Larval and adult brains

Apical organs are a well-known structure in almost all ciliated eumetazoan larvae, although their function is poorly known. A review of the literature indicates that this small ganglion is the "brain" of the early larva, and it seems probable that it represents the brain of the ancestral, holopelagic ancestor of all eumetazoans, the gastraea. This early brain is lost before or at metamorphosis in all groups. Protostomes (excluding phoronids and brachiopods) appear to have brains of dual origin. Their larvae develop a pair of cephalic ganglia at the episphere lateral to the apical organ, and these two ganglia become an important part of the adult brain. The episphere and the cerebral ganglia show Otx expression, whereas Hox gene expression has not been seen in this part of the brain. A ventral nervous system develops around the blastopore, which becomes divided into mouth and anus by fusion of the lateral blastopore lips. The circumblastoporal nerve ring becomes differentiated into a nerve ring around the mouth, becoming part of the adult brain, a pair of ventral nerve cords, in some cases differentiated into a chain of ganglia, and a ring around the anus. This part of the nervous system appears to be homologous with the oral nerve ring of cnidarians. This interpretation is supported by the expression of Hox genes around the cnidarian mouth and in the ventral nervous system of the protostomes. The development of phoronids, brachiopods, echinoderms, and enteropneusts does not lead to the formation of an episphere or to differentiation of cerebral ganglia. In general, a well-defined brain is lacking, and Hox genes are generally not expressed in the larval organs, although this has not been well studied.     

The evolution of the serotonergic nervous system

The pattern of development of the serotonergic nervous system is described from the larvae of ctenophores, platyhelminths, nemerteans, entoprocts, ectoprocts (bryozoans), molluscs, polychaetes, brachiopods, phoronids, echinoderms, enteropneusts and lampreys. The larval brain (apical ganglion) of spiralian protostomes (except nermerteans) generally has three serotonergic neurons and the lateral pair always innervates the ciliary band of the prototroch. In contrast, brachiopods, phoronids, echinoderms and enteropneusts have numerous serotonergic neurons in the apical ganglion from which the ciliary band is innervated. This pattern of development is much like the pattern seen in lamprey embryos and larvae, which leads the author to conclude that the serotonergic raphe system found in vertebrates originated in the larval brain of deuterostome invertebrates. Further, the neural tube of chordates appears to be derived, at least in part, from the ciliary band of deuterostome invertebrate larvae. The evidence shows no sign of a shift in the dorsal ventral orientation within the line leading to the chordates.     

Monophyly of brachiopods and phoronids: reconciliation of molecular evidence with Linnaean classification (the subphylum Phoroniformea nov.)

Molecular phylogenetic analyses of aligned 18S rDNA gene sequences from articulate and inarticulate brachiopods representing all major extant lineages, an enhanced set of phoronids and several unrelated protostome taxa, confirm previous indications that in such data, brachiopod and phoronids form a well-supported clade that (on previous evidence) is unambiguously affiliated with protostomes rather than deuterostomes. Within the brachiopod-phoronid clade, an association between phoronids and inarticulate brachiopods is moderately well supported, whilst a close relationship between phoronids and craniid inarticulates is weakly indicated. Brachiopod-phoronid monophyly is reconciled with the most recent Linnaean classification of brachiopods by abolition of the phylum Phoronida and rediagnosis of the phylum Brachiopoda to include tubiculous, shell-less forms. Recognition that brachiopods and phoronids are close genealogical allies of protostome phyla such as molluscs and annelids, but are much more distantly related to deuterostome phyla such as echinoderms and chordates, implies either (or both) that the morphology and ontogeny of blastopore, mesoderm and coelom formation have been widely misreported or misinterpreted, or that these characters have been subject to extensive homoplasy. This inference, if true, undermines virtually all morphology-based reconstructions of phylogeny made during the past century or more.     

A twist in time--the evolution of spiral cleavage in the light of animal phylogeny

Recent progress in reconstructing animal relationships enables us to draw a better picture of the evolution of important characters such as organ systems and developmental processes. By mapping these characters onto the phylogenetic framework, we can detect changes that have occurred in them during evolution. The spiral mode of development is a complex of characters that is present in many lineages, such as nemerteans, annelids, mollusks, and polyclad platyhelminthes. However, some of these lineages show variations of this general program in which sub-characters are modified without changing the overlying pattern. Recent molecular phylogenies suggest that spiral cleavage was lost, or at least has deviated from its original pattern, in more lineages than was previously thought (e.g., in rotifers, gastrotrichs, bryozoans, brachiopods, and phoronids). Here, I summarize recent progress in reconstructing the spiralian tree of life and discuss its significance for our understanding of the spiral-cleavage character complex. I conclude that more detailed knowledge of the development of spiralian taxa is necessary to understand the mechanisms behind these changes, and to understand the evolutionary changes and adaptations of spiralian embryos.     

Some aspects of spiralian development

Nielsen, C. 2010. Some aspects of spiralian development. —Acta Zoologica (Stockholm) 91 : 20–28 Spiralian development is not only a characteristic early cleavage pattern, with shifting orientations of the cleavage planes, but also highly conserved cell lineages, where the origin of several organs can be traced back to identifiable cells in the lineage. These patterns are well documented in annelids, molluscs, nemertines, and platyhelminths and are considered ancestral of a bilaterian clade including these phyla. Spiral cleavage has not been documented in ecdysozoans, and no trace of the spiral development pattern is seen in phoronids and brachiopods. Origin of the spatial organization in spiralian embryos is puzzling, but much of the information appears to be encoded in the developing oocyte. Fertilization and “pseudofertilization” apparently provides the information defining the secondary, anterior‐posterior body axis in many species. The central nervous system consists of three components: an apical organ, derived from the apical blastomeres 1a¹¹¹‐1d¹¹¹, which degenerates before or at metamorphosis; the cerebral ganglia derived from other blastomeres of the first micromere quartet and retained in the adult as a preoral part of the brain; and the originally circumblastoporal nerve cord, which has become differentiated into a perioral part of the brain, the paired or secondarily fused ventral nerve cords, and a small perianal nerve ring.     

Morphology of gametes of mollusks, echinoderms, and brachiopods in systematics and phylogeny

The morphology of eggs and sperm of echinoderms, mollusks, and brachiopods was studied and compared. The gametes of inarticulate brachiopods (two classes Lingulata and Craniata and two subphyla Linguliformea and Craniaformea) are shown to have significant morphological differences from those of articulate brachiopods (extant class Rhynchonellata, subphylum Rhynchonelliformea). Inarticulate brachiopods have similar sperm morphology to that of primitive brachiopods, bivalves and some polychaetes that have external fertilization. Sperm morphology of articulate brachiopods is similar to that of echinoderms, which are considered to be typical deuterostomate invertebrates. This similarity supports an early deviation of lophophore-bearing animals from Bilateria, before this lineage branched into Protostomia and Deuterostomia. Similar gamete morphology in Lingulata and Craniata supports the view that inarticulate brachiopods should be retained as a supraclass taxon for comparison with other Lophotrochozoa, in particular with phoronids, bryozoans, and mollusks. Based on the new data on the gamete morphology in inarticulate brachiopods, we propose the name Lingulophyles with the type genus Lingula, and for articulate brachiopods Coptothyrophyles with the type genus Coptothyris.     

Trochophora larvae: cell-lineages, ciliary bands, and body regions. 1. Annelida and Mollusca

The trochophora concept and the literature on cleavage patterns and differentiation of ectodermal structures in annelids ("polychaetes") and molluscs are reviewed. The early development shows some variation within both phyla, and the cephalopods have a highly modified development. Nevertheless, there are conspicuous similarities between the early development of the two phyla, related to the highly conserved spiral cleavage pattern. Apical and cerebral ganglia have almost identical origin in the two phyla, and the cell-lineage of the prototroch is identical, except for minor variations between species. The cell-lineage of the metatrochs is almost unknown, but the telotroch of annelids and the "telotroch" of the gastropod Patella originate from the 2d-cell, as does the gastrotroch in the few species which have been studied. The segmented annelid body, i.e. the region behind the peristome, develops through addition of new ectoderm from a ring of 2d-cells just in front of the telotroch. This whole region is thus derived from 2d-cells. Conversely, the mollusc body is covered by descendants of cells from both the C and D quadrants and a growth zone is not apparent. This supports the notion that the molluscs are not segmented like the annelids, and that the repeated structures seen in polyplacophorans and monoplacophorans do not represent a segmentation homologous to that of the annelids.     

The Essential Role of "Minor" Phyla in Molecular Studies of Animal Evolution

SYNOPSIS. Molecular studies have revealed many new hypothesesof metazoan evolution in recent years. Previously, using morphologicalmethods, it was difficult to relate "minor" animal groups representingmicroscopic metazoans to larger, more well known groups suchas arthropods, molluscs, and annelids. Molecular studies suggestthat acanthocephalans evolved from rotifers, that priapulidsshare common ancestry with all other molting animals (Ecdysozoa),and that flatworms, gnathostomulids and rotifers form a sistergroup to the remaining non-molting protostomes (Lophotrochozoa),together forming Spiralia. The lophophorate phyla (phoronids,brachiopods and bryozoans) appear as protostomes, allied withannelids and molluscs rather than with deuterostomes. Thesefindings present a very different view of metazoan evolution,and clearly show that small and simple animals do not necessarilyrepresent ancestral or primitive taxa.     

Comparison of the neuromuscular systems among actinotroch larvae: systematic and evolutionary implications

A comparative analysis of the larval and presumptive juvenile neuromuscular systems among actinotroch larvae was performed using confocal laser microscopy with probes for F-actin and serotonin. Currently, there are two main categories of larval nervous systems based on the origin of the nerve fibers that innervate the larval tentacles. Characteristics of the serotonergic cells of the larval apical ganglion and juvenile nervous system have remained relatively conserved, but the structure of the secondary (hood) sense organ and the juvenile tentacles has diversified among species. Differences in larval musculature are mainly associated with differences in hood morphology. The presumptive, juvenile neuromuscular system is either integrated or separated from that of the larva based on the origin of the juvenile tentacles. Among species, the juvenile tentacles are made by remodeling the larval tentacles, developed from a basal tentacular thickening, or developed as a completely separate set in the larva. Differentiation of the neuromuscular structures of the juvenile tentacles is more diverse than their outward morphological characteristics would suggest. Importance of these larval characters is discussed in terms of current problems that exist within phoronid systematics. Evolutionary implications of these morphological characters are discussed among the phoronids, brachiopods, and related bilaterians. Overall, the integration or separation of larval and juvenile neuromuscular characters may yield insights into the evolution of lophotrochozoan body plans.     

Rerooting the rDNA gene tree reveals phoronids to be ‘brachiopods without shells’; dangers of wide taxon samples in metazoan phylogenetics (Phoronida; Brachiopoda)

Molecular phylogenetics has resulted in conflicting accounts of the relationship between phoronids and brachiopods. Taxonomically comprehensive analyses of brachiopod and phoronid ribosomal DNA sequences (rDNAs) rooted with short‐branched mollusc sequences uniformly find that phoronids nest within brachiopods as the sister of the three extant inarticulate lineages. Here, this is called the ‘alternate’ topology because it does not match traditional, morphology‐based ideas. Many other analyses of protein‐coding genes and/or rDNAs place phoronids elsewhere, often as the sister group of all brachiopods, better matching ‘traditional’ ideas. However, these analyses generally are based on data from small selections of brachiopods and phoronids, include data from a wide range of other metazoan taxa, and are rooted with distant outgroups. Here, I show that outgroup rooting of brachiopods and phoronid rDNAs is unreliable, and instead find the root position with procedures that are free from all distortions caused by distantly related taxa, i.e. by Bayesian and maximum likelihood relaxed‐clock analyses of a purely ingroup alignment. All such analyses confirm the ‘alternate’ topology: phoronids belong within the Brachiopoda as the sister group of the inarticulates. In addition, nine factors are identified that (singly or in combination) can cause misreporting of the phylogenetic signal in wide taxon‐range analyses of both rDNA and amino acid sequence data. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012.     

Comparison of articulate brachiopod nuclear and mitochondrial gene trees leads to a clade-based redefinition of protostomes (Protostomozoa) and deuterostomes (Deuterostomozoa)

Nuclear and mtDNA sequences from selected short-looped terebratuloid (terebratulacean) articulate brachiopods yield congruent and genetically independent phylogenetic reconstructions by parsimony, neighbour-joining and maximum likelihood methods, suggesting that both sources of data are reliable guides to brachiopod species phylogeny. The present-day genealogical relationships and geographical distributions of the tested terebratuloid brachiopods are consistent with a tethyan dispersal and subsequent radiation. Concordance of nuclear and mitochondrial gene phylogenies reinforces previous indications that articulate brachiopods, inarticulate brachiopods, phoronids and ectoprocts cluster with other organisms generally regarded as protostomes. Since ontogeny and morphology in brachiopods, ectoprocts and phoronids depart in important respects from those features supposedly diagnostic of protostomes, this demonstrates that the operational definition of protostomy by the usual ontological characters must be misleading or unreliable. New, molecular, operational definitions are proposed to replace the traditional criteria for the recognition of protostomes and deuterostomes, and the clade-based terms ''Protostomoza'' and ''Deuterostomozoa'' are proposed to replace the existing term ''Protostomia'' and ''Deuterostomia''.     

Larval and Adult Characters in Animal Phytogeny

SYNOPSIS. Protostomes and deuterostomes can be characterizedby two completely separate sets of characters. Protostome charactersare downstream- collecting ciliary bands with compound ciliaon multiciliate cells and central nervous system with apicalbrain and ventral paired or fused cords; spiral cleavage isonly found in the protostome group Spiralia. Deuterostome charactersare upstream-collecting ciliary systems with separate ciliaon monociliate cells, dorsal central nervous system and lossof the larval apical organ in the adults, and archimery withprosome, mesosome and metasome. Only the ectoproct bryozoanslack all the justmentioned characters; they are referred tothe protostomes on basis of their metamorphosis which resemblesthat of entoprocts. The two bilateral groups have probably evolvedindependently from a radial, gastraea-like ancestor     

Phoronid phylogenetics (Brachiopoda; Phoronata): evidence from morphological cladistics,small and large subunit rDNA sequences,and mitochondrial cox1

A matrix of 24 morphodevelopmental characters and an alignment of small subunit (SSU) and large subunit (LSU) rDNA nuclear and cox1 mitochondrial gene sequences (～4500 sites) were compiled from up to 12 phoronids including most named taxa, but probably constituting only a portion of worldwide diversity. Morphological data were analysed by weighted parsimony; sequence data by maximum and Bayesian likelihood, both with Phoronis ovalis as the local outgroup. Morphological and sequence‐based phylogenies were similar, but not fully congruent. Phoronid rDNAs were almost free from mutational saturation, but cox1 showed strong saturation unless distant outgroups and P. ovalis were omitted, suggesting that many phoronid divergences are old (≥100 Myr). rDNA divergence between named phoronid taxa is generally substantial, but Phoronopsis harmeri (from Vladivostock) and Phoronopsis viridis (from California) are genetically close enough to be conspecific. In another alignment, of 24 taxa, phoronid rDNAs were combined with data from brachiopods and distant (molluscan) outgroups. The relative ages of divergence between phoronids and their brachiopod sister‐groups, of the split between the P. ovalis and non‐ovalis lineages, and of other phoronid splits, were estimated from this alignment with a Bayesian lognormal uncorrelated molecular clock model. Although confidence limits (95% highest probability density) are wide, the results are compatible with an Early Cambrian split between phoronids and brachiopods and with the Upper Devonian latest age suggested for the P. ovalis/non‐ovalis split by the putative phoronid ichnofossil, Talpina. Most other ingroup splits appear to be ～50–200 Myr old. Inclusion of phoronids with brachiopods in the crown clade pan‐Brachiopoda suggests that a distinctive metamorphosis and absence of mineralization are ancestral phoronid apomorphies. Worldwide diversity and possible associations between character‐states and life‐history attributes deserve comprehensive further study.     

The unique developmental program of the acoel flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a "duet" cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell-cell inductive interactions in these embryos.     

PHYLOGENETIC RELATIONSHIPS AMONG EXTANT BRACHIOPODS

Abstract— The monophyletic status of the Brachiopoda and phylogenetic relationships within the phylum have long been contentious issues for brachiopod systematists. The relationship of brachiopods to other lophophore-bearing taxa is also uncertain; results from recent morphological and molecular studies are in conflict. To test current hypotheses of relationship, a phylogenetic analysis was completed (using PAUP 3.1.1) with 112 morphological and embryological characters that vary among extant representatives of seven brachiopod superfamilies, using bryozoans, phoronids, pterobranchs and sipunculids as outgroups. In the range of analyses performed, brachiopod monophyly is well supported, particularly by characters of soft anatomy. Arguments concerning single or multiple origins of a bivalved shell are not relevant to recognizing brachiopods as a clade. Articulate monophyly is very strongly supported, but inarticulate monophyly receives relatively weak support. Unlike previous studies, the nature of uncertainties about the clade status of Inarticulata are detailed explicitly here, making them easier to test in the future. Calcareous inarticulates appear to share derived characters with the other inarticulates, while sharing many primitive characters with other calcareous brachiopods (the articulates). Experimental manipulation of the data matrix reveals potential sources of bias in previous hypotheses of brachiopod phylogeny. Although not tested explicitly, lophophorate monophyly is very tentatively supported. Molecular systematic studies of a diverse group of brachiopods and other lophophorates will be particularly welcome in providing a test of the conclusions presented here.     

Dumbbell-shaped gogiid clusters: the oldest evidence of secondary tiering for stalked echinoderms

Among 381 specimens of Cambrian stalked echinoderms from eastern Guizhou, China examined, several slabs ( n  = 19) contain either dumbbell-shaped or v-shaped echinoderm clusters. Four slabs of Globoeocrinus globules Zhao, Parsley & Peng, 2008 from the middle-upper part (Cambrian Series 3 portion) of the Kaili Formation are prepared to reveal the attachment sites. Articulated gogiid echinoderms are reported to be attached to both sides of inarticulate (organophosphatic) brachiopods; thus, allowing me to interpret that the larvae of these gogiids were capable of attaching to live benthic brachiopods. This study documents the one of the earliest examples of echinoderms employing secondary tiering, which elevates an organism higher into the benthic boundary layer. Many of the gogiid echinoderm pairs attached to a live brachiopod are similar in size, indicating they were from a single larval spatfall.     

Molecular paleobiological insights into the origin of the Brachiopoda

Most studies of brachiopod evolution have been based on their extensive fossil record, but molecular techniques, due to their independence from the rock record, can offer new insights into the evolution of a clade. Previous molecular phylogenetic hypotheses of brachiopod interrelationships place phoronids within the brachiopods as the sister group to the inarticulates, whereas morphological considerations suggest that Brachiopoda is a monophyletic group. Here, these hypotheses were tested with a molecular phylogenetic analysis of seven nuclear housekeeping genes combined with three ribosomal genes. The combined analysis finds brachiopods to be monophyletic, but with relatively weak support, and the craniid as the sister taxon of all other brachiopods. Phylogenetic-signal dissection suggests that the weak support is caused by the instability of the craniid, which is attracted to the phoronids. Analysis of slowly evolving sites results in a robustly supported monophyletic Brachiopoda and Inarticulata (Linguliformea+Craniiformea), which is regarded as the most likely topology for brachiopod interrelationships. The monophyly of Brachiopoda was further tested with microRNA-based phylogenetics, which are small, noncoding RNA genes whose presence and absence can be used to infer phylogenetic relationships. Two novel microRNAs were characterized supporting the monophyly of brachiopods. Congruence of the traditional molecular phylogenetic analysis, microRNAs, and morphological cladograms suggest that Brachiopoda is monophyletic with Phoronida as its likely sister group. Molecular clock analysis suggests that extant phoronids have a Paleozoic divergence despite their conservative morphology, and that the early brachiopod fossil record is robust, and is not affected by taphonomic factors relating to the late-Precambrian/early-Cambrian phosphogenic event.     

Nervous system development of the sea cucumber Stichopus japonicus

The nervous system development of the sea cucumber Stichopus japonicus was investigated to explore the development of the bilateral larval nervous system into the pentaradial adult form typical of echinoderms. The first nerve cells were detected in the apical region of epidermis in the late gastrula. In the auricularia larvae, nerve tracts were seen along the ciliary band. There was a pair of bilateral apical ganglia consisted of serotonergic nerve cells lined along the ciliary bands. During the transition to the doliolaria larvae, the nerve tracts rearranged together with the ciliary bands, but they were not segmented and remained continuous. The doliolaria larvae possessed nerves along the ciliary rings but strongly retained the features of auricularia larvae nerve pattern. The adult nervous system began to develop inside the doliolaria larvae before the larval nervous system disappears. None of the larval nervous system was observed to be incorporated into the adult nervous system with immunohistochemistry. Since S. japonicus are known to possess an ancestral mode of development for echinoderms, these results suggest that the larval nervous system and the adult nervous system were probably formed independently in the last common ancestor of echinoderms.     

Evaluating neurophylogenetic patterns in the larval nervous systems of brachiopods and their evolutionary significance to other bilaterian phyla

Recent structural analyses of invertebrate nervous systems have supported hypotheses stating that specific developmental and cytological aspects of larval and adult brains are conserved among bilaterian animals. Opposing views argue that structural similarities in larval nervous systems may be the result of convergent evolution and that the developmental diversity of adult brains is more indicative of several independent origins. Here, I use various cytological probes, confocal microscopy, and reconstruction techniques to investigate the cellular diversity within the larval nervous systems of Glottidia pyramidata and Terebratalia transversa (Brachiopoda). Neuronal cell types are compared among the rhynchonelliform, linguliform, and craniiform brachiopods as well as the phoronids. Although the respective larval types of the previously mentioned systematic groups clearly diverge in the neuroarchitecture of their larval apical organs (and nervous systems in general), a ground plan is proposed based on shared, centrally‐located, peptidergic neuronal cell types that can be compared with similar cell types in other lophotrochozoan phyla (bryozoans and spiralians). Assessing hierarchal levels of homology within and among the nervous systems of morphologically disparate phyla is challenging in that many phyla share early developmental signals that induce the specification of the neural ectoderm, clouding our ability to discern divergent larval and juvenile brain structure. Solving these problems will require a combined effort involving both traditional and more recent cytological techniques with a diversity of molecular probes that will better map the neuronal complexity of diverse invertebrate nervous systems. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot - fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method - monensin treatment - yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.