Oral-aboral Axis Research Articles

Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here, we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis, which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral–aboral axis of the polyp. Homeobox transcription factors and components of the TGF β , Notch, and FGF signalling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed, and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella, with implications for understanding the reproductive and regenerative biology of other cnidarian species.

The brain regulatory program predates central nervous system evolution

Understanding how brains evolved is critical to determine the origin(s) of centralized nervous systems. Brains are patterned along their anteroposterior axis by stripes of gene expression that appear to be conserved, suggesting brains are homologous. However, the striped expression is also part of the deeply conserved anteroposterior axial program. An emerging hypothesis is that similarities in brain patterning are convergent, arising through the repeated co-option of axial programs. To resolve whether shared brain neuronal programs likely reflect convergence or homology, we investigated the evolution of axial programs in neurogenesis. We show that the bilaterian anteroposterior program patterns the nerve net of the cnidarian Nematostella along the oral-aboral axis arguing that anteroposterior programs regionalized developing nervous systems in the cnidarian–bilaterian common ancestor prior to the emergence of brains. This finding rejects shared patterning as sufficient evidence to support brain homology and provides functional support for the plausibility that axial programs could be co-opted if nervous systems centralized in multiple lineages.

Sea anemone Frizzled receptors play partially redundant roles in oral-aboral axis patterning.

Canonical Wnt (cWnt) signalling is involved in a plethora of basic developmental processes such as endomesoderm specification, gastrulation and patterning the main body axis. To activate the signal, Wnt ligands form complexes with LRP5/6 and Frizzled receptors, which leads to nuclear translocation of β-catenin and a transcriptional response. In Bilateria, the expression of different Frizzled genes is often partially overlapping, and their functions are known to be redundant in several developmental contexts. Here, we demonstrate that all four Frizzled receptors take part in the cWnt-mediated oral-aboral axis patterning in the cnidarian Nematostella vectensis but show partially redundant functions. However, we do not see evidence for their involvement in the specification of the endoderm - an earlier event likely relying on maternal intracellular β-catenin signalling components. Finally, we demonstrate that the main Wnt ligands crucial for the early oral-aboral patterning are Wnt1, Wnt3 and Wnt4. Comparison of our data with knowledge from other models suggests that distinct but overlapping expression domains and partial functional redundancy of cnidarian and bilaterian Frizzled genes may represent a shared ancestral trait.

Understanding the development of oral epithelial organs through single cell transcriptomic analysis.

ABSTRACTDuring craniofacial development, the oral epithelium begins as a morphologically homogeneous tissue that gives rise to locally complex structures, including the teeth, salivary glands and taste buds. How the epithelium is initially patterned and specified to generate diverse cell types remains largely unknown. To elucidate the genetic programs that direct the formation of distinct oral epithelial populations, we mapped the transcriptional landscape of embryonic day 12 mouse mandibular epithelia at single cell resolution. Our analysis identified key transcription factors and gene regulatory networks that define different epithelial cell types. By examining the spatiotemporal patterning process along the oral-aboral axis, our results propose a model in which the dental field is progressively confined to its position by the formation of the aboral epithelium anteriorly and the non-dental oral epithelium posteriorly. Using our data, we also identified Ntrk2 as a proliferation driver in the forming incisor, contributing to its invagination. Together, our results provide a detailed transcriptional atlas of the embryonic mandibular epithelium, and unveil new genetic markers and regulators that are present during the specification of various oral epithelial structures.

From apolar gastrula to polarized larva: Embryonic development of a marine hydroid, Dynamena pumila.

In almost all metazoans examined to this respect, the axial patterning system based on canonical Wnt (cWnt) signaling operates throughout the course of development. In most metazoans, gastrulation is polar, and embryos develop morphological landmarks of axial polarity, such as blastopore under control/regulation from cWnt signaling. However, in many cnidarian species, gastrulation is morphologically apolar. The question remains whether сWnt signaling providing the establishment of a body axis controls morphogenetic processes involved in apolar gastrulation. In this study, we focused on the embryonic development of Dynamena pumila, a cnidarian species with apolar gastrulation. We thoroughly described cell behavior, proliferation, and ultrastructure and examined axial patterning in the embryos of this species. We revealed that the first signs of morphological polarity appear only after the end of gastrulation, while molecular prepatterning of the embryo does exist during gastrulation. We have shown experimentally that in D. pumila, the direction of the oral-aboral axis is highly robust against perturbations in cWnt activity. Our results suggest that morphogenetic processes are uncoupled from molecular axial patterning during gastrulation in D. pumila. Investigation of D. pumila might significantly expand our understanding of the ways in which morphological polarization and axial molecular patterning are linked in Metazoa.

Coelomic System of the Sea Urchin Strongylocentrotus pallidus (Echinodermata, Echinoidea) and the Issue of Coelomic Metamery in Echinoderms.

The representatives of the main phylogenetic clades of Bilateria are characterized by metamery. In Deuterostomia, metamery is presented in hemichordates and chordates. Sea urchins have 7 metameric coelomic rings located along the oral-aboral axis of the body. A similar coelomic metamery is also a sign of representatives of other classes of echinoderms. We hypothesize that the metameric coelomic rings in echinoderms are inherent of the common ancestor of Deuterostomia. Asymmetry in the structure of echinoderm coeloms is the result of ancestral forms lying on the right side of the body, which led to a reduction of the coeloms on the right side. During the sedentary lifestyle, the left-side coeloms formed metameric coelomic rings.

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

BackgroundThe Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor.ResultsHydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model.ConclusionsWe show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Epigenomic landscape of enhancer elements during Hydra head organizer formation

BackgroundAxis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra.ResultsTo gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity.ConclusionsThe activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3β inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

Wnt signaling determines body axis polarity in regenerating Hydra tissue fragments

How an animal establishes its body axis is a fundamental question in developmental biology. The freshwater cnidarian Hydra is an attractive model for studying axis formation because it is radially symmetric, with a single oral-aboral axis. It was recently proposed that the orientation of the new body axis in a regenerating Hydra polyp is determined by the oral-aboral orientation of the actin-myosin contractile processes (myonemes) in the animal’s outer epithelial layer. However, it remained unclear how the oral-aboral polarity of the body axis would be defined. As Wnt signaling is known to control axis polarity in Hydra and bilaterians, we hypothesized that it plays a role in axis formation during regeneration of Hydra tissue pieces. We tested this hypothesis using pharmacological perturbations and novel grafting experiments to set Wnt signaling and myoneme orientation perpendicular to each other to determine which controls axis formation. Our results demonstrate that Wnt signaling is the dominant encoder of axis orientation and polarity, in line with its conserved role in axial patterning.

Ancestral roles of atypical cadherins in planar cell polarity

Fat, Fat-like, and Dachsous family cadherins are giant proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterians. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of phylum Cnidaria a sister group of bilaterian. We found Hydra does not possess a true Fat homolog, but has homologs of Fat-like (HyFatl) and Dachsous (HyDs) that localize at the apical membrane of ectodermal epithelial cells and are planar polarized perpendicular to the oral-aboral axis of the animal. Using a knockdown approach we found that HyFatl is involved in local cell alignment and cell-cell adhesion, and that reduction of HyFatl leads to defects in tissue organization in the body column. Overexpression and knockdown experiments indicate that the intracellular domain (ICD) of HyFatl affects actin organization through proline-rich repeats. Thus, planar polarization of Fat-like and Dachsous cadherins has ancient, prebilaterian origins, and Fat-like cadherins have ancient roles in cell adhesion, spindle orientation, and tissue organization.

Neural crest cells require Meis2 for patterning the mandibular arch via the Sonic hedgehog pathway.

ABSTRACTCranial neural crest cells (cNCCs) originate in the anterior neural tube and populate pharyngeal arches in which they contribute to formation of bone and cartilage. This cell population also provides molecular signals for the development of tissues of non-neural crest origin, such as the tongue muscles, teeth enamel or gland epithelium. Here we show that the transcription factor Meis2 is expressed in the oral region of the first pharyngeal arch (PA1) and later in the tongue primordium. Conditional inactivation of Meis2 in cNCCs resulted in loss of Sonic hedgehog signalling in the oropharyngeal epithelium and impaired patterning of PA1 along the lateral–medial and oral–aboral axis. Failure of molecular specification of PA1, illustrated by altered expression of Hand1/2, Dlx5, Barx1, Gsc and other markers, led to hypoplastic tongue and ectopic ossification of the mandible. Meis2-mutant mice thus display craniofacial defects that are reminiscent of several human syndromes and patients with mutations in the Meis2 gene.

A novel bilateral grafting technique for studying patterning in Hydra

Control of patterning and the specification of body axes are fundamental aspects of animal development involving complex interactions between chemical, physical, and genetic signals. The freshwater polyp Hydra has long been recognized as a useful model system to address these questions due to its simple anatomy, optical transparency, and strong regenerative abilities, which enabled clever grafting experiments to alter and probe patterning. Reliable methods exist for the transplantation of small tissue pieces into the body column or the combination of sections cut perpendicular to the body axis, which can be used to examine oral-aboral gradients and axis induction potential of tissue fragments. However, existing methods do not allow researchers to probe questions of axis alignment and lateral information exchange. We therefore developed a technique to produce chimeric animals split longitudinally along the body axis of the animal by anesthetizing the animals with the terpene linalool and threading the donor pieces onto pairs of fine glass needles. Our novel approach can be applied to study questions in Hydra research that have thus far been inaccessible, including patterning processes acting perpendicular to the oral-aboral axis and the extent of lateral cell migration.

A cell-based boundary model of gastrulation by unipolar ingression in the hydrozoan cnidarian Clytia hemisphaerica

In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing presumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation.

Characterization of nAChRs in Nematostella vectensis supports neuronal and non-neuronal roles in the cnidarian\u2013bilaterian common ancestor

BackgroundNicotinic and muscarinic acetylcholine receptors likely evolved in the cnidarian–bilaterian common ancestor. Both receptor families are best known for their role at chemical synapses in bilaterian animals, but they also have described roles as non-neuronal signaling receptors within the bilaterians. It is not clear when either of the functions for nicotinic or muscarinic receptors evolved. Previous studies in cnidarians suggest that acetylcholine’s neuronal role existed prior to the cnidarian–bilaterian divergence, but did not address potential non-neuronal functions. To determine the origins of neuronal and non-neuronal functions of nicotinic acetylcholine receptors, we investigated the phylogenetic position of cnidarian acetylcholine receptors, characterized the spatiotemporal expression patterns of nicotinic receptors in N. vectensis, and compared pharmacological studies in N. vectensis to the previous work in other cnidarians.ResultsConsistent with described activity in other cnidarians, treatment with acetylcholine-induced tentacular contractions in the cnidarian sea anemone N. vectensis. Phylogenetic analysis suggests that the N. vectensis genome encodes 26 nicotinic (nAChRs) and no muscarinic (mAChRs) acetylcholine receptors and that nAChRs independently radiated in cnidarian and bilaterian linages. The namesake nAChR agonist, nicotine, induced tentacular contractions similar to those observed with acetylcholine, and the nAChR antagonist mecamylamine suppressed tentacular contractions induced by both acetylcholine and nicotine. This indicated that tentacle contractions are in fact mediated by nAChRs. Nicotine also induced the contraction of radial muscles, which contract as part of the peristaltic waves that propagate along the oral–aboral axis of the trunk. Radial contractions and peristaltic waves were suppressed by mecamylamine. The ability of nicotine to mimic acetylcholine responses, and of mecamylamine to suppress acetylcholine and nicotine-induced contractions, supports a neuronal function for acetylcholine in cnidarians. Examination of the spatiotemporal expression of N. vectensis nAChRs (NvnAChRs) during development and in juvenile polyps identified that NvnAChRs are expressed in neurons, muscles, gonads, and large domains known to be consistent with a role in developmental patterning. These patterns are consistent with nAChRs functioning in both a neuronal and non-neuronal capacity in N. vectensis.ConclusionOur data suggest that nAChR receptors functioned at chemical synapses in N. vectensis to regulate tentacle contraction. Similar responses to acetylcholine are well documented in cnidarians, suggesting that the neuronal function represents an ancestral role for nAChRs. Expression patterns of nAChRs are consistent with both neuronal and non-neuronal roles for acetylcholine in cnidarians. Together, these observations suggest that both neuronal and non-neuronal functions for the ancestral nAChRs were present in the cnidarian–bilaterian common ancestor. Thus, both roles described in bilaterian species likely arose at or near the base of nAChR evolution.

Molecular characterisation of a cellular conveyor belt in Clytia medusae

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a “cellular conveyor belt”. Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/β-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.

Differentiation of the Oral–Aboral Axis and Body Parts during Life Cycle Transitions in Scyphozoa

Abstract—Differentiation of body axes during formation of primary body plan is one of the most fundamental processes in metazoan development. How did body axes emerge in the course of animal evolution? How did developmental processes associated with axes differentiation evolve? The basal metazoans, representatives of the phylum Cnidaria, are valuable objects for resolving these questions, since they have an evolutionarily simple body plan and only one (oral-aboral) body axis. The high abundance and diversity of the modes of asexual reproduction is an interesting feature of this group. In this review, we summarized the data on the establishment of primary body axis and differentiation of main morphological structures during asexual reproduction in representatives of the class Scyphozoa. We found that the relative orientation of the parental and offspring body axes varies significantly and depends on the mode of asexual reproduction, and that the same body parts of the offspring polyp or medusa can be formed from different parts of the parental organism. The high plasticity of scyphozoan developmental processes can be explained by assuming that various types of asexual reproduction evolved from regeneration processes in this group. To test this assumption, it is necessary to study the molecular mechanisms of differentiation of the primary body axis and the body parts during regeneration and asexual reproduction of scyphozoans.

"Dorsal-Ventral" Genes Are Part of an Ancient Axial Patterning System: Evidence from Trichoplax adhaerens (Placozoa).

Placozoa are a morphologically simplistic group of marine animals found globally in tropical and subtropical environments. They consist of two named species, Trichoplax adhaerens and more recently Hoilungia hongkongensis, both with roughly six morphologically distinct cell types. With a sequenced genome, a limited number of cell types, and a simple flattened morphology, Trichoplax is an ideal model organism from which to explore the biology of an animal with a cellular complexity analagous to that of the earliest animals. Using a new approach for identification of gene expression patterns, this research looks at the relationship of Chordin/TgfΒ signaling and the axial patterning system of Placozoa. Our results suggest that placozoans have an oral–aboral axis similar to cnidarians and that the parahoxozoan ancestor (common ancestor of Placozoa and Cnidaria) was likely radially symmetric.

Hedgehog signaling patterns the oral-aboral axis of the mandibular arch.

Development of vertebrate jaws involves patterning neural crest-derived mesenchyme cells into distinct subpopulations along the proximal-distal and oral-aboral axes. Although the molecular mechanisms patterning the proximal-distal axis have been well studied, little is known regarding the mechanisms patterning the oral-aboral axis. Using unbiased single-cell RNA-seq analysis followed by in situ analysis of gene expression profiles, we show that Shh and Bmp4 signaling pathways are activated in a complementary pattern along the oral-aboral axis in mouse embryonic mandibular arch. Tissue-specific inactivation of hedgehog signaling in neural crest-derived mandibular mesenchyme led to expansion of BMP signaling activity to throughout the oral-aboral axis of the distal mandibular arch and subsequently duplication of dentary bone in the oral side of the mandible at the expense of tongue formation. Further studies indicate that hedgehog signaling acts through the Foxf1/2 transcription factors to specify the oral fate and pattern the oral-aboral axis of the mandibular mesenchyme.