The onset of whole-body regeneration in Botryllus schlosseri: morphological and molecular characterization

Colonial tunicates are the only chordates that regularly regenerate a fully functional whole body as part of their asexual life cycle, starting from specific epithelia and/or mesenchymal cells. In addition, in some species, whole-body regeneration (WBR) can also be triggered by extensive injuries, which deplete most of their tissues and organs and leave behind only small fragments of their body. In this manuscript, we characterized the onset of WBR in Botryllus schlosseri, one colonial tunicate long used as a laboratory model. We first analyzed the transcriptomic response to a WBR-triggering injury. Then, through morphological characterization, in vivo observations via time-lapse, vital dyes, and cell transplant assays, we started to reconstruct the dynamics of the cells triggering regeneration, highlighting an interplay between mesenchymal and epithelial cells. The dynamics described here suggest that WBR in B. schlosseri is initiated by extravascular tissue fragments derived from the injured individuals rather than particular populations of blood-borne cells, as has been described in closely related species. The morphological and molecular datasets here reported provide the background for future mechanistic studies of the WBR ontogenesis in B. schlosseri and allow to compare it with other regenerative processes occurring in other tunicate species and possibly independently evolved.

Morphological Study and 3D Reconstruction of the Larva of the Ascidian Halocynthia roretzi

The swimming larva represents the dispersal phase of ascidians, marine invertebrates belonging to tunicates. Due to its adhesive papillae, the larva searches the substrate, adheres to it, and undergoes metamorphosis, thereby becoming a sessile filter feeding animal. The larva anatomy has been described in detail in a few species, revealing a different degree of adult structure differentiation, called adultation. In the solitary ascidian Halocynthia roretzi, a species reared for commercial purposes, embryogenesis has been described in detail, but information on the larval anatomy is still lacking. Here, we describe it using a comparative approach, utilizing 3D reconstruction, as well as histological/TEM observations, with attention to its papillae. The larva is comparable to those of other solitary ascidians, such as Ciona intestinalis. However, it displays a higher level of adultation for the presence of the atrium, opened outside by means of the atrial siphon, and the peribranchial chambers. It does not reach the level of complexity of the larva of Botryllus schlosseri, a phylogenetically close colonial ascidian. Our study reveals that the papillae of H. roretzi, previously described as simple and conform, exhibit dynamic changes during settlement. This opens up new considerations on papillae morphology and evolution and deserves to be further investigated.

Whole body regeneration and developmental competition in two botryllid ascidians

Background Botryllid ascidians are a group of marine invertebrate chordates that are colonial and grow by repeated rounds of asexual reproduction to form a colony of individual bodies, called zooids, linked by a common vascular network. Two distinct processes are responsible for zooid regeneration. In the first, called blastogenesis, new zooids arise from a region of multipotent epithelium from a pre-existing zooid. In the second, called whole body regeneration (WBR), mobile cells in the vasculature coalesce and are the source of the new zooid. In some botryllid species, blastogenesis and WBR occur concurrently, while in others, blastogenesis is used exclusively for growth, while WBR only occurs following injury or exiting periods of dormancy. In species such as Botrylloides diegensis, injury induced WBR is triggered by the surgical isolation of a small piece of vasculature. However, Botryllus schlosseri has unique requirements that must be met for successful injury induced WBR. Our goal was to understand why there would be different requirements between these two species. Results While WBR in B. diegensis was robust, we found that in B. schlosseri, new zooid growth following injury is unlikely due to circulatory cells, but instead a result of ectopic development of tissues leftover from the blastogenic process. These tissues could be whole, damaged, or partially resorbed developing zooids, and we defined the minimal amount of vascular biomass to support ectopic regeneration. We did find a common theme between the two species: a competitive process exists which results in only a single zooid reaching maturity following injury. We utilized this phenomenon and found that competition is reversible and mediated by circulating factors and/or cells. Conclusions We propose that WBR does not occur in B. schlosseri and that the unique requirements defined in other studies only serve to increase the chances of ectopic development. This is likely a response to injury as we have discovered a vascular-based reversible competitive mechanism which ensures that only a single zooid completes development. This competition has been described in other species, but the unique response of B. schlosseri to injury provides a new model to study resource allocation and competition within an individual.

Complementary genomic and epigenomic adaptation to environmental heterogeneity

While adaptation is commonly thought to result from selection on DNA sequence-based variation, recent studies have highlighted an analogous epigenetic component as well. However, the extent to which these adaptive mechanisms to adaptation to environmental heterogeneity are redundant or complementary remains unclear. To address the underlying genetic and epigenetic mechanisms and their relationship underlying environmental adaptation, we screened the genomes and epigenomes of nine global populations of a predominately sessile marine invasive tunicate, Botryllus schlosseri. We detected clear population genetic and epigenetic differentiation, which were both significantly influenced by local environments, and the minimum annual sea surface temperature (T_min) was simultaneously identified as the top explanatory variable for both types of variation. However, there remain some degree of difference in population structure patterns between two levels, suggesting a certain level of autonomy in epigenetic variation. From the functional perspective, a set of functional genes and biological pathways were shared between two levels, indicating a conjoint contribution of genetic and epigenetic variation to environmental adaptation. Moreover, we also detected genetic- or epigenetic-specific genes/pathways in relation to a wide variety of core processes potentially underlying adaptation to local environmental factors, suggesting the partly independent relationship between two mechanisms. We infer that complementary genetic and epigenetic routes to adaptation are available in this system. Collectively, these mechanisms may facilitate population persistence under environmental changes and sustain successful invasions in novel but contrasting environments.

Botryllus schlosseri as a Unique Colonial Chordate Model for the Study and Modulation of Innate Immune Activity

Understanding the mechanisms that sustain immunological nonreactivity is essential for maintaining tissue in syngeneic and allogeneic settings, such as transplantation and pregnancy tolerance. While most transplantation rejections occur due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity. Botryllus schlosseri, a colonial tunicate, which is the nearest invertebrate group to the vertebrates, is devoid of T- and B-cell-based adaptive immunity. It has unique characteristics that make it a valuable model system for studying innate immunity mechanisms: (i) a natural allogeneic transplantation phenomenon that results in either fusion or rejection; (ii) whole animal regeneration and noninflammatory resorption on a weekly basis; (iii) allogeneic resorption which is comparable to human chronic rejection. Recent studies in B. schlosseri have led to the recognition of a molecular and cellular framework underlying the innate immunity loss of tolerance to allogeneic tissues. Additionally, B. schlosseri was developed as a model for studying hematopoietic stem cell (HSC) transplantation, and it provides further insights into the similarities between the HSC niches of human and B. schlosseri. In this review, we discuss why studying the molecular and cellular pathways that direct successful innate immune tolerance in B. schlosseri can provide novel insights into and potential modulations of these immune processes in humans.

Vascular Aging in the Invertebrate Chordate, Botryllus schlosseri

Vascular diseases affect over 1 billion people worldwide and are highly prevalent among the elderly, due to a progressive deterioration of the structure of vascular cells. Most of our understanding of these age-related cellular changes comes from in vitro studies on human cell lines. Further studies of the mechanisms underlying vascular aging in vivo are needed to provide insight into the pathobiology of age-associated vascular diseases, but are difficult to carry out on vertebrate model organisms. We are studying the effects of aging on the vasculature of the invertebrate chordate, Botryllus schlosseri. This extracorporeal vascular network of Botryllus is transparent and particularly amenable to imaging and manipulation. Here we use a combination of transcriptomics, immunostaining and live-imaging, as well as in vivo pharmacological treatments and regeneration assays to show that morphological, transcriptional, and functional age-associated changes within vascular cells are key hallmarks of aging in B. schlosseri, and occur independent of genotype. We show that age-associated changes in the cytoskeleton and the extracellular matrix reshape vascular cells into a flattened and elongated form and there are major changes in the structure of the basement membrane over time. The vessels narrow, reducing blood flow, and become less responsive to stimuli inducing vascular regression. The extracorporeal vasculature is highly regenerative following injury, and while age does not affect the regeneration potential, newly regenerated vascular cells maintain the same aged phenotype, suggesting that aging of the vasculature is a result of heritable epigenetic changes.

Assessing the Ecotoxicity of Copper and Polycyclic Aromatic Hydrocarbons: Comparison of Effects on Paracentrotus lividus and Botryllus schlosseri, as Alternative Bioassay Methods

Adult sea urchins and their embryos are ideal targets to investigate the medium- and long-term effects of various toxic agents, such as organic and inorganic pollutants, to forecast and mitigate their environmental effects. Similarly, small colonial tunicates such as Botryllid ascidians may reveal acute toxicity processes and permit quick responses for the management of contaminants impacting coastal waters, to preserve the functional integrity of marine ecosystems. This investigation compares the functional responses of two model invertebrates, the sea urchin Paracentrotus lividus and the sea squirt Botryllus schlosseri, to chronic and acute exposures to organic and inorganic toxic agents. Such heavy metals as copper produce both acute and chronic effects on marine biota, while polycyclic aromatic hydrocarbons (PAHs) mainly produce chronic effects at the concentrations ordinarily measured in marine coastal waters. Both models were tested over a range of concentrations of copper and PAHs. Copper triggered a clear effect in both species, producing a delay in the embryo development of P. lividus and a rapid death of sea squirts. B. schlosseri was less sensitive to PAHs than P. lividus. The results on both species may synergistically contribute to assess the toxicity of organic and inorganic compounds at various concentrations and different physiologic levels.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode

ABSTRACTBotryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications.To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer’s barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer’s fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer’s fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.