Search

BackgroundPhoronids, rhynchonelliform and linguliform brachiopods show striking similarities in their embryonic fate maps, in particular in their axis specification and regionalization. However, although brachiopod development has been studied in detail and demonstrated embryonic patterning as a causal factor of the gastrulation mode (protostomy vs deuterostomy), molecular descriptions are still missing in phoronids. To understand whether phoronids display underlying embryonic molecular mechanisms similar to those of brachiopods, here we report the expression patterns of anterior (otx,gsc,six3/6,nk2.1), posterior (cdx, bra) and endomesodermal (foxA,gata4/5/6,twist) markers during the development of the protostomic phoronidPhoronopsis harmeri.ResultsThe transcription factorsfoxA, gata4/5/6andcdxshow conserved expression in patterning the development and regionalization of the phoronid embryonic gut, withfoxAexpressed in the presumptive foregut,gata4/5/6demarcating the midgut andcdxconfined to the hindgut. Furthermore,six3/6,usually a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria. Surprisingly,brachyury, a transcription factor often associated with gastrulation movements and mouth and hindgut development, seems not to be involved with these patterning events in phoronids.ConclusionsOur description and comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarity to that of rhynchonelliform brachiopods, which is likely related to their shared protostomic mode of development. Despite these similarities, the phoronidPh. harmerialso shows particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

Bilaterian photoreceptor cells are characterized by the expression of opsins, signal transduction genes, and ion channels, which together facilitate behavioral responses to light. We have previously identified a ciliary opsin gene from the brachiopod Terebratalia transversa, whose expression in gastrula stage embryos coincides with a photoresponse behavior, suggesting the presence of a functional phototransduction system in these early embryos. To further evaluate the potential for light reception in these embryos, we surveyed transcriptome data to identify phototransduction genes and evaluated their expression. In addition to the previously described ciliary opsin gene, we have identified two Go-class opsins that are also expressed in gastrula stage embryos. Representative members from all classes of Gα-protein genes were also expressed, with a Gα12-class gene being localized in the same anterior ectodermal domain as the opsin transcripts. Both CNG-class and TRP-class ion channels were expressed in the gastrula stage embryos, as were GRK and arrestin genes, which are associated with inhibition of rhodopsin activity. Taken together, these data support the presence of a functional phototransduction system in the early brachiopod embryo.

The mouth opening of bilaterian animals develops either separate from (deuterostomy) or connected to (protostomy) the embryonic blastopore, the site of endomesoderm internalization. Although this distinction preluded the classification of bilaterian animals in Deuterostomia and Protostomia, and has influenced major scenarios of bilaterian evolution, the developmental basis for the appearance of these different embryonic patterns remains unclear. To identify the underlying mechanisms, we compared the development of two brachiopod species that show deuterostomy (Novocrania anomala) and protostomy (Terebratalia transversa), respectively. We show that the differential activity of Wnt signalling, together with the timing and location of mesoderm formation, correlate with the differential behaviour and fate of the blastopore. We further assess these principles in the spiral-cleaving group Annelida, and propose that the developmental relationships of mouth and blastoporal openings are secondary by-products of variations in axial and mesoderm development. This challenges the previous evolutionary emphasis on extant blastoporal behaviours to explain the origin and diversification of bilaterian animals.

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.

Although we have advanced in our understanding of the molecular mechanisms intrinsic to the morphogenesis of chordate embryos, the question of how individual developmental events are integrated to generate the final morphological form is still unresolved. Microscopic observation is a pivotal tool in developmental biology, both for determining the normal course of events and for contrasting this with the results of experimental and pathological perturbations. Since embryonic development takes place in three dimensions over time, to fully understand the events required to build an embryo we must investigate embryo morphogenesis in multiple dimensions in situ. Recent advances in the isolation of naturally fluorescent proteins, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos using optical imaging. These technologies allow direct visual access to complex events as they happen in their native environment, and thus provide greater insights into cell behaviors operating during embryonic development. Since most fluorescent protein probes and modes of data acquisition are common across species, we have chosen the mouse and the ascidian, two model organisms at opposite ends of the chordate clade, to review the use of some of the current genetically-encoded fluorescent proteins and their visualization in vivo in living embryos for the generation of high-resolution imaging data.

Thanks to their transparent and rapidly developing mosaic embryos, ascidians (or sea squirts) have been a model system for embryological studies for over a century. Recently, ascidians have entered the postgenomic era, with the sequencing of the Ciona intestinalis genome and the accumulation of molecular resources that rival those available for fruit flies and mice. One strength of ascidians as a model system is their close similarity to vertebrates. Literature reporting molecular homologies between vertebrate and ascidian tissues has flourished over the past 15 years, since the first ascidian genes were cloned. However, it should not be forgotten that ascidians diverged from the lineage leading to vertebrates over 500 million years ago. Here, we review the main similarities and differences so far identified, at the molecular level, between ascidian and vertebrate tissues and discuss the evolution of the compact ascidian genome.

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona. genesis 43:136–147, 2005. © 2005 Wiley-Liss, Inc.

The Mollusca represent one of the most morphologically diverse animal phyla, prompting a variety of hypotheses on relationships between the major lineages within the phylum based upon morphological, developmental, and paleontological data. Analyses of small-ribosomal RNA (SSU rRNA) gene sequence have provided limited resolution of higher-level relationships within the Mollusca. Recent analyses suggest large-subunit (LSU) rRNA gene sequences are useful in resolving deep-level metazoan relationships, particularly when combined with SSU sequence. To this end, LSU (approximately 3.5 kb in length) and SSU (approximately 2 kb) sequences were collected for 33 taxa representing the major lineages within the Mollusca to improve resolution of intraphyletic relationships. Although the LSU and combined LSU+SSU datasets appear to hold potential for resolving branching order within the recognized molluscan classes, low bootstrap support was found for relationships between the major lineages within the Mollusca. LSU+SSU sequences also showed significant levels of rate heterogeneity between molluscan lineages. The Polyplacophora, Gastropoda, and Cephalopoda were each recovered as monophyletic clades with the LSU+SSU dataset. While the Bivalvia were not recovered as monophyletic clade in analyses of the SSU, LSU, or LSU+SSU, the Shimodaira-Hasegawa test showed that likelihood scores for these results did not differ significantly from topologies where the Bivalvia were monophyletic. Analyses of LSU sequences strongly contradict the widely accepted Diasoma hypotheses that bivalves and scaphopods are closely related to one another. The data are consistent with recent morphological and SSU analyses suggesting scaphopods are more closely related to gastropods and cephalopods than to bivalves. The dataset also presents the first published DNA sequences from a neomeniomorph aplacophoran, a group considered critical to our understanding of the origin and early radiation of the Mollusca.

Bryozoans, or moss animals, are small colonial organisms that possess a suspension-feeding apparatus called a lophophore. Traditionally, this "phylum" has been grouped with brachiopods and phoronids because of the feeding structure. Available molecular and morphological data refute this notion of a monophyletic "Lophophorata." Alternative hypotheses place bryozoans either at the base of the Lophotrochozoa or basal to the Lophotrochozoa/Ecdysozoa split. Surprisingly, the only molecular data bearing on this issue are from the 18S nuclear ribosomal gene. Here we report the results of a Hox gene survey using degenerate polymerase chain reaction primers in a gymnolaemate bryozoan, Bugula turrita. Putative orthologs to both the Post2 and the Lox5 genes were found, suggesting that bryozoans are not a basal protostome group but closely allied to other lophotrochozoan taxa. We also found the first definitive evidence of two Deformed/Hox4 class genes in a nonvertebrate animal.

Background The primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the eukaryotes. While the role for melatonin in cyclic behavior via interactions with the circadian clock has only been reported in vertebrates, comparative research has shown that the transcription-translation loops of the animal circadian clock likely date to the cnidarian-bilaterian ancestor, leaving open significant questions about the evolutionary origin of melatonin signaling in circadian behavior by interacting with the molecular clock. Results Expression of melatonin in adult anemones showed peak expression at the end of light period (zeitgeber time (ZT) = 12) when cultured under diel conditions, coinciding with expression of genes and enzyme activity for members of the melatonin synthesis pathway (tryptophan hydroxylase and hydroxyindol-O-methyltransferase), which also showed rhythmic expression. During embryogenesis and juvenile stages, melatonin showed cyclic oscillations in concentration, peaking in midday. Spatial (in situ hybridization) and quantitative (real-time PCR) transcription of clock genes during development of N. vectensis showed these ‘clock’ genes are expressed early in the development, prior to rhythmic oscillations, suggesting functions independent of a function in the circadian clock. Finally, time-course studies revealed that animals transferred from diel conditions to constant darkness lose circadian expression for most of the clock genes within 4 days, which can be reset by melatonin supplementation. Conclusions Our results support an ancient role for melatonin in the circadian behavior of animals by showing cyclic expression of this hormone under diel conditions, light-dependent oscillations in genes in the melatonin synthesis pathway, and the function of melatonin in initiating expression of circadian clock genes in the cnidarian N. vectensis. The differences in expression melatonin and the circadian clock gene network in the adult stage when compared with developmental stages of N. vectensis suggests new research directions to characterize stage-specific mechanisms of circadian clock function in animals.

Underlying any analysis on the evolution of development is a phylogenetic framework, whether explicitly stated or implied. As such, differing views on phylogenetic relationships lead to variable interpretations of how developmental mechanisms have changed through time. Over the past decade, many long-standing hypotheses about animal evolution have been questioned causing substantial changes in the assumed phylogenetic framework underlying comparative developmental studies. Current hypotheses about early metazoan history suggest that three, not two, major lineages of bilateral animals originated in the Precambrian: the Deuterostomes (e.g., seastars, acorn worms, and vertebrates), the Ecdysozoans (e.g., nematodes and arthropods), and the Lophotrochozoans (e.g., annelids, mollusks, and lophophorates). Although information in Hox-genes bears directly on our understanding of early metazoan evolution and the formation of body plans, research effort has been focused primarily on two taxa, insects and ve...

Background The contribution of cell proliferation to regeneration varies greatly between different metazoan models. Planarians rely on pluripotent neoblasts and amphibian limb regeneration depends upon formation of a proliferative blastema, while regeneration in Hydra can occur in the absence of cell proliferation. Recently, the cnidarian Nematostella vectensis has shown potential as a model for studies of regeneration because of the ability to conduct comparative studies of patterning during embryonic development, asexual reproduction, and regeneration. The present study investigates the pattern of cell proliferation during the regeneration of oral structures and the role of cell proliferation in this process. Results In intact polyps, cell proliferation is observed in both ectodermal and endodermal tissues throughout the entire oral-aboral axis, including in the tentacles and physa. Following bisection, there is initially little change in proliferation at the wound site of the aboral fragment, however, beginning 18 to 24 hours after amputation there is a dramatic increase in cell proliferation at the wound site in the aboral fragment. This elevated level of proliferation is maintained throughout the course or regeneration of oral structures, including the tentacles, the mouth, and the pharynx. Treatments with the cell proliferation inhibitors hydroxyurea and nocodazole demonstrate that cell proliferation is indispensable for the regeneration of oral structures. Although inhibition of regeneration by nocodazole was generally irreversible, secondary amputation reinitiates cell proliferation and regeneration. Conclusions The study has found that high levels of cell proliferation characterize the regeneration of oral structures in Nematostella, and that this cell proliferation is necessary for the proper progression of regeneration. Thus, while cell proliferation contributes to regeneration of oral structures in both Nematostella and Hydra, Nematostella lacks the ability to undergo the compensatory morphallactic mode of regeneration that characterizes Hydra. Our results are consistent with amputation activating a quiescent population of mitotically competent stem cells in spatial proximity to the wound site, which form the regenerated structures.

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription.

Acid-sensing ion channels (ASICs) constitute a family of neuron-specific voltage-insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mechanosensation, and modulation of synaptic transmission. However, the role protons play in mediating the effects of ASICs remains elusive. We have examined ASICs from the ascidian Ciona intestinalis, a simple chordate organism whose nervous system in the larval stage exhibits high similarity to that of higher vertebrates. We found that the ascidian genome contains a single ASIC gene that gives rise to two splice forms analogous to the mammalian ASIC1 and ASIC2. CiASIC is expressed in most neurons of the larva but is absent in the adult. Despite high sequence similarity with mammalian counterparts, CiASIC is proton-insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton sensitivity is conferred by accessory proteins or particular factors present only in Ciona neurons. Down-regulation of the CiASIC transcript by double-stranded RNA disrupted the regular pattern of larval swimming, implying that proton-independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a highly conserved channel distinctive of chordate nervous systems and show that protons are not essential for ASIC function.

Of the three major bilaterian clades, Lophotrochozoa has the greatest diversity and disparity of body forms and is the least understood in terms of phylogenetic history. Within this clade, small nuclear ribosomal subunit (SSU or 18S) studies have failed to provide resolution and other molecular markers have insufficient taxon sampling. To examine relationships within Lophotrochozoa, we collected and complied complete SSU data and nearly complete (>90%) large nuclear ribosomal subunit (LSU or 28S) data totaling approximately 5 kb per taxon, for 36 lophotrochozoans. Results of LSU and combined SSU + LSU likelihood analyses provide topologies more consistent with morphological data than analyses of SSU data alone. Namely, most phyla recognized on morphological grounds are recovered as monophyletic entities when the LSU data is considered (contra SSU data alone). These new data show with significant support that “Lophophorata” (traditionally recognized to include Brachiopoda, Phoronida, and Bryozoa) is not a monophyletic entity. Further, the data suggest that Platyzoa is real and may be derived within lophotrochozans rather than a basal or sister taxon. The recently discovered Cycliophora are allied to entoprocts, consistent with their initial placement based on morphology. Additional evidence for Syndermata (i.e., Rotifera + Acanthocephala) is also found. Although relationships among groups with trochophore-like larvae could not be resolved and nodal support values are generally low, the addition of LSU data is a considerable advance in our understanding of lophotrochozoan phylogeny from the molecular perspective.

DNA sequencing and phylogenetic analyses were conducted to investigate evolutionary relationships between taxa within the metazoan clade Lophotrochozoa. Chapter 1 presents an introduction to phylogenetics of the Metazoa and the clade Lophotrochozoa. Chapter 2 analyzes higher level relationships between the major groups within the phylum Mollusca using sequences of the nuclear ribosomal large-subunit RNA gene (LSD rDNA). Results presented provide the first molecular evidence for a close relationship between the Scaphopoda and Cephalopoda. Phylogenetic trees with this topology were found to have likelihood scores significantly better than those for phylogenies constrained to fit the Diasoma hypothesis grouping Scaphopoda and Bivalvia as sister taxa. Chapter 3 utilizes LSU rDNA sequences to analyze relationships between diverse phyla within the clade Lophotrochozoa. LSU rDNA sequences were found to provide greater resolution than has been provided by previous analyses of the nuclear small-subunit ribosomal RNA gene (SSU rDNA). Analysis ofLSU rDNA sequences recovered the monophyly of several phyla, such as Mollusca and Anelida, whose members are found to be paraphyletic using SSU rDNA sequences alone. Results also suggest that the clade Platyzoa, including rotifers and platyhelminthes, may have arisen within the Lophotrochozoa, rather than as a sister group to lophotrochozoans. Chapter 4 investigates the Hox gene complement of the bryozoan Bugula turrita. Six Hox genes were recovered, including an ortholog of the posterior class gene Post2, which is a synapomorphy for the Lophotrochozoa. The identification of a Post2 ortholog provides evidence of a close relationship between the Bryozoa and other lophotrochozoan phyla.

Background Axial elongation is a key morphogenetic process that serves to shape developing organisms. Tail extension in the ascidian larva represents a striking example of this process, wherein paraxially positioned muscle cells undergo elongation and differentiation independent of the segmentation process that characterizes the formation of paraxial mesoderm in vertebrates. Investigating the cell behaviors underlying the morphogenesis of muscle in ascidians may therefore reveal the evolutionarily conserved mechanisms operating during this process. Methodology/Principle Findings A live cell imaging approach utilizing subcellularly-localized fluorescent proteins was employed to investigate muscle cell behaviors during tail extension in the ascidian Ciona intestinalis. Changes in the position and morphology of individual muscle cells were analyzed in vivo in wild type embryos undergoing tail extension and in embryos in which muscle development was perturbed. Muscle cells were observed to undergo elongation in the absence of positional reorganization. Furthermore, high-speed high-resolution live imaging revealed that the onset and progression of tail extension were characterized by the presence of dynamic and polarized actin-based protrusive activity at the plasma membrane of individual muscle cells. Conclusions/Significance Our results demonstrate that in the Ciona muscle, tissue elongation resulted from gradual and coordinated changes in cell geometry and not from changes in cell topology. Proper formation of muscle cells was found to be necessary not only for muscle tissue elongation, but also more generally for completion of tail extension. Based upon the characterized dynamic changes in cell morphology and plasma membrane protrusive activity, a three-phase model is proposed to describe the cell behavior operating during muscle morphogenesis in the ascidian embryo.