Gene expression in bryozoan larvae suggest a fundamental importance of pre-patterned blastemic cells in the bryozoan life-cycle

<p>Abstract</p> <p>Background</p> <p>Bryozoa is a clade of aquatic protostomes. The bryozoan life cycle typically comprises a larval stage, which metamorphoses into a sessile adult that proliferates by asexual budding to form colonies. The homology of bryozoan larvae with other protostome larvae is enigmatic. Bryozoan larvae exhibit blastemic tissues that contribute to build the adult during morphogenesis. However, it remains unclear if the cells of these tissues are pre-determined according to their future fate or if the cells are undifferentiated, pluripotent stem cells. Gene expression studies can help to identify molecular patterning of larval and adult tissues and enlighten the evolution of bryozoan life cycle stages.</p> <p>Results</p> <p>We investigated the spatial expression of 13 developmental genes in the larval stage of the gymnolaemate bryozoan <it>Bugula neritina</it>. We found most genes expressed in discrete regions in larval blastemic tissues that form definitive components of the adult body plan. Only two of the 13 genes, <it>BnTropomyosin </it>and <it>BnFoxAB</it>, were exclusively expressed in larval tissues that are discarded during metamorphosis.</p> <p>Conclusions</p> <p>Our results suggest that the larval blastemas in <it>Bugula </it>are pre-patterned according to their future fate in the adult. The gene expression patterns indicate that some of the bryozoan blastemas can be interpreted to correspond to homologous adult tissues of other animals. This study challenges an earlier proposed view that metazoan larvae share homologous undifferentiated "set-aside cells", and instead points to an independent origin of the bryozoan larval stage with respect to other lophotrochozoans.</p

Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification

<p>Abstract</p> <p>Background</p> <p>Nervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx), which have highly conserved functions in neural specification in bilaterian animals.</p> <p>Results</p> <p>Using next generation sequencing, the first draft of the genome of the ctenophore <it>Mnemiopsis leidyi </it>has been generated. The Lhx genes in all animals are represented by seven subfamilies (<it>Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8</it>, and <it>LMO</it>) of which four were found to be represented in the ctenophore lineage (<it>Lhx1/5, Lhx3/4, Lmx</it>, and <it>Islet</it>). Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons) than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount <it>in situ </it>hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells.</p> <p>Conclusion</p> <p>This research gives us a first look at neural cell type specification in the ctenophore <it>M. leidyi</it>. Within <it>M. leidyi</it>, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more diversified complements of neural and non-neural cell types in later evolving animals.</p

A cleavage clock regulates features of lineage-specific differentiation in the development of a basal branching metazoan, the ctenophore Mnemiopsis leidyi

Background: An important question in experimental embryology is to understand how the developmental potential responsible for the generation of distinct cell types is spatially segregated over developmental time. Classical embryological work showed that ctenophores, a group of gelatinous marine invertebrates that arose early in animal evolution, display a highly stereotyped pattern of early development and a precocious specification of blastomere fates. Here we investigate the role of autonomous cell specification and the developmental timing of two distinct ctenophore cell types (motile compound comb-plate-like cilia and light-emitting photocytes) in embryos of the lobate ctenophore, Mnemiopsis leidyi. Results: In Mnemiopsis, 9 h after fertilization, comb plate cilia differentiate into derivatives of the E lineage, while the bioluminescent capability begins in derivatives of the M lineage. Arresting cleavage with cytochalasin B at the 1-, 2- or 4-cell stage does not result in blastomere death; however, no visible differentiation of the comb-plate-like cilia or bioluminescence was observed. Cleavage arrest at the 8- or 16-cell stage, in contrast, results in the expression of both differentiation products. Fate-mapping experiments indicate that only the lineages of cells that normally express these markers in an autonomous fashion during normal development express these traits in cleavage-arrested 8- and 16-cell stage embryos. Lineages that form comb plates in a non-autonomous fashion (derivatives of the M lineage) do not. Timed actinomycin D and puromycin treatments show that transcription and translation are required for comb formation and suggest that the segregated material might be necessary for activation of the appropriate genes. Interestingly, even in the absence of cytokinesis, differentiation markers appear to be activated at the correct times. Treatments with a DNA synthesis inhibitor, aphidicolin, show that the number of nuclear divisions, and perhaps the DNA to cytoplasmic ratio, are critical for the appearance of lineage-specific differentiation. Conclusion: Our work corroborates previous studies demonstrating that the cleavage program is causally involved in the spatial segregation and/or activation of factors that give rise to distinct cell types in ctenophore development. These factors are segregated independently to the appropriate lineage at the 8- and the 16-cell stages and have features of a clock, such that comb-plate-like cilia and light-emitting photoproteins appear at roughly the same developmental time in cleavage-arrested embryos as they do in untreated embryos. Nuclear division, which possibly affects DNA-cytoplasmic ratios, appears to be important in the timing of differentiation markers. Evidence suggests that the 60-cell stage, just prior to gastrulation, is the time of zygotic gene activation. Such cleavage-clock-regulated phenomena appear to be widespread amongst the Metazoa and these cellular and molecular developmental mechanisms probably evolved early in metazoan evolution

Ciliary photoreceptors in the cerebral eyes of a protostome larva

Background: Eyes in bilaterian metazoans have been described as being composed of either ciliary or rhabdomeric photoreceptors. Phylogenetic distribution, as well as distinct morphologies and characteristic deployment of different photopigments (ciliary vs. rhabdomeric opsins) and transduction pathways argue for the co-existence of both of these two photoreceptor types in the last common bilaterian ancestor. Both receptor types exist throughout the Bilateria, but only vertebrates are thought to use ciliary photoreceptors for directional light detection in cerebral eyes, while all other invertebrate bilaterians studied utilize rhabdomeric photoreceptors for this purpose. In protostomes, ciliary photoreceptors that express c-opsin have been described only from a nonvisual deep-brain photoreceptor. Their homology with vertebrate rods and cones of the human eye has been hypothesized to represent a unique functional transition from non-visual to visual roles in the vertebrate lineage. Results: To test the hypothesis that protostome cerebral eyes employ exclusively rhabdomeric photoreceptors, we investigated the ultrastructure of the larval eyes in the brachiopod Terebratalia transversa. We show that these pigment-cup eyes consist of a lens cell and a shading pigment cell, both of which are putative photoreceptors, deploying a modified, enlarged cilium for light perception, and have axonal connections to the larval brain. Our investigation of the gene expression patterns of c-opsin, Pax6 and otx in these eyes confirms that the larval eye spots of brachiopods are cerebral eyes that deploy ciliary type photoreceptors for directional light detection. Interestingly, c-opsin is also expressed during early embryogenesis in all potential apical neural cells, becoming restricted to the anterior neuroectoderm, before expression is initiated in the photoreceptor cells of the eyes. Coincident with the expression of c-opsin in the presumptive neuroectoderm, we found that middle gastrula stage embryos display a positive photoresponse behavior, in the absence of a discrete shading pigment or axonal connections between cells. Conclusions: Our results indicate that the dichotomy in the deployment of ciliary and rhabdomeric photoreceptors for directional light detection is not as clear-cut as previously thought. Analyses of brachiopod larval eyes demonstrate that the utilization of c-opsin expressing ciliary photoreceptors in cerebral eyes is not limited to vertebrates. The presence of ciliary photoreceptor-based eyes in protostomes suggests that the transition between non-visual and visual functions of photoreceptors has been more evolutionarily labile than previously recognized, and that co-option of ciliary and rhabdomeric photoreceptor cell types for directional light detection has occurred multiple times during animal evolution. In addition, positive photoresponse behavior in gastrula stage embryos suggests that a discrete shading pigment is not requisite for directional photoreception in metazoans. Scanning photoreception of light intensities mediating cell-autonomous changes of ciliary movement may represent an ancient mechanism for regulating locomotory behavior, and is likely to have existed prior to the evolution of eye-mediated directional light detection employing axonal connections to effector cells and a discreet shading pigment

Development of the larval anterior neurogenic domains of Terebratalia transversa (Brachiopoda) provides insights into the diversification of larval apical organs and the spiralian nervous system

<p>Abstract</p> <p>Background</p> <p>Larval features such as the apical organ, apical ciliary tuft, and ciliated bands often complicate the evaluation of hypotheses regarding the origin of the adult bilaterian nervous system. Understanding how neurogenic domains form within the bilaterian head and larval apical organ requires expression data from animals that exhibit aspects of both centralized and diffuse nervous systems at different life history stages. Here, we describe the expression of eight neural-related genes during the larval development of the brachiopod, <it>Terebratalia transversa</it>.</p> <p>Results</p> <p>Radially symmetric gastrulae broadly express <it>Tt-Six3/6 </it>and <it>Tt-hbn </it>in the animal cap ectoderm. <it>Tt-NK2.1 </it>and <it>Tt-otp </it>are restricted to a central subset of these cells, and <it>Tt-fez </it>and <it>Tt-FoxQ2 </it>expression domains are already asymmetric at this stage. As gastrulation proceeds, the spatial expression of these genes is split between two anterior ectodermal domains, a more dorsal region comprised of <it>Tt-Six3/6, Tt-fez, Tt-FoxQ2</it>, and <it>Tt-otp </it>expression domains, and an anterior ventral domain demarcated by <it>Tt-hbn </it>and <it>Tt-NK2.1 </it>expression. More posteriorly, the latter domains are bordered by <it>Tt-FoxG </it>expression in the region of the transverse ciliated band. <it>Tt-synaptotagmin 1 </it>is expressed throughout the anterior neural ectoderm. All genes are expressed late into larval development. The basiepithelial larval nervous system includes three neurogenic domains comprised of the more dorsal apical organ and a ventral cell cluster in the apical lobe as well as a mid-ventral band of neurons in the mantle lobe. <it>Tt-otp </it>is the only gene expressed in numerous flask-shaped cells of the apical organ and in a subset of neurons in the mantle lobe.</p> <p>Conclusions</p> <p>Our expression data for <it>Tt-Six3/6, Tt-FoxQ2</it>, and <it>Tt-otp </it>confirm some aspects of bilaterian-wide conservation of spatial partitioning within anterior neurogenic domains and also suggest a common origin for central <it>otp</it>-positive cell types within the larval apical organs of spiralians. However, the field of sensory neurons within the larval apical organ of <it>Terebratalia </it>is broader and composed of more cells relative to those of other spiralian larvae. These cellular differences are mirrored in the broader spatial and temporal expression patterns of <it>Tt-FoxQ2 </it>and <it>Tt-otp</it>. Corresponding differences in the expression of <it>Tt-hbn, Tt-NK2.1</it>, and <it>Tt-FoxG </it>are also observed relative to their respective domains within the cerebral ganglia of spiralians. Based on these data we argue that the anterior region of the bilaterian stem species included <it>Six3/6, NK2.1, otp, hbn, fez</it>, and <it>FoxQ2 </it>expression domains that were subsequently modified within larval and adult neural tissues of protostome and deuterostome animals.</p

A comprehensive fate map by intracellular injection of identified blastomeres in the marine polychaete Capitella teleta

<p>Abstract</p> <p>Background</p> <p>The polychaete annelid <it>Capitella teleta </it>(formerly <it>Capitella </it>sp. I) develops by spiral cleavage and has been the focus of several recent developmental studies aided by a fully sequenced genome. Fate mapping in polychaetes has lagged behind other spiralian taxa, because of technical limitations.</p> <p>Results</p> <p>To generate a modern fate map for <it>C. teleta</it>, we injected 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) into individual identified blastomeres through fourth-quartet micromere formation. Confocal laser scanning microscopy at single-cell resolution was used to characterize blastomere fates during larval stages. Our results corroborate previous observations from classic studies, and show a number of similarities with other spiralian fate maps, including unique and stereotypic fates for individual blastomeres, presence of four discrete body domains arising from the A, B, C and D cell quadrants, generation of anterior ectoderm from first quartet micromeres, and contributions to trunk ectoderm and ventral nerve cord by the 2d somatoblast. Of particular interest are several instances in which the <it>C. teleta </it>fate map deviates from other spiralian fate maps. For example, we identified four to seven distinct origins of mesoderm, all ectomesodermal. In addition, the left and right mesodermal bands arise from 3d and 3c, respectively, whereas 4d generates a small number of trunk muscle cells, the primordial germ cells and the anus. We identified a complex set of blastomere contributions to the posterior gut in <it>C. teleta</it>, which establishes the most complete map of posterior gut territories to date.</p> <p>Conclusions</p> <p>Our detailed cellular descriptions reveal previously underappreciated complexity in the ontogenetic contributions to several spiralian larval tissues, including the mesoderm, nervous system and gut. The formation of the mesodermal bands by 3c and 3d is in stark contrast to other spiralians, in which 4d generates the mesodermal bands. The results of this study provide a framework for future phylogenetic comparisons and functional analyses of cell-fate specification.</p

The evolutionary diversification of LSF and Grainyhead transcription factors preceded the radiation of basal animal lineages

<p>Abstract</p> <p>Background</p> <p>The transcription factors of the LSF/Grainyhead (GRH) family are characterized by the possession of a distinctive DNA-binding domain that bears no clear relationship to other known DNA-binding domains, with the possible exception of the p53 core domain. In triploblastic animals, the LSF and GRH subfamilies have diverged extensively with respect to their biological roles, general expression patterns, and mechanism of DNA binding. For example, <it>Grainyhead </it>(GRH) homologs are expressed primarily in the epidermis, and they appear to play an ancient role in maintaining the epidermal barrier. By contrast, LSF homologs are more widely expressed, and they regulate general cellular functions such as cell cycle progression and survival in addition to cell-lineage specific gene expression.</p> <p>Results</p> <p>To illuminate the early evolution of this family and reconstruct the functional divergence of LSF and GRH, we compared homologs from 18 phylogenetically diverse taxa, including four basal animals (<it>Nematostella vectensis</it>, <it>Vallicula multiformis</it>, <it>Trichoplax adhaerens</it>, and <it>Amphimedon queenslandica</it>), a choanoflagellate (<it>Monosiga brevicollis</it>) and several fungi. Phylogenetic and bioinformatic analyses of these sequences indicate that (1) the LSF/GRH gene family originated prior to the animal-fungal divergence, and (2) the functional diversification of the LSF and GRH subfamilies occurred prior to the divergence between sponges and eumetazoans. Aspects of the domain architecture of LSF/GRH proteins are well conserved between fungi, choanoflagellates, and metazoans, though within the Metazoa, the LSF and GRH families are clearly distinct. We failed to identify a convincing LSF/GRH homolog in the sequenced genomes of the algae <it>Volvox carteri </it>and <it>Chlamydomonas reinhardtii </it>or the amoebozoan <it>Dictyostelium purpureum</it>. Interestingly, the ancestral GRH locus has become split into two separate loci in the sea anemone <it>Nematostella</it>, with one locus encoding a DNA binding domain and the other locus encoding the dimerization domain.</p> <p>Conclusions</p> <p>In metazoans, LSF and GRH proteins play a number of roles that are essential to achieving and maintaining multicellularity. It is now clear that this protein family already existed in the unicellular ancestor of animals, choanoflagellates, and fungi. However, the diversification of distinct LSF and GRH subfamilies appears to be a metazoan invention. Given the conserved role of GRH in maintaining epithelial integrity in vertebrates, insects, and nematodes, it is noteworthy that the evolutionary origin of Grh appears roughly coincident with the evolutionary origin of the epithelium.</p

Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation

Background: The Sox genes, a family of transcription factors characterized by the presence of a high mobility group (HMG) box domain, are among the central groups of developmental regulators in the animal kingdom. They are indispensable in progenitor cell fate determination, and various Sox family members are involved in managing the critical balance between stem cells and differentiating cells. There are 20 mammalian Sox genes that are divided into five major groups (B, C, D, E, and F). True Sox genes have been identified in all animal lineages but not outside Metazoa, indicating that this gene family arose at the origin of the animals. Whole-genome sequencing of the lobate ctenophore Mnemiopsis leidyi allowed us to examine the full complement and expression of the Sox gene family in this early-branching animal lineage. Results: Our phylogenetic analyses of the Sox gene family were generally in agreement with previous studies and placed five of the six Mnemiopsis Sox genes into one of the major Sox groups: SoxB (MleSox1), SoxC (MleSox2), SoxE (MleSox3, MleSox4), and SoxF (MleSox5), with one unclassified gene (MleSox6). We investigated the expression of five out of six Mnemiopsis Sox genes during early development. Expression patterns determined through in situ hybridization generally revealed spatially restricted Sox expression patterns in somatic cells within zones of cell proliferation, as determined by EdU staining. These zones were located in the apical sense organ, upper tentacle bulbs, and developing comb rows in Mnemiopsis, and coincide with similar zones identified in the cydippid ctenophore Pleurobrachia. Conclusions: Our results are consistent with the established role of multiple Sox genes in the maintenance of stem cell pools. Both similarities and differences in juvenile cydippid stage expression patterns between Mnemiopsis Sox genes and their orthologs from Pleurobrachia highlight the importance of using multiple species to characterize the evolution of development within a given phylum. In light of recent phylogenetic evidence that Ctenophora is the earliest-branching animal lineage, our results are consistent with the hypothesis that the ancient primary function of Sox family genes was to regulate the maintenance of stem cells and function in cell fate determination