Yale J. Passamaneck

Ciliary photoreceptors in the cerebral eyes of a protostome larva

BackgroundEyes 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 non-visual 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.ResultsTo 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.ConclusionsOur 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

BackgroundLarval 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, Terebratalia transversa.ResultsRadially symmetric gastrulae broadly express Tt-Six3/6 and Tt-hbn in the animal cap ectoderm. Tt-NK2.1 and Tt-otp are restricted to a central subset of these cells, and Tt-fez and Tt-FoxQ2 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 Tt-Six3/6, Tt-fez, Tt-FoxQ2, and Tt-otp expression domains, and an anterior ventral domain demarcated by Tt-hbn and Tt-NK2.1 expression. More posteriorly, the latter domains are bordered by Tt-FoxG expression in the region of the transverse ciliated band. Tt-synaptotagmin 1 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. Tt-otp is the only gene expressed in numerous flask-shaped cells of the apical organ and in a subset of neurons in the mantle lobe.ConclusionsOur expression data for Tt-Six3/6, Tt-FoxQ2, and Tt-otp confirm some aspects of bilaterian-wide conservation of spatial partitioning within anterior neurogenic domains and also suggest a common origin for central otp-positive cell types within the larval apical organs of spiralians. However, the field of sensory neurons within the larval apical organ of Terebratalia 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 Tt-FoxQ2 and Tt-otp. Corresponding differences in the expression of Tt-hbn, Tt-NK2.1, and Tt-FoxG 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 Six3/6, NK2.1, otp, hbn, fez, and FoxQ2 expression domains that were subsequently modified within larval and adult neural tissues of protostome and deuterostome animals.

Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis

BackgroundThe 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.ResultsIn 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.ConclusionsThe 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.

The developmental basis for the recurrent evolution of deuterostomy and protostomy

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.

Mesodermal gene expression during the embryonic and larval development of the articulate brachiopod Terebratalia transversa

BackgroundBrachiopods undergo radial cleavage, which is distinct from the stereotyped development of closely related spiralian taxa. The mesoderm has been inferred to derive from the archenteron walls following gastrulation, and the primary mesoderm derivative in the larva is a complex musculature. To investigate the specification and differentiation of the mesoderm in the articulate brachiopod Terebratalia transversa, we have identified orthologs of genes involved in mesoderm development in other taxa and investigated their spatial and temporal expression during the embryonic and larval development of T. transversa.ResultsOrthologs of 17 developmental regulatory genes with roles in the development of the mesoderm in other bilaterian animals were found to be expressed in the developing mesoderm of T. transversa. Five genes, Tt.twist, Tt.GATA456, Tt.dachshund, Tt.mPrx, and Tt.NK1, were found to have expression throughout the archenteron wall at the radial gastrula stage, shortly after the initiation of gastrulation. Three additional genes, Tt.Pax1/9, Tt.MyoD, and Tt.Six1/2, showed expression at this stage in only a portion of the archenteron wall. Tt.eya, Tt.FoxC, Tt.FoxF, Tt.Mox, Tt.paraxis, Tt.Limpet, and Tt.Mef2 all showed initial mesodermal expression during later gastrula or early larval stages. At the late larval stage, Tt.dachshund, Tt.Limpet, and Tt.Mef2 showed expression in nearly all mesoderm cells, while all other genes were localized to specific regions of the mesoderm. Tt.FoxD and Tt.noggin both showed expression in the ventral mesoderm at the larval stages, with gastrula expression patterns in the archenteron roof and blastopore lip, respectively.ConclusionsExpression analyses support conserved roles for developmental regulators in the specification and differentiation of the mesoderm during the development of T. transversa. Expression of multiple mesodermal factors in the archenteron wall during gastrulation supports previous morphological observations that this region gives rise to larval mesoderm. Localized expression domains during gastrulation and larval development evidence early regionalization of the mesoderm and provide a basis for hypotheses regarding the molecular regulation underlying the complex system of musculature observed in the larva.

Clustered brachiopod Hox genes are not expressed collinearly and are associated with lophotrochozoan novelties

Significance Hox genes pattern the anteroposterior axis of all animals that have left and right body sides. In many animals, Hox genes are clustered along the chromosomes and expressed in spatial and temporal order. This coordinated regulation is thought to have preserved the cluster through a developmental constraint. Our study of the genomic organization and the embryonic spatial and temporal expression of Hox genes in sessile marine animals called lampshells (brachiopods) shows that along with having a broken Hox cluster, they lack both temporal and spatial collinearity. Furthermore, we present molecular evidence that the hard tissues (chaetae and shells) of segmented worms, mollusks, and brachiopods share a common origin that dates back to the Early Cambrian. Temporal collinearity is often considered the main force preserving Hox gene clusters in animal genomes. Studies that combine genomic and gene expression data are scarce, however, particularly in invertebrates like the Lophotrochozoa. As a result, the temporal collinearity hypothesis is currently built on poorly supported foundations. Here we characterize the complement, cluster, and expression of Hox genes in two brachiopod species, Terebratalia transversa and Novocrania anomala. T. transversa has a split cluster with 10 genes (lab, pb, Hox3, Dfd, Scr, Lox5, Antp, Lox4, Post2, and Post1), whereas N. anomala has 9 genes (apparently missing Post1). Our in situ hybridization, real-time quantitative PCR, and stage-specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 (in both brachiopods), and Dfd (in both brachiopods) show staggered mesodermal expression. Thus, our findings support the idea that temporal collinearity might contribute to keeping Hox genes clustered. Remarkably, expression of the Hox genes in both brachiopod species demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of Hox genes, together with Arx, Zic, and Notch pathway components in chaetae and shell fields in brachiopods, mollusks, and annelids provide molecular evidence supporting the conservation of the molecular basis for these lophotrochozoan hallmarks.

Aryl hydrocarbon receptor (AHR) in the cnidarian Nematostella vectensis: comparative expression, protein interactions, and ligand binding

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachealess). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, β-naphthoflavone). Together, our data suggest that AHR’s original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian–bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates.

Evidence for a Phototransduction Cascade in an Early Brachiopod Embryo

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.

Brachiopods possess a split Hox cluster with signs of spatial, but not temporal collinearity

Temporal collinearity is often regarded as the force preserving Hox clusters in vertebrate genomes. Studies that combine genomic and gene expression data in invertebrates would allow generalizing this observation across all animals, but are scarce, particularly within Lophotrochozoa (e.g., snails and segmented worms). Here, we use two brachiopod species –Terebratalia transversa, Novocrania anomala– to characterize the complement, cluster and expression of their Hox genes. T. transversa has an ordered, split cluster with ten genes (lab, pb, Hox3, dfd, scr, lox5, antp, lox4, post2, post1), while N. anomala has nine (missing post1). Our in situ hybridization, qPCR and stage specific transcriptomic analyses show that brachiopod Hox genes are neither strictly temporally nor spatially collinear; only pb (in T. transversa), Hox3 and dfd (in both brachiopods) show staggered mesodermal expression. The spatial expression of the Hox genes in both brachiopod species correlates with their morphology and demonstrates cooption of Hox genes in the chaetae and shell fields, two major lophotrochozoan morphological novelties. The shared and specific expression of a subset of Hox genes, Arx and Zic orthologs in chaetae and shell-fields between brachiopods, mollusks, and annelids supports the deep conservation of the molecular basis forming these lophotrochozoan hallmarks. Our findings challenge that collinearity alone preserves lophotrochozoan Hox clusters, indicating that additional genomic traits need to be considered in understanding Hox evolution.Hox genes are often clustered in animal genomes and exhibit spatial and/or temporal collinearity. It is generally believed that temporal collinearity is the major force preserving Hox clusters. However, studies combining genomic and gene expression analyses of Hox genes are scarce, particularly within Spiralia and Lophotrochozoa (e.g. mollusks, segmented worms, and flatworms). Here, we use two brachiopod species --Terebratalia transversa and Novocrania anomala-- that respectively belong to the two major brachiopod lineages to characterize their Hox complement, the presence of a Hox cluster, and the temporal and spatial expression of their Hox genes. We demonstrate that the Hox complement consists of ten Hox genes in T. transversa (lab, pb, Hox3, dfd, scr, lox5, antp, lox4, post2 and post1) and nine in N. anomala (missing post1). Additionally, T. transversa has an ordered, split Hox cluster. Expression analyses reveal that Hox genes are neither temporally nor spatially collinear, and only the genes pb (in T. transversa), Hox3 and dfd (in both brachiopods) show staggered expression in the mesoderm. Remarkably, lab, scr, antp and post1 are associated with the development of the chaetae and shell-forming epithelium, as also observed in annelid chaetae and mollusk shell fields. This, together with the expression of Arx homeobox, supports the deep conservation of the molecular basis for chaetae formation and shell patterning in Lophotrochozoa. Our findings challenge the current evolutionary scenario that (temporal) collinearity is the major mechanism preserving Hox clusters, and suggest that Hox genes were involved in the evolution of lophotrochozoan novelties.