Postembryonic development and male paedomorphosis in Osedax (Siboglinidae, Annelida).

Most species of the bone-devouring marine annelid, Osedax, display distinct sexual dimorphism with macroscopic sedentary females rooted in bones and free-living microscopic dwarf males. The paedomorphic male resembles the non-feeding metatrochophore larva in size, presence of eight pairs of chaetae, and a head ciliation potentially representing a residual prototroch. The male development may thus uniquely reiterate and validate the theoretical heterochrony process "progenesis", which suggests that an accelerated sexual maturation and early arrest of somatic growth can lead to a miniaturized and paedomorphic adult. In this study, we describe the postembryonic larval and juvenile organogenesis of Osedax japonicus to test for a potential synchronous arrest of somatic growth during male development. Five postembryonic stages could be distinguished, resembling day one to five in the larval development at 10°C: (0D) first cleavage of fertilized eggs (embryos undergo unequal spiral cleavage), (1D) pre-trochophore, with apical organ, (2D) early trochophore, + prototroch, brain, circumesophageal connectives and subesophageal commissure, (3D) trochophore, + telotroch, four ventral nerves, (4D) early metatrochophore, + protonephridia, dorsal and terminal sensory organs, (5D) metatrochophore, + two ventral paratrochs, mid-ventral nerve, posterior trunk commissure, two dorsal nerves; competent for metamorphosis. The larval development largely mirrors that of other lecithotrophic annelid larvae but does not show continuous chaetogenesis or full gut development. Additionally, O. japonicus larvae exhibit an unpaired, mid-dorsal, sensory organ. Female individuals shed their larval traits during metamorphosis and continue organogenesis (including circulatory system) and extensive growth for 2-3 weeks before developing oocytes. In contrast, males develop sperm within a day of metamorphosis and display a synchronous metamorphic arrest in neural and muscular development, retaining a large portion of larval features post metamorphosis. Our findings hereby substantiate male miniaturization in Osedax to be the outcome of an early and synchronous offset of somatic development, fitting the theoretical process "progenesis". This may be the first compelling morpho-developmental exemplification of a progenetic origin of a microscopic body plan. The presented morphological staging system will further serve as a framework for future examination of molecular patterns and pathways determining Osedax development.

Sifting through the mud: A tale of building the annelid Capitella teleta for EvoDevo studies.

Over the last few decades, the annelid Capitella teleta has been used increasingly as a study system for investigations of development and regeneration. Its favorable properties include an ability to continuously maintain a laboratory culture, availability of a sequenced genome, a stereotypic cleavage program of early development, substantial regeneration abilities, and established experimental and functional genomics techniques. With this review I tell of my adventure of establishing the Capitella teleta as an emerging model and share examples of a few of the contributions our work has made to the fields of evo-devo and developmental biology. I highlight examples of conservation in developmental programs as well as surprising deviations from existing paradigms that highlight the importance of leveraging biological diversity to shift thinking in the field. The story for each study system is unique, and every animal has its own advantages and disadvantages as an experimental system. Just like most progress in science, it takes strategy, hard work and determination to develop tools and resources for a less studied animal, but luck and serendipity also play a role. I include a few narratives to personalize the science, share details of the story that are not included in typical publications, and provide perspective for investigators who are interested in developing their own study organism.

Regeneration in the Segmented Annelid Capitella teleta.

The segmented worms, or annelids, are a clade within the Lophotrochozoa, one of the three bilaterian superclades. Annelids have long been models for regeneration studies due to their impressive regenerative abilities. Furthermore, the group exhibits variation in adult regeneration abilities with some species able to replace anterior segments, posterior segments, both or neither. Successful regeneration includes regrowth of complex organ systems, including the centralized nervous system, gut, musculature, nephridia and gonads. Here, regenerative capabilities of the annelid Capitella teleta are reviewed. C. teleta exhibits robust posterior regeneration and benefits from having an available sequenced genome and functional genomic tools available to study the molecular and cellular control of the regeneration response. The highly stereotypic developmental program of C. teleta provides opportunities to study adult regeneration and generate robust comparisons between development and regeneration.

Functional evidence that Activin/Nodal signaling is required for establishing the dorsal-ventral axis in the annelid Capitella teleta.

The TGF-ß superfamily comprises two distinct branches: the Activin/Nodal and BMP pathways. During development, signaling by this superfamily regulates a variety of embryological processes, and it has a conserved role in patterning the dorsal-ventral body axis. Recent studies show that BMP signaling establishes the dorsal-ventral axis in some mollusks. However, previous pharmacological inhibition studies in the annelid Capitella teleta, a sister clade to the mollusks, suggests that the dorsal-ventral axis is patterned via Activin/Nodal signaling. Here, we determine the role of both the Activin/Nodal and BMP pathways as they function in Capitella axis patterning. Antisense morpholino oligonucleotides were targeted to Ct-Smad2/3 and Ct-Smad1/5/8, transcription factors specific to the Activin/Nodal and BMP pathways, respectively. Following microinjection of zygotes, resulting morphant larvae were scored for axial anomalies. We demonstrate that the Activin/Nodal pathway of the TGF-ß superfamily, but not the BMP pathway, is the primary dorsal-ventral patterning signal in Capitella These results demonstrate variation in the molecular control of axis patterning across spiralians, despite sharing a conserved cleavage program. We suggest that these findings represent an example of developmental system drift.

Activin/Nodal signaling mediates dorsal-ventral axis formation before third quartet formation in embryos of the annelid Chaetopterus pergamentaceus.

BACKGROUND: The clade of protostome animals known as the Spiralia (e.g., mollusks, annelids, nemerteans and polyclad flatworms) shares a highly conserved program of early development. This includes shared arrangement of cells in the early-stage embryo and fates of descendant cells into embryonic quadrants. In spiralian embryos, a single cell in the D quadrant functions as an embryonic organizer to pattern the body axes. The precise timing of the organizing signal and its cellular identity varies among spiralians. Previous experiments in the annelid Chaetopterus pergamentaceus Cuvier, 1830 demonstrated that the D quadrant possesses an organizing role in body axes formation; however, the molecular signal and exact cellular identity of the organizer were unknown. RESULTS: In this study, the timing of the signal and the specific signaling pathway that mediates organizing activity in C. pergamentaceus was investigated through short exposures to chemical inhibitors during early cleavage stages. Chemical interference of the Activin/Nodal pathway but not the BMP or MAPK pathways results in larvae that lack a detectable dorsal-ventral axis. Furthermore, these data show that the duration of organizing activity encompasses the 16 cell stage and is completed before the 32 cell stage. CONCLUSIONS: The timing and molecular signaling pathway of the C. pergamentaceus organizer is comparable to that of another annelid, Capitella teleta, whose organizing signal is required through the 16 cell stage and localizes to micromere 2d. Since C. pergamentaceus is an early branching annelid, these data in conjunction with functional genomic investigations in C. teleta hint that the ancestral state of annelid dorsal-ventral axis patterning involved an organizing signal that occurs one to two cell divisions earlier than the organizing signal identified in mollusks, and that the signal is mediated by Activin/Nodal signaling. Our findings have significant evolutionary implications within the Spiralia, and furthermore suggest that global body patterning mechanisms may not be as conserved across bilaterians as was previously thought.

Genes with spiralian-specific protein motifs are expressed in spiralian ciliary bands.

Spiralia is a large, ancient and diverse clade of animals, with a conserved early developmental program but diverse larval and adult morphologies. One trait shared by many spiralians is the presence of ciliary bands used for locomotion and feeding. To learn more about spiralian-specific traits we have examined the expression of 20 genes with protein motifs that are strongly conserved within the Spiralia, but not detectable outside of it. Here, we show that two of these are specifically expressed in the main ciliary band of the mollusc Tritia (also known as Ilyanassa). Their expression patterns in representative species from five more spiralian phyla-the annelids, nemerteans, phoronids, brachiopods and rotifers-show that at least one of these, lophotrochin, has a conserved and specific role in particular ciliated structures, most consistently in ciliary bands. These results highlight the potential importance of lineage-specific genes or protein motifs for understanding traits shared across ancient lineages.

Functional role of pax6 during eye and nervous system development in the annelid Capitella teleta.

The transcription factor Pax6 is an important regulator of early animal development. Loss of function mutations of pax6 in a range of animals result in a reduction or complete loss of the eye, a reduction of a subset of neurons, and defects in axon growth. There are no studies focusing on the role of pax6 during development of any lophotrochozoan representative, however, expression of pax6 in the developing eye and nervous system in a number of species suggest that pax6 plays a highly conserved role in eye and nervous system formation. We investigated the functional role of pax6 during development of the marine annelid Capitella teleta. Expression of pax6 transcripts in C. teleta larvae is similar to patterns found in other animals, with distinct subdomains in the brain and ventral nerve cord as well as in the larval and juvenile eye. To perturb pax6 function, two different splice-blocking morpholinos and a translation-blocking morpholino were used. Larvae resulting from microinjections with either splice-blocking morpholino show a reduction of the pax6 transcript. Development of both the larval eyes and the central nervous system architecture are highly disrupted following microinjection of each of the three morpholinos. The less severe phenotype observed when only the homeodomain is disrupted suggests that presence of the paired domain is sufficient for partial function of the Pax6 protein. Preliminary downstream target analysis confirms disruption in expression of some components of the retinal gene regulatory network, as well as disruption of genes involved in nervous system development. Results from this study, taken together with studies from other species, reveal an evolutionarily conserved role for pax6 in eye and neural specification and development.

Investigation into the cellular origins of posterior regeneration in the annelid Capitella teleta.

Many animals can regenerate, although there is great diversity in regenerative capabilities. A major question in regenerative biology is determining the cellular source of newly formed tissue. The polychaete annelid, Capitella teleta, can regenerate posterior segments following transverse amputation. However, the source, behavior and molecular characteristics of the cells that form new tissue during regeneration are largely unknown. Using an indirect cell tracking method involving 5'-ethynyl-2'-deoxyuridine (EdU) incorporation, we show that cell migration occurs during C. teleta posterior regeneration. Expression of the multipotency/germ line marker CapI-vasa led us to hypothesize that stem cells originate from a multipotent progenitor cell (MPC) cluster, migrate through the coelomic cavity, and contribute to regeneration of tissue. We show that the capacity for posterior regeneration and segment formation is greater with than without the MPC cluster. Finally, we propose a working model of posterior regeneration in C. teleta. This work is the first in C. teleta that addresses the potential source of cells contributing to posterior regeneration, and may provide clues as to why some animals are highly successful regenerators.

Regeneration of the germline in the annelid Capitella teleta.

The germline is essential for sexual reproduction and survival of the species. In many metazoans, the developmental potential to generate a distinct germline is segregated from somatic cell lineages early in embryogenesis, suggesting that the unique features of the germline must be established from its onset. Previous studies suggest that germ cells cannot regenerate once removed from the embryo, but few animals have been experimentally tested. We investigated the ability of the germline to regenerate in a lophotrochozoan, the segmented worm Capitella teleta, which has a stereotyped cell lineage program by deleting the germline precursor (cell 3D) in early stage embryos using an infrared laser. Larvae and juveniles resulting from germline deletions were examined for presence of multipotent progenitor cells (MPCs), stem cells that form the germ cells and somatic stem cells. In contrast to control deletions of a non-germline macromere, most larvae resulting from deletion of cell 3D lacked MPCs as assayed by expression of germline markers CapI-vasa, CapI-nanos and Ct-piwi1, but showed persistent expression of these markers in the somatic posterior growth zone. However, approximately 13% of experimental larvae had MPCs, indicative of some germline regeneration. In contrast, by two weeks post-metamorphosis, all juveniles resulting from deletion of cell 3D had MPCs, as detected by CapI-vasa expression. Furthermore, when raised to adulthood, most animals developed reproductive structures and were fertile. In another set of deletions, both the D quadrant mesodermal and germline progenitors were removed. These juveniles also regenerated MPCs. Surprisingly, this deletion caused substantial ectopic expression of CapI-vasa and CapI-nanos in other larval tissues. Our results indicate that C. teleta can regenerate the germline following removal of the germline progenitors in the early embryo. The dramatic difference in ability to regenerate the germline between the larval and adult stages suggests that there are two distinct compensation events at two phases of the life cycle: a regulative event in the early stage larva and a stem cell transition event after metamorphosis, when the animals are capable of substantial body regeneration.

An organizing role for the TGF-ß signaling pathway in axes formation of the annelid Capitella teleta.

Embryonic organizers are signaling centers that coordinate developmental events within an embryo. Localized to either an individual cell or group of cells, embryonic organizing activity induces the specification of other cells in the embryo and can influence formation of body axes. In the spiralian Capitella teleta, previous cell deletion studies have shown that organizing activity is localized to a single cell, 2d, and this cell induces the formation of the dorsal-ventral axis and bilateral symmetry. In this study, we attempt to identify the signaling pathway responsible for the organizing activity of 2d. Embryos at stages when organizing activity is occurring were exposed to various small molecule inhibitors that selectively inhibited either the Activin/Nodal or the BMP branch of the TGF-ß signaling pathway. Embryos were then raised to larval stages, and scored for axial anomalies analogous to 2d ablated phenotypes. Our results show that interference with the Activin/Nodal pathway through a short three hour exposure to the inhibitor SB431542 results in larvae that lack bilateral symmetry and a detectable dorsal-ventral axis. However, interference with the BMP signaling pathway through exposure to the inhibitors DMH1 and dorsomorphin dihydrochloride does not appear to play a role in specification by 2d of the dorsal-ventral axis or bilateral symmetry. Our findings highlight species differences in how the molecular architecture of the conserved TGF-ß superfamily signaling pathway components was utilized to mediate the organizing activity signal during early spiralian development.

Spatiotemporal regulation of nervous system development in the annelid Capitella teleta.

BACKGROUND: How nervous systems evolved remains an unresolved question. Previous studies in vertebrates and arthropods revealed that homologous genes regulate important neurogenic processes such as cell proliferation and differentiation. However, the mechanisms through which such homologs regulate neurogenesis across different bilaterian clades are variable, making inferences about nervous system evolution difficult. A better understanding of neurogenesis in the third major bilaterian clade, Spiralia, would greatly contribute to our ability to deduce the ancestral mechanism of neurogenesis. RESULTS: Using whole-mount in situ hybridization, we examined spatiotemporal gene expression for homologs of soxB, musashi, prospero, achaete-scute, neurogenin, and neuroD in embryos and larvae of the spiralian annelid Capitella teleta, which has a central nervous system (CNS) comprising a brain and ventral nerve cord. For all homologs examined, we found expression in the neuroectoderm and/or CNS during neurogenesis. Furthermore, the onset of expression and localization within the developing neural tissue for each of these genes indicates putative roles in separate phases of neurogenesis, e.g., in neural precursor cells (NPCs) versus in cells that have exited the cell cycle. Ct-soxB1, Ct-soxB, and Ct-ngn are the earliest genes expressed in surface cells in the anterior and ventral neuroectoderm, while Ct-ash1 expression initiates slightly later in surface neuroectoderm. Ct-pros is expressed in single cells in neural and non-neural ectoderm, while Ct-msi and Ct-neuroD are localized to differentiating neural cells in the brain and ventral nerve cord. CONCLUSIONS: These results suggest that the genes investigated in this article are involved in a neurogenic gene regulatory network in C. teleta. We propose that Ct-SoxB1, Ct-SoxB, and Ct-Ngn are involved in maintaining NPCs in a proliferative state. Ct-Pros may function in division of NPCs, Ct-Ash1 may promote cell cycle exit and ingression of NPC daughter cells, and Ct-NeuroD and Ct-Msi may control neuronal differentiation. Our results support the idea of a common genetic toolkit driving neural development whose molecular architecture has been rearranged within and across clades during evolution. Future functional studies should help elucidate the role of these homologs during C. teleta neurogenesis and identify which aspects of bilaterian neurogenesis may have been ancestral or were derived within Spiralia.

Annelid models I: Capitella teleta.

Investigating development in a wide variety of organisms allows researchers to take advantage of both diverse and conserved evolutionary solutions that animals have made to solve a range of biological problems. Next generation sequencing and functional genomic techniques can now be applied to many animals, providing opportunities to study organisms whose biology offers advantages for tracking particular questions. Capitella teleta is an segmented annelid worm whose phylogenetic position, stable genome, environmental resiliency, early development and regeneration capabilities position it to provide insights to its evolutionary success. This review discusses attributes of C. teleta that make it amenable for a variety of development and regeneration studies.

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta.

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.

Regulative capacity for eye formation by first quartet micromeres of the polychaete Capitella teleta.

The stereotypic cleavage pattern shared by spiralian embryos provides unique opportunities to compare mechanisms of cell fate specification of homologous blastomeres, and can give insights into how changes in fate may have influenced the evolution of novel structures and morphological diversity. The potential of cells to undergo regulation and the timing of cell fate specification were investigated during early development in the polychaete annelid, Capitella teleta. Targeted laser deletions of the first quartet micromeres were performed, with a focus on the eye-forming cells 1a and 1c. Most of the larvae resulting from deletion of the 1a or 1c micromeres lack both the pigment cell and sensory cell of the eye as predicted by the C. teleta fate map. In a minority of cases, however, both left and right larval eye spots develop, suggesting that other blastomeres within the embryo regulate for loss of these cells. Deletion of the 1a and 1c derivatives, 1a(1) or 1c(1), also largely result in larvae with one pigment spot, although there are larvae with two eye spots, suggesting that the ability to regulate for loss of an eye-generating cell persists for an additional cell cycle. Cell deletion in conjunction with intracellular labeling indicates that all four quadrants retain the ability to generate eyes, including those that normally do not. Deletion of all four first quartet micromeres provides evidence that only the first quartet micromeres have eye-forming potential. Additionally, in contrast to the right side of the head where larval and adult eye sensory cells are derived from the same cell (1c), on the left side, the larval and adult eye sensory cells are generated by different embryonic lineages. We hypothesize that cell-cell interactions and cell position are important for regulative ability in Capitella. To our knowledge, this is one of the first detailed deletion studies of the first quartet micromeres and the first convincing example of regulation in polychaetes, which are often thought to be non-regulative in nature.

Nervous system development in lecithotrophic larval and juvenile stages of the annelid Capitella teleta.

BACKGROUND: Reconstructing the evolutionary history of nervous systems requires an understanding of their architecture and development across diverse taxa. The spiralians encompass diverse body plans and organ systems, and within the spiralians, annelids exhibit a variety of morphologies, life histories, feeding modes and associated nervous systems, making them an ideal group for studying evolution of nervous systems. RESULTS: We describe nervous system development in the annelid Capitella teleta (Blake JA, Grassle JP, Eckelbarger KJ. Capitella teleta, a new species designation for the opportunistic and experimental Capitella sp. I, with a review of the literature for confirmed records. Zoosymposia. 2009;2:25-53) using whole-mount in situ hybridization for a synaptotagmin 1 homolog, nuclear stains, and cross-reactive antibodies against acetylated α-tubulin, 5-HT and FMRFamide. Capitella teleta is member of the Sedentaria (Struck TH, Paul C, Hill N, Hartmann S, Hosel C, Kube M, et al. Phylogenomic analyses unravel annelid evolution. Nature. 2011;471:95-8) and has an indirectly-developing, lecithotrophic larva. The nervous system of C. teleta shares many features with other annelids, including a brain and a ladder-like ventral nerve cord with five connectives, reiterated commissures, and pairs of peripheral nerves. Development of the nervous system begins with the first neurons differentiating in the brain, and follows a temporal order from central to peripheral and from anterior to posterior. Similar to other annelids, neurons with serotonin-like-immunoreactivity (5HT-LIR) and FMRFamide-like-immunoreactivity (FMRF-LIR) are found throughout the brain and ventral nerve cord. A small number of larval-specific neurons and neurites are present, but are visible only after the central nervous system begins to form. These larval neurons are not visible after metamorphosis while the rest of the nervous system is largely unchanged in juveniles. CONCLUSIONS: Most of the nervous system that forms during larvogenesis in C. teleta persists into the juvenile stage. The first neurons differentiate in the brain, which contrasts with the early formation of peripheral, larval-specific neurons found in some spiralian taxa with planktotrophic larvae. Our study provides a clear indication that certain shared features among annelids - e.g., five connectives in the ventral nerve cord - are only visible during larval stages in particular species, emphasizing the need to include developmental data in ancestral character state reconstructions. The data provided in this paper will serve as an important comparative reference for understanding evolution of nervous systems, and as a framework for future molecular studies of development.

Molecular conservation of metazoan gut formation: evidence from expression of endomesoderm genes in Capitella teleta (Annelida).

BACKGROUND: Metazoan digestive systems develop from derivatives of ectoderm, endoderm and mesoderm, and vary in the relative contribution of each germ layer across taxa and between gut regions. In a small number of well-studied model systems, gene regulatory networks specify endoderm and mesoderm of the gut within a bipotential germ layer precursor, the endomesoderm. Few studies have examined expression of endomesoderm genes outside of those models, and thus, it is unknown whether molecular specification of gut formation is broadly conserved. In this study, we utilize a sequenced genome and comprehensive fate map to correlate the expression patterns of six transcription factors with embryonic germ layers and gut subregions during early development in Capitella teleta. RESULTS: The genome of C. teleta contains the five core genes of the sea urchin endomesoderm specification network. Here, we extend a previous study and characterize expression patterns of three network orthologs and three additional genes by in situ hybridization during cleavage and gastrulation stages and during formation of distinct gut subregions. In cleavage stage embryos, Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a are expressed in all four macromeres, the endoderm precursors. Ct-otx, Ct-blimp1, and Ct-nkx2.1a are also expressed in presumptive endoderm of gastrulae and later during midgut development. Additional gut-specific expression patterns include Ct-otx, Ct-bra, Ct-foxAB and Ct-gsc in oral ectoderm; Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a in the foregut; and both Ct-bra and Ct-nkx2.1a in the hindgut. CONCLUSIONS: Identification of core sea urchin endomesoderm genes in C. teleta indicates they are present in all three bilaterian superclades. Expression of Ct-otx, Ct-blimp1 and Ct-bra, combined with previously published Ct-foxA and Ct-gataB1 patterns, provide the most comprehensive comparison of these five orthologs from a single species within Spiralia. Each ortholog is likely involved in endoderm specification and midgut development, and several may be essential for establishment of the oral ectoderm, foregut and hindgut, including specification of ectodermal and mesodermal contributions. When the five core genes are compared across the Metazoa, their conserved expression patterns suggest that 'gut gene' networks evolved to specify distinct digestive system subregions, regardless of species-specific differences in gut architecture or germ layer contributions within each subregion.

Variation in spiralian development: insights from polychaetes.

Spiralian development is characterized by the conservation of spindle orientation and cell geometry during early cleavage stages, as well as features of the ultimate fates of identified cells. This complex set of characters is shared by a number of animal lineages including nemerteans, polyclad platyhelminthes, annelids and mollusks. How a similar, highly stereotypical cleavage program can give rise to such diversity of larval and adult forms has intrigued researchers for many years. This review summarizes recent data from polychaete annelids, and highlights both conservation and variation in the cellular and molecular mechanisms that guide the spiral cleavage developmental program. There is a specific focus on comparisons of fate maps, patterns of cleavage, mechanisms of cell fate specification, organizing activity, and differences in molecular patterning. Some of the differences in early development represent intra-clade variation within annelids, and others hint at differences between annelids and other taxa. Because much of the classic work on spiralians has focused on mollusks, these new data from annelids have expanded our knowledge about the evolutionary flexibility in spiralian development and potentially its role in body plan evolution.

An organizing activity is required for head patterning and cell fate specification in the polychaete annelid Capitella teleta: new insights into cell-cell signaling in Lophotrochozoa.

Many lophotrochozoans (i.e., molluscs, annelids, nemerteans, and polyclad flatworms) display a well-conserved early developmental program called spiral cleavage that contrasts with the high diversity of adult body forms present in this group. Due to this stereotypical development, each cell can be uniquely identified and its lineage history known following intracellular injection of lineage tracers. Cell deletion experiments performed mainly in molluscs have demonstrated that one or two cells associated with the endomesodermal lineage represent an embryonic organizer of subsequent development and are causally involved in cell fate and body patterning. Utilizing the published fate map of the spiral-cleaving annelid Capitella teleta, we used infrared laser cell deletions to dissect the role of individual cells on the patterning of the larval body. Thirteen uniquely identifiable individual blastomeres and two double cell combination deletions were studied to assess larval phenotypes by scoring multiple morphological structures and cell type-specific molecular markers differentially expressed along the antero-posterior and dorso-ventral axes. Surprisingly, our results show that in C. teleta, the cellular identity of the "organizing cell" and the timing of the organizing activity are different from that of other spiralians. retain-->In C. teleta, the ectodermal primary somatoblast, 2d, is the key cell responsible for organizing activity during early embryonic development, and is necessary for bilateral symmetry and dorso-ventral axis organization of the head as well as neural, foregut and mesoderm tissue formation. Furthermore, we show that the ERK/MAPK signaling pathway does not appear to be involved in organizing activity in retain-->C. teleta. This contrasts with data from molluscs and the molecular mechanism suggested for another polychaete, Hydroides elegans, highlighting likely molecular level variation among spiralian embryos. These results reinforce the idea that an embryonic organizing activity is present across spiralians. Our data also emphasize the developmental variation within lophotrochozoans, and may ultimately provide insight into the role of developmental processes in the evolution of diverse body forms in metazoans.

Insights into bilaterian evolution from three spiralian genomes.

Current genomic perspectives on animal diversity neglect two prominent phyla, the molluscs and annelids, that together account for nearly one-third of known marine species and are important both ecologically and as experimental systems in classical embryology. Here we describe the draft genomes of the owl limpet (Lottia gigantea), a marine polychaete (Capitella teleta) and a freshwater leech (Helobdella robusta), and compare them with other animal genomes to investigate the origin and diversification of bilaterians from a genomic perspective. We find that the genome organization, gene structure and functional content of these species are more similar to those of some invertebrate deuterostome genomes (for example, amphioxus and sea urchin) than those of other protostomes that have been sequenced to date (flies, nematodes and flatworms). The conservation of these genomic features enables us to expand the inventory of genes present in the last common bilaterian ancestor, establish the tripartite diversification of bilaterians using multiple genomic characteristics and identify ancient conserved long- and short-range genetic linkages across metazoans. Superimposed on this broadly conserved pan-bilaterian background we find examples of lineage-specific genome evolution, including varying rates of rearrangement, intron gain and loss, expansions and contractions of gene families, and the evolution of clade-specific genes that produce the unique content of each genome.

Expression of the pair-rule gene homologs runt, Pax3/7, even-skipped-1 and even-skipped-2 during larval and juvenile development of the polychaete annelid Capitella teleta does not support a role in segmentation.

BACKGROUND: Annelids and arthropods each possess a segmented body. Whether this similarity represents an evolutionary convergence or inheritance from a common segmented ancestor is the subject of ongoing investigation. METHODS: To investigate whether annelids and arthropods share molecular components that control segmentation, we isolated orthologs of the Drosophila melanogaster pair-rule genes, runt, paired (Pax3/7) and eve, from the polychaete annelid Capitella teleta and used whole mount in situ hybridization to characterize their expression patterns. RESULTS: When segments first appear, expression of the single C. teleta runt ortholog is only detected in the brain. Later, Ct-runt is expressed in the ventral nerve cord, foregut and hindgut. Analysis of Pax genes in the C. teleta genome reveals the presence of a single Pax3/7 ortholog. Ct-Pax3/7 is initially detected in the mid-body prior to segmentation, but is restricted to two longitudinal bands in the ventral ectoderm. Each of the two C. teleta eve orthologs has a unique and complex expression pattern, although there is partial overlap in several tissues. Prior to and during segment formation, Ct-eve1 and Ct-eve2 are both expressed in the bilaterial pair of mesoteloblasts, while Ct-eve1 is expressed in the descendant mesodermal band cells. At later stages, Ct-eve2 is expressed in the central and peripheral nervous system, and in mesoderm along the dorsal midline. In late stage larvae and adults, Ct-eve1 and Ct-eve2 are expressed in the posterior growth zone. CONCLUSIONS: C. teleta eve, Pax3/7 and runt homologs all have distinct expression patterns and share expression domains with homologs from other bilaterians. None of the pair-rule orthologs examined in C. teleta exhibit segmental or pair-rule stripes of expression in the ectoderm or mesoderm, consistent with an independent origin of segmentation between annelids and arthropods.