Going where traditional markers have not gone before: utility of and promise for RAD sequencing in marine invertebrate phylogeography and population genomics.

Characterization of large numbers of single-nucleotide polymorphisms (SNPs) throughout a genome has the power to refine the understanding of population demographic history and to identify genomic regions under selection in natural populations. To this end, population genomic approaches that harness the power of next-generation sequencing to understand the ecology and evolution of marine invertebrates represent a boon to test long-standing questions in marine biology and conservation. We employed restriction-site-associated DNA sequencing (RAD-seq) to identify SNPs in natural populations of the sea anemone Nematostella vectensis, an emerging cnidarian model with a broad geographic range in estuarine habitats in North and South America, and portions of England. We identified hundreds of SNP-containing tags in thousands of RAD loci from 30 barcoded individuals inhabiting four locations from Nova Scotia to South Carolina. Population genomic analyses using high-confidence SNPs resulted in a highly-resolved phylogeography, a result not achieved in previous studies using traditional markers. Plots of locus-specific FST against heterozygosity suggest that a majority of polymorphic sites are neutral, with a smaller proportion suggesting evidence for balancing selection. Loci inferred to be under balancing selection were mapped to the genome, where 90% were located in gene bodies, indicating potential targets of selection. The results from analyses with and without a reference genome supported similar conclusions, further highlighting RAD-seq as a method that can be efficiently applied to species lacking existing genomic resources. We discuss the utility of RAD-seq approaches in burgeoning Nematostella research as well as in other cnidarian species, particularly corals and jellyfishes, to determine phylogeographic relationships of populations and identify regions of the genome undergoing selection.

Ventralization of an indirect developing hemichordate by NiCl2 suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms).

One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl2), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl2 disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior-posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl2 sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl2 sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan.

Vestigial prototroch in a basal nemertean, Carinoma tremaphoros (Nemertea; Palaeonemertea).

Nemerteans have been alleged to belong to a protostome clade called the Trochozoa that includes mollusks, annelids, sipunculids, echiurids, and kamptozoans and is characterized by, among other things, the trochophore larva. The trochophore possesses a prototroch, a preoral belt of specialized ciliary cells, derived from the trochoblast cells. Nemertea is the only trochozoan phylum for which presence of the trochophore larva possessing a prototroch had never been shown. However, so little is known about nemertean larval development that comparing it with development of other trochozoans is difficult. Development in the nemertean clade Pilidiophora is via a highly specialized planktonic larva, the pilidium, and most of the larval body is lost during a drastic metamorphosis. Other nemerteans (hoplonemerteans and palaeonemerteans) lack a pilidium, and their development is direct, forming either an encapsulated or planktonic "planuliform" larva, producing a juvenile without a dramatic change in body plan. We show that early in the development of a member of a basal nemertean assemblage, the palaeonemertean Carinoma tremaphoros, large squamous cells cover the entire larval surface except for the apical and posterior regions. Although apical and posterior cells continue to divide, the large surface cells cleavage arrest and form a contorted preoral belt. Based on its position, cell lineage, and fate, we suggest that this belt corresponds to the prototroch of other trochozoans. Lack of differential ciliation obscures the presence of the prototroch in Carinoma, but differentiation of the trochoblasts is clearly manifested in their permanent cleavage arrest and ultimate degenerative fate. Our results allow a meaningful comparison between the development of nemerteans and other trochozoans. We review previous hypotheses of the evolution of nemertean development and suggest that a trochophore-like larva is plesiomorphic for nemerteans while a pilidium type of development with drastic metamorphosis is derived.

Hox gene duplication and deployment in the annelid leech Helobdella.

The segmented leeches are members of the phylum Annelida within the Lophotrochozoa. Here, we describe the isolation of a new Hox gene, Lox18, in the leech Helobdella triserialis. Phylogenetic analysis indicates that Lox18 is a Deformed (Dfd) ortholog. H. triserialis has at least two Dfd orthologs, Lox18 and the previously described Lox6 (Kourakis et al. 1997; Wong and Macagno 1998), indicating that these genes duplicated after the last common ancestor of annelids and arthropods. Although the temporal appearance of Lox18 message is similar to that of Lox6, the spatial pattern is different. Lox18 does not have a sharply defined anterior border of expression in the second neuromere of the subesophageal ganglion of the central nervous system (CNS) as does Lox6, but is expressed uniformly in a small subset of cells in the longitudinal connectives and lateral roots in every segment of the CNS along the entire anterior-posterior (AP) axis. Even though Lox18 shares greater sequence similarity within the homeodomain and flanking regions to Drosophila Dfd than to the previously isolated Lox6, its expression pattern suggests that its function has diverged from the ancestral Hox function. Previous sampling has indicated that the last common ancestor of protostomes and deuterostomes had as many as 10 clustered Hox genes representing distinct paralogy groups (Irvine et al. 1997; de Rosa et al. 1999); leech Hox genes may have undergone subsequent and independent cluster or genome-wide duplication. These results point to the need for total genome level understanding for key members of the Lophotrochozoa.

The spatial and temporal expression of Ch-en, the engrailed gene in the polychaete Chaetopterus, does not support a role in body axis segmentation.

We are interested in understanding whether the annelids and arthropods shared a common segmented ancestor and have approached this question by characterizing the expression pattern of the segment polarity gene engrailed (en) in a basal annelid, the polychaete Chaetopterus. We have isolated an en gene, Ch-en, from a Chaetopterus cDNA library. Genomic Southern blotting suggests that this is the only en class gene in this animal. The predicted protein sequence of the 1.2-kb cDNA clone contains all five domains characteristic of en proteins in other taxa, including the en class homeobox. Whole-mount in situ hybridization reveals that Ch-en is expressed throughout larval life in a complex spatial and temporal pattern. The Ch-en transcript is initially detected in a small number of neurons associated with the apical organ and in the posterior portion of the prototrochophore. At later stages, Ch-en is expressed in distinct patterns in the three segmented body regions (A, B, and C) of Chaetopterus. In all segments, Ch-en is expressed in a small set of segmentally iterated cells in the CNS. In the A region, Ch-en is also expressed in a small group of mesodermal cells at the base of the chaetal sacs. In the B region, Ch-en is initially expressed broadly in the mesoderm that then resolves into one band/segment coincident with morphological segmentation. The mesodermal expression in the B region is located in the anterior region of each segment, as defined by the position of ganglia in the ventral nerve cord, and is involved in the morphogenesis of segment-specific feeding structures late in larval life. We observe banded mesodermal and ectodermal staining in an anterior-posterior sequence in the C region. We do not observe a segment polarity pattern of expression of Ch-en in the ectoderm, as is observed in arthropods.

Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi.

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required.

Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava.

Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister-phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect-developing representative of the enteropneust hemichordates, Ptychodera flava. Single blastomeres were iontophoretically labeled with Dil at the 2- through 16-cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct-developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect-developing echinoids. The 16-celled embryo contains eight animal "mesomeres," four slightly larger "macromeres," and four somewhat smaller vegetal "micromeres." The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct-developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two- or the four-cell stage are capable of forming normal-appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.

Regulation and regeneration in the ctenophore Mnemiopsis leidyi.

Lobate ctenophores (tentaculates) generally exhibit a remarkable ability to regenerate missing structures as adults. On the other hand, their embryos exhibit a highly mosaic behavior when cut into halves or when specific cells are ablated. These deficient embryos do not exhibit embryonic regulation, and generate incomplete adult body plans. Under certain conditions, however, these deficient animals are subsequently able to replace the missing structures during the adult phase in a process referred to as "post-regeneration." We have determined that successful post-regeneration can be predicted on the basis of a modified polar coordinate model, and the rules of intercalary regeneration, as defined by French et al. (V. French, P. J. Bryant, and S. V. Bryant, 1976, Science 193, 969-981.) The model makes certain assumptions about the organization of the ctenophore body plan that fit well with what we have determined on the basis of cell lineage fates maps, and their twofold rotational ("biradial") symmetry. The results suggest that cells composing the ctenophore adult body plan possess positional information, which is utilized to reconstruct the adult body plan. More specifically, we have found that the progeny of three specific cell lineages are required to support post-regeneration of the comb rows (the e(1), e(2), and m(1) micromeres). Furthermore, post-regeneration of the comb rows involves a suite of cell-cell inductive interactions, which are similar to those that take place during their embryonic formation. The significance of these findings is discussed in terms of the organization of the ctenophore body plan, and the mechanisms involved in cell fate specification. This situation is also contrasted with that of the atentaculate ctenophores, which are unable to undergo post-regeneration.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Combined-method phylogenetic analysis of Hox and ParaHox genes of the metazoa.

The clustered Hox genes show a conserved role in patterning the body axis of bilaterian metazoans. Increasingly, a broader phylogenetic sampling of non-model system organisms is being examined to detect a correlation, if any, between Hox gene evolution, and body plan innovations. To assess how Hox gene expression and function evolve with changing cluster arrangements, we must be able to reliably assign gene orthologies between Hox genes. Recent evidence suggests that a four-gene proto-Hox cluster duplicated to form the precursor of the present cluster and an additional sister-cluster, the ParaHox group. Here, phylogenetic methods are used to determine Hox-gene orthologies and to infer probable clustering events leading to the current bilaterian Hox complement. This analysis supports the ParaHox hypothesis and gives first confirmation that ind (intermediate neuroblasts defective) is an anterior ParaHox ortholog from protostomes. This analysis supports a proto-Hox cluster of four genes in which the central-class member of the ParaHox cluster may have been lost. It is also proposed here that ancestral diploblasts had central-class members of both Hox and ParaHox clusters. Primitive Hox gene ancestors are estimated by phylogenetic methods and found to have no strong affinity to any particular class of extant Hox members.

Expression patterns of anterior Hox genes in the polychaete Chaetopterus: correlation with morphological boundaries.

Expression patterns for five Hox genes were examined by whole-mount in situ hybridization in larvae of Chaetopterus, a polychaete annelid with a tagmatized axial body plan. Phylogenetic analysis demonstrates that these genes are orthologs of the Drosophila genes labial, proboscipedia, zen, Deformed, and Sex combs reduced and are termed CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4, and CH-Hox5, respectively. Expression studies reveal a biphasic expression pattern. In early larval stages, well before any indications of segmental organization exist, a novel pattern of expression in bilateral posterior proliferating cell populations, corresponding to the teloblasts, was detected for each of the genes, with CH-Hox1 and CH-Hox2 expressed before the remaining three. In middle larval stages, all five genes are expressed in bilateral strips along the ventral midline, corresponding with the developing ventral nerve cord. In addition, CH-Hox1 and CH-Hox2 show strong expression at the foregut-midgut boundary. By late larval stages the expression is generally confined to the ventral CNS and ectoderm of the anterior parapodia. Anterior boundaries of expression are "colinear," at later larval stages, with CH-Hox2 expressed most rostrally, in the first segment, and anterior expression boundaries for CH-Hox1, CH-Hox3, CH-Hox4, and CH-Hox5 in segments 2, 3, 4, and 5, respectively. Like vertebrates and spiders, but unlike insects, CH-Hox3 participates in this colinear axial expression pattern. CH-Hox1 and CH-Hox2 have distinct posterior boundaries of expression in the ninth segment, which corresponds to a major morphological boundary, and may reflect a reorganization of Hox gene regulation related to the evolutionary reorganization of the Chaetopterus body plan.

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.

Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages.

Ctenophores are marine invertebrates that develop rapidly and directly into juvenile adults. They are likely to be the simplest metazoans possessing definitive muscle cells and are possibly the sister group to the Bilateria. All ctenophore embryos display a highly stereotyped, phylum-specific pattern of development in which every cell can be identified by its lineage history. We generated a cell lineage fate map for Mnemiopsis leidyi by injecting fluorescent lineage tracers into individual blastomeres up through the 60-cell stage. The adult ctenophore body plan is composed of four nearly identical quadrants organized along the oral-aboral axis. Each of the four quadrants is derived largely from one cell of the four-cell-stage embryo. At the eight-cell stage each quadrant contains a single E ("end") and M ("middle") blastomere. Subsequently, micromeres are formed first at the aboral pole and later at the oral pole. The ctene rows, apical organ, and tentacle apparatus are complex structures that are generated by both E and M blastomere lineages from all four quadrants. All muscle cells are derived from micromeres born at the oral pole of endomesodermal precursors (2M and 3E macromeres). While the development of the four quadrants is similar, diagonally opposed quadrants share more similarities than adjacent quadrants. Adult ctenophores possess two diagonally opposed endodermal anal canals that open at the base of the apical organ. These two structures are derived from the two diagonally opposed 2M/ macromeres. The two opposing 2M/ macromeres generated a unique set of circumpharyngeal muscle cells, but do not contribute to the anal canals. No other lineages displayed such diagonal asymmetries. Clones from each blastomere yielded regular, but not completely invariant patterns of descendents. Ectodermal descendents normally, but not always, remained within their corresponding quadrants. On the other hand, endodermal and mesodermal progeny dispersed throughout the body. The variability in the exact complements of adult structures, along with previously published cell deletion experiments, demonstrates that cell interactions are required for normal cell fate determination. Ctenophore embryos, like those of many bilaterian phyla (e.g., spiralians, nematodes, and echinoids), display a highly stereotyped cleavage program in which some, but not all, blastomeres are determined at the time of their birth. The results suggest that mesodermal tissues originally evolved from endoderm tissue.

Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria.

Among the bilaterally symmetrical, triploblastic animals (the Bilateria), a conserved set of developmental regulatory genes are known to function in patterning the anterior-posterior (AP) axis. This set includes the well-studied Hox cluster genes, and the recently described genes of the ParaHox cluster, which is believed to be the evolutionary sister of the Hox cluster (Brooke et al. 1998). The conserved role of these axial patterning genes in animals as diverse as frogs and flies is believed to reflect an underlying homology (i.e., all bilaterians derive from a common ancestor which possessed an AP axis and the developmental mechanisms responsible for patterning the axis). However, the origin and early evolution of Hox genes and ParaHox genes remain obscure. Repeated attempts have been made to reconstruct the early evolution of Hox genes by analyzing data from the triphoblastic animals, the Bilateria (Schubert et al. 1993; Zhang and Nei 1996). A more precise dating of Hox origins has been elusive due to a lack of sufficient information from outgroup taxa such as the phylum Cnidaria (corals, hydras, jellyfishes, and sea anemones). In combination with outgroup taxa, another potential source of information about Hox origins is outgroup genes (e.g., the genes of the ParaHox cluster). In this article, we present cDNA sequences of two Hox-like genes (anthox2 and anthox6) from the sea anemone, Nematostella vectensis. Phylogenetic analysis indicates that anthox2 (= Cnox2) is homologous to the GSX class of ParaHox genes, and anthox6 is homologous to the anterior class of Hox genes. Therefore, the origin of Hox genes and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria and predated the evolution of the anterior-posterior axis of bilaterian animals. Our analysis also suggests that the central Hox class was invented in the bilaterian lineage, subsequent to their split from the Cnidaria.

Larval ontogenetic stages of Chaetopterus: developmental heterochrony in the evolution of chaetopterid polychaetes.

Seven post-gastrulation larval stages are described for the sedentary polychaete Chaetopterus. Analysis of larval anatomy and morphology through ontogeny reveals significant differences in the temporal sequence of segmentation, and in the character of segments formed, from the typical embryological pattern described for other polychaete families, such as nereidids or spionids. When compared in alternative phylogenetic schemes, these differences represent significant developmental heterochrony, among other evolutionary transitions, which has arisen in the chaetopterid lineage. The heterochrony is correlated with the extreme morphological regionalization along the anterior-posterior body axis, a feature that is also characteristic of chaetopterids.

The cell lineage of a polyclad turbellarian embryo reveals close similarity to coelomate spiralians.

Recent molecular evidence suggests the turbellarian Platyhelminthes may represent the extant basal members of the Spiralia and therefore probably exhibit ancient features of the spiralian developmental program. The stereotypic quartet spiral cleavage pattern of the polyclad turbellarian embryo, among other features, indicates that this group may be closely related to the ancestral flatworm; however, polyclad embryos have been the subject of few experimental studies. Here we report the results of a cell lineage analysis of the embryo of the polyclad Hoploplana inquilina based on microinjection of DiI into cleavage-stage blastomeres following formation of each of the four quartets of micromeres. The first quartet gives rise to most of the lateral and anterior ectoderm of the Müller's larva; the second quartet forms largely dorsal and ventral ectoderm as well as the circular muscles; the third quartet forms only small clones of ectoderm; and only the 4d cell of the fourth quartet contributes to larval structure, forming the longitudinal muscles, mesenchyme, and probably endoderm. Our results demonstrate a striking similarity between the cell lineages of polyclad and higher spiralian embryos, in which the four quadrants also bear the same relationships to the larval axes and give rise to comparable larval structures, including derivation of mesoderm from both ectodermal (2b) and endodermal precursors (4d).

Conservation of the spiralian developmental program: cell lineage of the nemertean, Cerebratulus lacteus.

Lineage tracers were injected into individual blastomeres in embryos of the indirect-developing nemertean Cerebratulus lacteus through the formation of the fourth quartet of micromeres. Subsequent development was followed to the formation of feeding pilidium larvae to establish their ultimate fates. Results showed that these blastomeres have unique fates, and their clones give rise to highly predictable regions of the larval body. As in other spiralians, four discrete cell quadrants can be identified. For the most part, their identities are homologous to the typical spiralian A, B, C, and D cell quadrants. In some respects their fates differ from the typical spiralian fate map; however, these can be understood in terms of simple modifications of the early cleavage program. Unlike most spiralians, the first quartet micromeres in the eight-celled embryo are larger than the corresponding vegetal macromeres, and generate most of the larval ectoderm. All four of these micromeres contribute to the apical organ and generate four bilaterally situated domains of ectoderm, where the progeny of the 1a and 1d micromeres lie to the left of the median plane while those of 1b and 1c lie to the right. Unlike the progeny of the first quartet, those of the second quartet are situated in left (2a), ventral (2b), right (2c), and dorsal (2d) positions. The third quartet micromeres generate clones situated in a bilaterally symmetrical fashion similar to those of the first quartet. The alternating axial relationships exhibited by successive micromere quartets are a characteristic of spiralian development. Unlike other spiralian larvae possessing a ciliary band, the pilidium larval ciliary band is formed by all blastomeres of the first and second micromere quartets, as well as 3c and 3d. Ectomesoderm is derived from two blastomeres (3a and 3b), which give rise to the extensive array of the larval muscle cells. C. lacteus also possesses a true mesentoblast (4d) which gives rise to a pair of mesodermal bandlets, and scattered mesenchymal cells. The dual origin of the mesoderm, as both ectomesoderm and endomesoderm, appears to be a condition present in all spiralians. The gut is formed by all the fourth quartet micromeres as well as the vegetal macromeres (4A, 4B, 4C, 4D). Despite differences in the determination of axial properties and some modifications in quadrant fates, nemerteans appear to be constructed on the typical spiralian developmental platform.

The evolution of the Hox cluster: insights from outgroups.

Two burgeoning research trends are helping to reconstruct the evolution of the Hox cluster with greater detail and clarity. First, Hox genes are being studied in a broader phylogenetic sampling of taxa: the past year has witnessed important new data from teleost fishes, onychophorans, myriapods, polychaetes, glossiphoniid leeches, ribbon worms, and sea anemones. Second, commonly accepted notions of animal relationships are being challenged by alternative phylogenetic hypotheses that are causing us to rethink the evolutionary relationships of important metazoan lineages, especially arthropods, annelids, nematodes, and platyhelminthes.