Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii.

BACKGROUND: Restorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered. RESULTS: We generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process. CONCLUSIONS: In this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

Cooption of regulatory modules for tektin paralogs during ciliary band formation in a marine annelid larva.

Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.

Allometric scaling of interspecific RNA transcript abundance to extend the use of metatranscriptomics in characterizing complex communities.

Metatranscriptomics allows profiling of community mRNA and rRNA transcript abundance under certain environmental conditions. However, variations in the proportion of RNA transcripts across different community size structures remain less explained, thus limiting the possible applications of metatranscriptomics in community studies. Here, we extended the assumptions of the growth-rate hypothesis (GRH) and the metabolic theory of ecology (MTE) to validate the allometric scaling of interspecific RNA transcript (mRNA and rRNA) abundance through metatranscriptomic analysis of mock communities consisting of model organisms. The results suggest that body size imposes significant constraints on RNA transcript abundance. Interestingly, the relationship between the total mitochondrial transcript abundance (mRNA and rRNA slopes were -0.30 and -0.28, respectively) and body size aligned with the MTE assumptions with slopes close to -», while the nuclear transcripts displayed much steeper slopes (mRNA and rRNA slopes were -0.33 and -0.40, respectively). The assumed temperature dependence was not observed in this study. At the gene level, the allometric slopes range from 0 to -1. Overall, the above results showed that larger individuals have lesser RNA transcript abundance per tissue mass than smaller ones regardless of temperature. Analyses of field-collected microcrustacean zooplankton samples demonstrated that the correction of size effect, using the allometric exponents derived from the model organism mock community, explains better the patterns of interspecific RNA transcripts abundance within the metatranscriptome. Integrating allometry with metatranscriptomics can extend the use of RNA transcript reads in estimating ecological processes within complex communities.

The Nereid on the rise: Platynereis as a model system.

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community.

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.

Taxon-specific expansion and loss of tektins inform metazoan ciliary diversity.

BACKGROUND: Cilia and flagella are complex cellular structures thought to have first evolved in a last ciliated eukaryotic ancestor due to the conserved 9 + 2 microtubule doublet structure of the axoneme and associated proteins. The Tektin family of coiled-coil domain containing proteins was previously identified in cilia of organisms as diverse as green algae and sea urchin. While studies have shown that some Tektins are necessary for ciliary function, there has been no comprehensive phylogenetic survey of tektin genes. To fill this gap, we sampled tektin sequences broadly among metazoan and unicellular lineages in order to determine how the tektin gene complements evolved in over 100 different extant species. RESULTS: Using Bayesian and Maximum Likelihood analyses, we have ascertained with high confidence that all metazoan tektins arose from a single ancestral tektin gene in the last common ancestor of metazoans and choanoflagellates. Gene duplications gave rise to two tektin genes in the metazoan ancestor, and a subsequent expansion to three and four tektin genes in early bilaterian ancestors. While all four tektin genes remained highly conserved in most deuterostome and spiralian species surveyed, most tektin genes in ecdysozoans are highly derived with extensive gene loss in several lineages including nematodes and some crustaceans. In addition, while tektin-1, - 2, and - 4 have remained as single copy genes in most lineages, tektin-3/5 has been duplicated independently several times, notably at the base of the spiralian, vertebrate and hymenopteran (Ecdysozoa) clades. CONCLUSIONS: We provide a solid description of tektin evolution supporting one, two, three, and four ancestral tektin genes in a holozoan, metazoan, bilaterian, and nephrozoan ancestor, respectively. The isolated presence of tektin in a cryptophyte and a chlorophyte branch invokes events of horizontal gene transfer, and that the last common ciliated eukaryotic ancestor lacked a tektin gene. Reconstructing the evolutionary history of the tektin complement in each extant metazoan species enabled us to pinpoint lineage specific expansions and losses. Our analysis will help to direct future studies on Tektin function, and how gain and loss of tektin genes might have contributed to the evolution of various types of cilia and flagella.

PdumBase: a transcriptome database and research tool for Platynereis dumerilii and early development of other metazoans.

BACKGROUND: The marine polychaete annelid Platynereis dumerilii has recently emerged as a prominent organism for the study of development, evolution, stem cells, regeneration, marine ecology, chronobiology and neurobiology within metazoans. Its phylogenetic position within the spiralian/ lophotrochozoan clade, the comparatively high conservation of ancestral features in the Platynereis genome, and experimental access to any stage within its life cycle, make Platynereis an important model for elucidating the complex regulatory and functional molecular mechanisms governing early development, later organogenesis, and various features of its larval and adult life. High resolution RNA-seq gene expression data obtained from specific developmental stages can be used to dissect early developmental mechanisms. However, the potential for discovery of these mechanisms relies on tools to search, retrieve, and compare genome-wide information within Platynereis, and across other metazoan taxa. RESULTS: To facilitate exploration and discovery by the broader scientific community, we have developed a web-based, searchable online research tool, PdumBase, featuring the first comprehensive transcriptome database for Platynereis dumerilii during early stages of development (2 h ~ 14 h). Our database also includes additional stages over the P. dumerilii life cycle and provides access to the expression data of 17,213 genes (31,806 transcripts) along with annotation information sourced from Swiss-Prot, Gene Ontology, KEGG pathways, Pfam domains, TmHMM, SingleP, and EggNOG orthology. Expression data for each gene includes the stage, the normalized FPKM, the raw read counts, and information that can be leveraged for statistical analyses of differential gene expression and the construction of genome-wide co-expression networks. In addition, PdumBase offers early stage transcriptome expression data from five further species as a valuable resource for investigators interested in comparing early development in different organisms. To understand conservation of Platynereis gene models and to validate gene annotation, most Platynereis gene models include a comprehensive phylogenetic analysis across 18 species representing diverse metazoan taxa. CONCLUSIONS: PdumBase represents the first online resource for the early developmental transcriptome of Platynereis dumerilii. It serves as a research platform for discovery and exploration of gene expression during early stages, throughout the Platynereis life cycle, and enables comparison to other model organisms. PdumBase is freely available at http://pdumbase.gdcb.iastate.edu .

The asymmetric cell division machinery in the spiral-cleaving egg and embryo of the marine annelid Platynereis dumerilii.

BACKGROUND: Over one third of all animal phyla utilize a mode of early embryogenesis called 'spiral cleavage' to divide the fertilized egg into embryonic cells with different cell fates. This mode is characterized by a series of invariant, stereotypic, asymmetric cell divisions (ACDs) that generates cells of different size and defined position within the early embryo. Astonishingly, very little is known about the underlying molecular machinery to orchestrate these ACDs in spiral-cleaving embryos. Here we identify, for the first time, cohorts of factors that may contribute to early embryonic ACDs in a spiralian embryo. RESULTS: To do so we analyzed stage-specific transcriptome data in eggs and early embryos of the spiralian annelid Platynereis dumerilii for the expression of over 50 candidate genes that are involved in (1) establishing cortical domains such as the partitioning defective (par) genes, (2) directing spindle orientation, (3) conveying polarity cues including crumbs and scribble, and (4) maintaining cell-cell adhesion between embryonic cells. In general, each of these cohorts of genes are co-expressed exhibiting high levels of transcripts in the oocyte and fertilized single-celled embryo, with progressively lower levels at later stages. Interestingly, a small number of key factors within each ACD module show different expression profiles with increased early zygotic expression suggesting distinct regulatory functions. In addition, our analysis discovered several highly co-expressed genes that have been associated with specialized neural cell-cell recognition functions in the nervous system. The high maternal contribution of these 'neural' adhesion complexes indicates novel general adhesion functions during early embryogenesis. CONCLUSIONS: Spiralian embryos are champions of ACD generating embryonic cells of different size with astonishing accuracy. Our results suggest that the molecular machinery for ACD is already stored as maternal transcripts in the oocyte. Thus, the spiralian egg can be viewed as a totipotent yet highly specialized cell that evolved to execute fast and precise ACDs during spiral cleaving stages. Our survey identifies cohorts of factors in P. dumerilii that are candidates for these molecular mechanisms and their regulation, and sets the stage for a functional dissection of ACD in a spiral-cleaving embryo.

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.

BACKGROUND: Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. RESULTS: Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP) w37 ]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. CONCLUSION: We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders.

A transcriptional blueprint for a spiral-cleaving embryo.

BACKGROUND: The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RESULTS: RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. CONCLUSIONS: Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

Structure, phylogeny, and expression of the frizzled-related gene family in the lophotrochozoan annelid Platynereis dumerilii.

BACKGROUND: Wnt signaling pathways are highly conserved signal transduction pathways important for axis formation, cell fate specification, and organogenesis throughout metazoan development. Within the various Wnt pathways, the frizzled transmembrane receptors (Fzs) and secreted frizzled-related proteins (sFRPs) play central roles in receiving and antagonizing Wnt signals, respectively. Despite their importance, very little is known about the frizzled-related gene family (fzs & sfrps) in lophotrochozoans, especially during early stages of spiralian development. Here we ascertain the frizzled-related gene complement in six lophotrochozoan species, and determine their spatial and temporal expression pattern during early embryogenesis and larval stages of the marine annelid Platynereis dumerilii. RESULTS: Phylogenetic analyses confirm conserved homologs for four frizzled receptors (Fz1/2/7, Fz4, Fz5/8, Fz9/10) and sFRP1/2/5 in five of six lophotrochozoan species. The sfrp3/4 gene is conserved in one, divergent in two, and evidently lost in three lophotrochozoan species. Three novel fz-related genes (fzCRD1-3) are unique to Platynereis. Transcriptional profiling and in situ hybridization identified high maternal expression of fz1/2/7, expression of fz9/10 and fz1/2/7 within animal and dorsal cell lineages after the 32-cell stage, localization of fz5/8, sfrp1/2/5, and fzCRD-1 to animal-pole cell lineages after the 80-cell stage, and no expression for fz4, sfrp3/4, and fzCRD-2, and -3 in early Platynereis embryos. In later larval stages, all frizzled-related genes are expressed in distinct patterns preferentially in the anterior hemisphere and less in the developing trunk. CONCLUSIONS: Lophotrochozoans have retained a generally conserved ancestral bilaterian frizzled-related gene complement (four Fzs and two sFRPs). Maternal expression of fz1/2/7, and animal lineage-specific expression of fz5/8 and sfrp1/2/5 in early embryos of Platynereis suggest evolutionary conserved roles of these genes to perform Wnt pathway functions during early cleavage stages, and the early establishment of a Wnt inhibitory center at the animal pole, respectively. Numerous frizzled receptor-expressing cells and embryonic territories were identified that might indicate competence to receive Wnt signals during annelid development. An anterior bias for frizzled-related gene expression in embryos and larvae might point to a polarity of Wnt patterning systems along the anterior-posterior axis of this annelid.

Expression of the wnt gene complement in a spiral-cleaving embryo and trochophore larva.

The highly conserved wnt gene family has roles in developmental processes ranging from axis formation to cell fate determination. The polychaete Platynereis dumerilii has retained 12 of the 13 ancient wnt subfamilies and is a good model system to study the roles of the wnt ligands in spiralian development. While it has been shown that Platynereis uses a global beta-catenin-mediated binary cell fate specification module in development, the early roles of the 12 wnt genes present in Platynereis are unknown. Transcriptional profiling by RNA-Seq during early development and whole-mount in situ hybridization of embryo and larval stages were used to determine the temporal and spatial regulation of the wnt complement in Platynereis. None of the 12 wnt transcripts were maternally provided at significant levels. In pregastrula embryos, zygotic wntA, wnt4, and wnt5 transcripts exhibited distinctive patterns of differential gene expression. In contrast, in trochophore larvae, all 12 wnt ligands were expressed and each had a distinct expression pattern. While three wnt ligands were expressed in early development, none were expressed in the right place for a widespread role in beta-catenin-mediated binary specification in early Platynereis development. However, the expression patterns of the wnt ligands suggest the presence of numerous wnt signaling centers, with the most prominent being a bias for staggered posterior wnt expression in trochophore larvae. The similarity to wnt expression domains in cnidarians around the blastopore and the tail organizer in chordates supports a hypothesis of a common evolutionary origin of posterior organizing centers.

Animal development: an ancient ß-catenin switch?

High and low nuclear levels of the conserved transcriptional regulator ß-catenin distinguish multiple sister cell fates to specify endoderm and mesoderm during early embryogenesis in a chordate embryo.

Whole genome duplications and expansion of the vertebrate GATA transcription factor gene family.

BACKGROUND: GATA transcription factors influence many developmental processes, including the specification of embryonic germ layers. The GATA gene family has significantly expanded in many animal lineages: whereas diverse cnidarians have only one GATA transcription factor, six GATA genes have been identified in many vertebrates, five in many insects, and eleven to thirteen in Caenorhabditis nematodes. All bilaterian animal genomes have at least one member each of two classes, GATA123 and GATA456. RESULTS: We have identified one GATA123 gene and one GATA456 gene from the genomic sequence of two invertebrate deuterostomes, a cephalochordate (Branchiostoma floridae) and a hemichordate (Saccoglossus kowalevskii). We also have confirmed the presence of six GATA genes in all vertebrate genomes, as well as additional GATA genes in teleost fish. Analyses of conserved sequence motifs and of changes to the exon-intron structure, and molecular phylogenetic analyses of these deuterostome GATA genes support their origin from two ancestral deuterostome genes, one GATA 123 and one GATA456. Comparison of the conserved genomic organization across vertebrates identified eighteen paralogous gene families linked to multiple vertebrate GATA genes (GATA paralogons), providing the strongest evidence yet for expansion of vertebrate GATA gene families via genome duplication events. CONCLUSION: From our analysis, we infer the evolutionary birth order and relationships among vertebrate GATA transcription factors, and define their expansion via multiple rounds of whole genome duplication events. As the genomes of four independent invertebrate deuterostome lineages contain single copy GATA123 and GATA456 genes, we infer that the 0R (pre-genome duplication) invertebrate deuterostome ancestor also had two GATA genes, one of each class. Synteny analyses identify duplications of paralogous chromosomal regions (paralogons), from single ancestral vertebrate GATA123 and GATA456 chromosomes to four paralogons after the first round of vertebrate genome duplication, to seven paralogons after the second round of vertebrate genome duplication, and to fourteen paralogons after the fish-specific 3R genome duplication. The evolutionary analysis of GATA gene origins and relationships may inform understanding vertebrate GATA factor redundancies and specializations.

The evolution of protostome GATA factors: molecular phylogenetics, synteny, and intron/exon structure reveal orthologous relationships.

BACKGROUND: Invertebrate and vertebrate GATA transcription factors play important roles in ectoderm and mesendoderm development, as well as in cardiovascular and blood cell fate specification. However, the assignment of evolutionarily conserved roles to GATA homologs requires a detailed framework of orthologous relationships. Although two distinct classes, GATA123 and GATA456, have been unambiguously recognized among deuterostome GATA genes, it has been difficult to resolve exact orthologous relationships among protostome homologs. Protostome GATA genes are often present in multiple copies within any one genome, and rapidly evolving gene sequences have obscured orthology among arthropod and nematode GATA homologs. In addition, a lack of taxonomic sampling has prevented a stepwise reconstruction of protostome GATA gene family evolution. RESULTS: We have identified the complete GATA complement (53 genes) from a diverse sampling of protostome genomes, including six arthropods, three lophotrochozoans, and two nematodes. Reciprocal best hit BLAST analysis suggested orthology of these GATA genes to either the ancestral bilaterian GATA123 or the GATA456 class. Using molecular phylogenetic analyses of gene sequences, together with conserved synteny and comparisons of intron/exon structure, we inferred the evolutionary relationships among these 53 protostome GATA homologs. In particular, we resolved the orthology and evolutionary birth order of all arthropod GATA homologs including the highly divergent Drosophila GATA genes. CONCLUSION: Our combined analyses confirm that all protostome GATA transcription factor genes are members of either the GATA123 or GATA456 class, and indicate that there have been multiple protostome-specific duplications of GATA456 homologs. Three GATA456 genes exhibit linkage in multiple protostome species, suggesting that this gene cluster arose by tandem duplications from an ancestral GATA456 gene. Within arthropods this GATA456 cluster appears orthologous and widely conserved. Furthermore, the intron/exon structures of the arthropod GATA456 orthologs suggest a distinct order of gene duplication events. At present, however, the evolutionary relationship to similarly linked GATA456 paralogs in lophotrochozoans remains unclear. Our study shows how sampling of additional genomic data, especially from less derived and interspersed protostome taxa, can be used to resolve the orthologous relationships within more divergent gene families.

beta-Catenin asymmetries after all animal/vegetal- oriented cell divisions in Platynereis dumerilii embryos mediate binary cell-fate specification.

In response to Wnt signaling during animal development, beta-catenin accumulates in nuclei to mediate the transcriptional activation of target genes. Here, we show that a highly conserved beta-catenin in the annelid Platynereis dumerilii exhibits a reiterative, nearly universal embryonic pattern of nuclear accumulation remarkably similar to that observed in the nematode Caenorhabditis elegans. Platynereis exhibits beta-catenin sister-cell asymmetries after all cell divisions that occur along the animal/vegetal axis beginning early in embryogenesis, but not after two transverse divisions that establish bilateral symmetry in the trunk. Moreover, ectopic activation of nuclear beta-catenin accumulation in Platynereis causes animal-pole sister cells, which normally have low nuclear beta-catenin levels, to adopt the fate of their vegetal-pole sisters, which normally have high nuclear beta-catenin levels. The presence of reiterative and functionally important beta-catenin asymmetries in two distantly related animal phyla suggests an ancient metazoan origin of a beta-catenin-mediated binary cell-fate specification module.

Ectoderm- and endomesoderm-specific GATA transcription factors in the marine annelid Platynereis dumerilli.

The GATA family of transcription factors appears to retain conserved roles in early germ layer patterning in most, if not all, animals; however, the number and structure of GATA factor genes varies substantially when different animal genomes are compared. Thus, the origin and relationships of invertebrate and vertebrate GATA factors, and their involvement in animal germ layer evolution, are unclear. We identified two highly conserved GATA factor genes in a marine annelid, the polychaete Platynereis dumerilii. A phylogenetic analysis indicates that the two Platynereis GATA factors are orthologous to the GATA1/2/3 and GATA4/5/6 subfamilies present in vertebrates. We also identified conserved motifs within each GATA class, and assigned the divergent Caenorhabditiselegans and Drosophila melanogaster GATA factor genes to the vertebrate classes. Similar to their vertebrate homologs, PdGATA123 mRNA expression was restricted to ectoderm, whereas PdGATA456 was detected only in endomesoderm. Finally, we identified in genome databases one GATA factor gene in each of two distantly related cnidarians that include motifs from both bilaterian GATA factor classes. Our results show that distinct orthologs of the two vertebrate GATA factor classes exist in a protostome invertebrate, suggesting that bilaterian GATA factors originated from GATA1/2/3 and 4/5/6 ancestral orthologs. Moreover, our results indicate that the GATA gene duplication and the functional divergence that led to these two ancestral GATA factor genes occurred after the split of the bilaterian stem group from the cnidarians.

Cell polarity and the cytoskeleton in the Caenorhabditis elegans zygote.

The anterior-posterior axis of the Caenorhabditis elegans zygote forms shortly after fertilization when the sperm pronucleus and its associated centrosomal asters provide a cue that establishes the anterior-posterior (AP) body axis. In response to this cue, the microfilament cytoskeleton polarizes the distribution of a group of widely conserved, cortically localized regulators called the PAR proteins, which are required for the first mitotic division to be asymmetric. These asymmetries include a posterior displacement of the first mitotic spindle and the differential segregation of cell-fate determinants to the anterior and posterior daughters produced by the first cleavage of the zygote. Here we review recent advances in our understanding of the mechanisms that polarize the one-cell zygote to generate an AP axis of asymmetry.

Protein evolution: structure-function relationships of the oncogene beta-catenin in the evolution of multicellular animals.

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the 'natural experiments' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.