 1998, 1999; van 1974). Some species do not form an apical tuft, and in many den Biggelaar, 1996; van den Biggelaar and Guerrier, 1983; other spiralians all four quadrants (1a–1d) generate the Verdonk and Cather, 1983; Verdonk and van den Biggelaar, apical tuft, such as the polychaete, Chaetopterus variope- 1983). datus (Henry and Martindale, 1987) and the nemertean, C. lacteus (Henry and Martindale, 1998). In others like H. Apical tuft formation inquilina, only 1a and 1c generate this structure (Boyer et al., 1998).InSabellaria cementarum, the 1d micromere Intracellular cell lineage analyses allow the identification inherits an inhibitor from the second polar lobe that prevents of subtle differences in the fates of homologous blastomeres this cell from contributing to the formation of the apical that are not strictly correlated with differences in cell size. organ (normally formed by 1a–1c derivatives; Render, Our results show that only the 1c1 and 1d1 micromeres give 1983). The mechanisms that control these fate decisions rise to the apical sensory organ in C. apiculata. In the are not known; however, it is obvious that first-quartet scaphopod Dentalium dentale, this structure is also formed contributions to the formation of the apical organ vary by 1c and 1d derivatives (van Dongen and Geilenkirchen, widely among different spiralians.

Fig. 5. Summary diagram illustrating the ectodermal clones derived from each of the first- through third-quartet micromeres, as indicated. All larvae are illustrated with the apical, anterior ends oriented toward the top of the figure. For each micromere clone, dorsal (left of figure) and ventral (right of figure) views are presented. Red label indicates the distribution of external tissues and the ocelli if labeled. Purple label indicates the distribution of deeper ectodermal tissues within the stomodeum. 154 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160

Prototroch formation posed of two to three irregular rows of differentially ciliated trochoblasts (C. polii; Kowalevsky, 1883); I. rissoi (Heath, Molluscan trochophore larvae have gone through multi- 1899); L. asellus (Christiansen, 1954); C. apiculata (herein, ple independent modification events producing diverse see Fig. 2B). Gastropod larvae (e.g., P. vulgata) have two larval types, such as the pericalymma and veliger larvae, rows of prototrochal cells (28–40 cells total) derived from that involve changes in the primary locomotory organ, the three distinct cell lineages that later form a third row, the prototroch. Prototrochs in molluscan larvae can either be accessory trochoblasts (Dictus and Damen, 1997; Damen completely absent or have one, two, three, or numerous and Dictus, 1994; Patten, 1886; Fig. 2C). Various scapho- prominent rows of ciliated cells (Figs. 2B–F). Chiton pod veligers have three (e.g., Antalis entail, Wanninger and trochophore larvae posses a unique prototroch that is com- Haszprunar, 2001) and up to four or six (e.g., Dentale, Lacaz-Duthiers, 1856, pl. 7; Figs. 1–9) rows of prototrochal ciliated bands that later form the specialized locomotory and feeding velum (Fig. 2D). The primary trochoblasts are derived from 1q2 micromeres, accessory trochoblasts are derived from 1q1 derivatives, and secondary trochoblasts are derived from second-quartet micromeres (2q). The primary trochoblasts are autonomously specified, while the accessory and secondary trochoblasts require the inductive influ- ence of the D quadrant for normal development (Damen and Dictus, 1996). The caudofoveat larvae (Chaetoderma nitid- ulum) has been reported to posses three rows of trochoblasts based on an unpublished drawing by Dr. Gunnar Gustafsson (Nielsen, 2001), whereas only one row is known from the solenogaster larvae such as in Epimenia babai (Baba, 1938; Okusu, 2002), Neomenia carinata (Thompson, 1960), and Nematomenia banyulensis (Pruvot, 1890; Fig. 2E). Al- though the exact cell lineage is still not understood for the protobranch bivalves, some species such as Nucula proxima (Drew, 1899a), Acila castrensis (Zardus and Morse, 1998), and Yoldia limatua (Drew, 1899b) have three rows of ciliated cells that are situated in the similar position as the trochoblasts (Fig. 2F). In chitons, the prototroch is composed of two to three irregular rows of trochoblasts that are composed of 36 different cells (16 primary + 8 accessory + 12 secondary, Heath, 1899). The 1q2 gives rise to four trochoblasts in each quadrant, 1q1 gives rise to two cells in each quadrant, and 2q gives rise to four cells in each quadrant except for in the

Fig. 7. Relative contributions of the first and second quartet derivatives to the formation of the ocelli. All trochophore larvae are shown from the ventral surface, with the apical, anterior end located toward the top of the figure. Circled regions in B, D, F, and H indicate the exact positions of the corresponding ocelli shown in A, C, E, and G, respectively. (A–B) Corresponding high-magnification DIC and fluorescence light micrographs showing the contribution of the first-quartet micromere 1a to the formation of ectoderm and prototroch cells. Although superficial labeled cells lie close to the eye, the left ocellus is not labeled in B. (C–D) Corresponding high- magnification DIC and fluorescence light micrographs showing the contribution of the first quartet micromere 1c to the formation of ectoderm and prototroch cells. Note that the right ocellus is not labeled in D. (E, F) Corresponding high-magnification DIC and fluorescence light micrographs showing the contribution of the second-quartet micromere 2a to the formation of ectoderm, prototroch cells, and the left ocellus. Note that the left ocellus is labeled in F. (G, H) Corresponding high-magnification DIC and fluorescence light micrographs showing the contribution of the second- quartet micromere 2c to the formation of ectoderm, prototroch cells, and the right ocellus. Note that the right ocellus is labeled in H. All labels are the same as those used in Fig. 4. Scale bar equals 50 Am. J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 155

Fig. 8. Summary diagram illustrating the ectodermal and endodermal clones derived from each of the first- (1A–1D) and third-quartet macromeres (3A–3D), as indicated. All larvae are illustrated with the anterior ends oriented toward the top of the figure. For each micromere clone, dorsal (left of figure) and ventral (right of figure) views are presented. Red label indicates the distribution of external ectodermal tissues and the ocelli if labeled. Purple label indicates the distribution of deeper ectodermal tissues within the stomodeum and endodermal derivatives. 156 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160

Fig. 9. Diagram showing the lineage relationships and ultimate fates of each blastomere through the 24-cell stage (third-quartet formation).

D quadrant (in agreement with Heath, 1899). These are the cells by 3d (Fig. 4L). Other molluscs (Fig. 1B) such as the same cells that give rise to the prototroch in P. vulgata, solenogasters (and protobranch bivalves) have pericalymma indicating that subtle changes in the morphogenesis of the larvae with an enlarged larval ectodermal envelope called prototroch are derived from a common embryological origin the ‘‘test’’ within which the definitive adult structures (Patten, 1886). One unique feature in C. apiculata is the develop. The test cells are completely ciliated and arise at apparent contribution to the formation of a few prototrochal the animal pole. Because of their position, and the fact that J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 157 they are cast off, ingested, or absorbed at metamorphosis, only cells that have the developmental potential to give rise they are suggested to be homologous to the trochoblasts of to eyes, and because first-quartet micromeres are required trochophore larvae (Baba, 1938; Okusu, 2002). No accurate for the establishment of dorsoventral polarity (D quadrant cell lineage analysis has been performed on these forms, so formation). the homology of the test cells is still uncertain. Lineage Our work shows that chitons are different from other analysis in the paleonemertean Carinoma tremaphoros has spiralians because the eyes are normally derived from revealed the presence of prototroch cells for the first time in second-quartet derivatives. Together, these findings suggest any nemertean (Masalokova et al., 2004). that changes have occurred in the spatial and temporal induction of the eyes in these species. When van den Eye development and D quadrant induction Biggelaar (1996) deleted the first-quartet micromeres in the chiton Acanthochiton crinitus, no eyes formed and The most surprising modification of the spiralian fate map dorsoventral polarization was prevented. Because a fate in chitons is the origin of larval ocelli. Cell lineage experi- map has not been generated for A. crinitus, it was impos- ments performed on other molluscs (Ilyanassa obsoleta, sible to determine whether eyes did not form because they Render, 1991, 1997; L. stagnalis, Verdonk, 1965); poly- were generated from cells that had been deleted, or, because chaete worms (C. variopedatus, Henry and Martindale, eyes required the inductive interaction with the D quadrant 1987); nemerteans (Maslakova et al., 2004); and polyclad that did not form without first-quartet induction. Our results flatworms (Boyer et al., 1998) all indicate that larval ocelli suggest that the developmental potential to form eyes in are derived from first-quartet micromere derivatives of the A chitons is segregated to second-quartet derivatives and these and C quadrants (1a, 1c). This makes sense as first-quartet cells require the inductive interaction of the D quadrant that derivatives in all spiralians studied to date give rise to is set up by first-quartet micromere-third-quartet macromere pretrochal derivatives of the episphere and anterior rows of interactions. It is likely that the quadrant-specific differences the prototroch. The subepidermal, posterior location of the in contributions to the apical organ and prototroch are also eyes in chitons in the hyposphere suggests that first-quartet due to alterations in blastomere competence and the induc- derivatives migrated posteriorly to their final location. Our tive interactions with the D quadrant macromeres. It would cell lineage results indicate that, in fact, the larval ocelli are be interesting to delete second-quartet micromeres to see if generated from the second-quartet derivatives, 2a and 2c. first-quartet micromeres retain the developmental potential Thus, in one sense, the eyes of chitons are not strictly to give rise to larval ocelli. These hypotheses can be tested homologous with the eyes of other spiralians, although it experimentally and provide the hope of combining experi- has been argued that all larval eyes are homologous and were mental embryological techniques to understand the changes present in the ‘‘urbilaterian’’ (Arendt et al., 2002). that have occurred in cell fate determination in the spiralian If ones uses Van Valen’s (1982) definition of homology clade. as ‘‘resemblance caused by a continuity of information,’’ then the eyes of chitons could, in fact, be homologous with Shell formation those of other spiralians. In chitons, the developmental mechanism giving rise to eyes might simply be modified It has been suggested that the calcium carbonate spicules such that the developmental potential to give rise to eyes is of aplacophorans and polyplacophorans represent precur- segregated to a different set of cells. A fair amount of sors to the conchiferon shell (Salvini-Plawen, 1985; Haas, information is known about eye development in spiralians, 1981). These spicules would have fused to form polypla- particularly gastropod molluscs. Cell lineage and cell dele- cophoran shell plates, which then ultimately fused to form tion experiments have shown that the eyes normally arise the conchiferan shell (Haas, 1981). However, ontogenetic from first-quartet derivatives 1a and 1c, and that no other studies to support this hypothesis are lacking and the cells other than first-quartet micromeres are capable of homology among spicules/plates/shells and the polarity in generating larval ocelli (Sweet, 1998; Verdonk and van which they evolved is still a controversial issue (Eernisse den Biggelaar, 1983; Henry and Martindale, 1999).In and Reynolds, 1994). Aplacophoran and polyplacophoran equal-cleaving molluscs, including chitons, as well as in spicules have been suggested to be a more ‘‘primitive’’ type nemerteans, the D quadrant is determined by inductive of calcium carbonate deposition than that found in other interactions from first-quartet micromeres (Henry, 2002; molluscs. The mineral composition of aplacophoran and Martindale et al., 1985; van den Biggelaar, 1977, 1996; polyplacophoran spicules are generally known to be an van den Biggelaar and Guerrier, 1979). Subsequently, in aragonitic form of calcium carbonate (Haas, 1981; Carter both equal- and unequal-cleaving species, the 3D-quadrant and Hall, 1990; Scheltema, 1988). The formation of spi- macromere induces the first-quartet cells 1a and 1c to form cules in both groups begins with invagination of a single eyes (Clement, 1962; Lambert and Nagy, 2001; Sweet, basal secreting cell (BSC), within which calcium carbonate 1998). Thus, eyes do not form in equal-cleaving gastropod is precipitated extracellularly (Haas, 1981). As the spicules molluscs following the deletion of first-quartet micromeres continue to grow and protrude from the epithelium, the for two reasons, because first-quartet micromeres are the neighboring cells (NCs) surrounding the BSC secrete an 158 J.Q. Henry et al. / Developmental Biology 272 (2004) 145–160 organic pellicle onto the spicules, thus forming a closed Acknowledgments crystallization chamber. Most polyplacophorans have been observed to have this organic pellicle, while only some We would like to thank the faculty and students of the aplacophorans have been reported to have this structure MBL Embryology course for facilitating these studies. This (Haas, 1981). work was supported in part by NIH/NEI grant EY09844 to Heath (1899) claimed that the primary somatoblast (2d) JQH (JJH) and NSF grants to MQM. Also special thanks to contributed to the shell-forming region in Ischnochiton. Cell Woods Hole Oceanographic Institution, Drs. Ame´lie H. lineage studies in other molluscs have shown that the adult Scheltema, Gonzalo Giribet and Kenneth M. Halanych for shell is generated primarily from the 2d micromere, with their support for AO. The NSF PEET grant (#9521930) to less extensive contributions from 2a, 2b,and 2c and in some A.H. Scheltema supported the graduate education and cases 3c micromeres. 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