Comparing dormancy in two distantly related tunicates reveals morphological, molecular, and ecological convergences and repeated co-option.

Many asexually-propagating marine invertebrates can survive extreme environmental conditions by developing dormant structures, i.e., morphologically simplified bodies that retain the capacity to completely regenerate a functional adult when conditions return to normal. Here, we examine the environmental, morphological, and molecular characteristics of dormancy in two distantly related clonal tunicate species: Polyandrocarpa zorritensis and Clavelina lepadiformis. In both species, we report that the dormant structures are able to withstand harsher temperature and salinity conditions compared to the adults. The dormant structures are the dominant forms these species employ to survive adverse conditions when the zooids themselves cannot survive. While previous work shows C. lepadiformis dormant stage is present in winters in the Atlantic Ocean and summers in the Mediterranean, this study is the first to show a year-round presence of P. zorritensis dormant forms in NW Italy, even in the late winter when all zooids have disappeared. By finely controlling the entry and exit of dormancy in laboratory-reared individuals, we were able to select and characterize the morphology of dormant structures associated with their transcriptome dynamics. In both species, we identified putative stem and nutritive cells in structures that resemble the earliest stages of asexual propagation. By characterizing gene expression during dormancy and regeneration into the adult body plan (i.e., germination), we observed that genes which control dormancy and environmental sensing in other metazoans, notably HIF-α and insulin signaling genes, are also expressed in tunicate dormancy. Germination-related genes in these two species, such as the retinoic acid pathway, are also found in other unrelated clonal tunicates during asexual development. These results are suggestive of repeated co-option of conserved eco-physiological and regeneration programs for the origin of novel dormancy-germination processes across distantly related animal taxa.

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.

From the individual to the colony: Marine invertebrates as models to understand levels of biological organization.

The developmental and evolutionary principles of coloniality in marine animals remain largely unexplored. Although many common traits have evolved independently in different groups of colonial animals, questions about their significance for colonial life histories remain unanswered. In 2018 (Nov. 25 - Dec. 8), the inaugural course on the Evolution of Coloniality and Modularity took place at the Center for Marine Biology of the University of São Paulo (CEBIMAR-USP), Brazil. During the intensive two-week graduate-level course, we addressed some of the historical ideas about animal coloniality by focal studies in bryozoans, tunicates, cnidarians, and sponges. We discussed many historical hypotheses and ways to test these using both extant and paleontological data, and we carried direct observations of animal colonies in the different phyla to address questions about coloniality. We covered topics related to multi-level selection theory and studied colonial traits, including modular miniaturization, polymorphism, brooding, and allorecognition. Course participants carried out short research projects using local species of animals to address questions on allorecognition and regeneration in ascidians and sponges, fusion and chimerism in anthoathecate hydrozoans, and evolution of polymorphism in bryozoans. Although many questions remain unanswered, this course served as a foundation to continue to develop a developmental and evolutionary synthesis of clonal and modular development in colonial marine organisms.

Coloniality, clonality, and modularity in animals: The elephant in the room.

Nearly half of the animal phyla contain species that propagate asexually via agametic reproduction, often forming colonies of genetically identical modules, that is, ramets, zooids, or polyps. Clonal reproduction, colony formation, and modular organization have important consequences for many aspects of organismal biology. Theories in ecology, evolution, and development are often based on unitary and, mainly, strictly sexually reproducing organisms, and though colonial animals dominate many marine ecosystems and habitats, recognized concepts for the study of clonal species are often lacking. In this review, we present an overview of the study of colonial and clonal animals, from the historic interests in this subject to modern research in a range of topics, including immunology, stem cell biology, aging, biogeography, and ecology. We attempt to portray the fundamental questions lying behind the biology of colonial animals, focusing on how colonial animals challenge several dogmas in biology as well as the remaining puzzles still to be answered, of which there are many.

The eventful history of nonembryonic development in tunicates.

Tunicates encompass a large group of marine filter-feeding animals and more than half of them are able to reproduce asexually by a particular form of nonembryonic development (NED) generally called budding. The phylogeny of tunicates suggests that asexual reproduction is an evolutionarily plastic trait, a view that is further reinforced by the fact that budding mechanisms differ from one species to another, involving nonhomologous tissues and cells. In this review, we explore more than 150 years of literature to provide an overview of NED diversity and we present a comparative picture of budding tissues across tunicates. Based on the phylogenetic relationships between budding and nonbudding species, we hypothesize that NED diversity is the result of seven independent acquisitions and subsequent diversifications in the course of tunicate evolution. While this scenario represents the state-of-the-art of our current knowledge, we point out gray areas that need to be further explored to refine our understanding of tunicate phylogeny and NED. Tunicates, with their plastic evolution and diversity of budding, represent an ideal playground for evolutionary developmental biologists to unravel the genetic and molecular mechanisms regulating nonembryonic development, as well as to better understand how such a profound innovation in life-history has evolved in numerous metazoans.

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.

Putative stem cells in the hemolymph and in the intestinal submucosa of the solitary ascidian Styela plicata.

BACKGROUND: In various ascidian species, circulating stem cells have been documented to be involved in asexual reproduction and whole-body regeneration. Studies of these cell population(s) are mainly restricted to colonial species. Here, we investigate the occurrence of circulating stem cells in the solitary Styela plicata, a member of the Styelidae, a family with at least two independent origins of coloniality. RESULTS: Using flow cytometry, we characterized a population of circulating putative stem cells (CPSCs) in S. plicata and determined two gates likely enriched with CPSCs based on morphology and aldehyde dehydrogenase (ALDH) activity. We found an ALDH + cell population with low granularity, suggesting a stem-like state. In an attempt to uncover putative CPSCs niches in S. plicata, we performed a histological survey for hemoblast-like cells, followed by immunohistochemistry with stem cell and proliferation markers. The intestinal submucosa (IS) showed high cellular proliferation levels and high frequency of undifferentiated cells and histological and ultrastructural analyses revealed the presence of hemoblast aggregations in the IS suggesting a possible niche. Finally, we document the first ontogenetic appearance of distinct metamorphic circulatory mesenchyme cells, which precedes the emergence of juvenile hemocytes. CONCLUSIONS: We find CPSCs in the hemolymph of the solitary ascidian Styela plicata, presumably involved in the regenerative capacity of this species. The presence of proliferating and undifferentiated mesenchymal cells suggests IS as a possible niche.

Novel budding mode in Polyandrocarpa zorritensis: a model for comparative studies on asexual development and whole body regeneration.

BACKGROUND: In tunicates, the capacity to build an adult body via non-embryonic development (NED), i.e., asexual budding and whole body regeneration, has been gained or lost several times across the whole subphylum. A recent phylogeny of the family Styelidae revealed an independent acquisition of NED in the colonial species Polyandrocarpa zorritensis and highlighted a novel budding mode. In this paper, we provide the first detailed characterization of the asexual life cycle of P. zorritensis. RESULTS: Bud formation occurs along a tubular protrusion of the adult epidermis, the stolon, in a vascularized area defined as budding nest. The bud arises through a folding of the epithelia of the stolon with the contribution of undifferentiated mesenchymal cells. This previously unreported mode of bud onset leads to the formation of a double vesicle, which starts to develop into a zooid through morphogenetic mechanisms common to other Styelidae. The budding nest can also continue to accumulate nutrients and develop into a round-shaped structure, designated as spherule, which represents a dormant form able to survive low temperatures. CONCLUSIONS: To understand the mechanisms of NED and their evolution, it is fundamental to start from a robust phylogenetic framework in order to select relevant species to compare. The anatomical description of P. zorritensis NED provides the foundation for future comparative studies on plasticity of budding and regeneration in tunicates.

Expression of Hox, Cdx, and Six3/6 genes in the hoplonemertean Pantinonemertes californiensis offers insight into the evolution of maximally indirect development in the phylum Nemertea.

BACKGROUND: Maximally indirect development via a pilidium larva is unique to the pilidiophoran clade of phylum Nemertea. All other nemerteans have more or less direct development. The origin of pilidial development with disjunct invaginated juvenile rudiments and catastrophic metamorphosis remains poorly understood. While basal members of the phylum, the Palaeonemertea, do not appear to have ever had a pilidium, certain similarity exists in the development of the Pilidiophora and the sister clade, the Hoplonemertea. It is unclear whether this similarity represents the homology and whether pilidial development evolved before or after pilidiophorans diverged from hoplonemerteans. To gain insight into these questions, we examined the expression of Hox, Cdx, and Six3/6 genes in the development of the hoplonemertean Pantinonemertes californiensis and expression of Six3/6 in the pilidium of Micrura alaskensis. To further characterize the function of larval structures showing expression of these genes, we examined the serotonergic nervous system and cell proliferation in P. californiensis. RESULTS: We show that Hox and Cdx genes, which pattern the pilidial imaginal discs giving rise to the juvenile trunk, are expressed in paired posterior epidermal invaginations in P. californiensis larvae. We also show that Six3/6 patterns both the pilidial cephalic discs, which give rise to the juvenile head, and a pair of anterior epidermal invaginations in hoplonemertean development. We show that anterior invaginations in larval P. californiensis are associated with a pair of serotonergic neurons, and thus may have a role in the development of the juvenile nervous system. This is similar to the role of cephalic discs in pilidiophoran development. Finally, we show that four zones of high cell proliferation correspond to the paired invaginations in P. californiensis, suggesting that these invaginations may play a similar role in the development of the hoplonemertean juvenile to the role of imaginal discs in the pilidium, which also exhibit high rates of cell proliferation. CONCLUSIONS: Expression of Hox, Cdx, and Six3/6 genes supports the homology between the imaginal discs of the pilidium and the paired larval invaginations in hoplonemerteans. This suggests that invaginated juvenile rudiments (possible precursors to pilidial imaginal discs) may have been present in the most recent common ancestor of the Pilidiophora and Hoplonemertea.

Hox genes pattern the anterior-posterior axis of the juvenile but not the larva in a maximally indirect developing invertebrate, Micrura alaskensis (Nemertea).

BACKGROUND: The pilidium larva is a novel body plan that arose within a single clade in the phylum Nemertea - the Pilidiophora. While the sister clade of the Pilidiophora and the basal nemerteans develop directly, pilidiophorans have a long-lived planktotrophic larva with a body plan distinctly different from that of the juvenile. Uniquely, the pilidiophoran juvenile develops inside the larva from several discrete rudiments. The orientation of the juvenile with respect to the larval body varies within the Pilidiophora, which suggests that the larval and juvenile anteroposterior (AP) axes are patterned differently. In order to gain insight into the evolutionary origins of the pilidium larva and the mechanisms underlying this implied axial uncoupling, we examined the expression of the Hox genes during development of the pilidiophoran Micrura alaskensis. RESULTS: We identified sequences of nine Hox genes and the ParaHox gene caudal through a combination of transcriptome analysis and molecular cloning, and determined their expression pattern during development using in situ hybridization in whole-mounted larvae. We found that Hox genes are first expressed long after the pilidium is fully formed and functional. The Hox genes are expressed in apparently overlapping domains along the AP axis of the developing juvenile in a subset of the rudiments that give rise to the juvenile trunk. Hox genes are not expressed in the larval body at any stage of development. CONCLUSIONS: While the Hox genes pattern the juvenile pilidiophoran, the pilidial body, which appears to be an evolutionary novelty, must be patterned by some mechanism other than the Hox genes. Although the pilidiophoran juvenile develops from separate rudiments with no obvious relationship to the embryonic formation of the larva, the Hox genes appear to exhibit canonical expression along the juvenile AP axis. This suggests that the Hox patterning system can maintain conserved function even when widely decoupled from early polarity established in the egg.

The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity.

A fundamental question in brain development is how precursor cells generate a diverse group of neural progeny in an ordered manner. Drosophila neuroblasts sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor (Cas). Hb is necessary and sufficient to specify early-born temporal identity and, thus, Hb downregulation is essential for specification of later-born progeny. Here, we show that distal antenna (dan) and distal antenna-related (danr), encoding pipsqueak motif DNA-binding domain protein family members, are detected in all neuroblasts during the Hb-to-Cas expression window. Dan and Danr are required for timely downregulation of Hb in neuroblasts and for limiting the number of early-born neurons. Dan and Danr function independently of Seven-up (Svp), an orphan nuclear receptor known to repress Hb expression in neuroblasts, because Dan, Danr and Svp do not regulate each other and dan danr svp triple mutants have increased early-born neurons compared with either dan danr or svp mutants. Interestingly, misexpression of Hb can induce Dan and Svp expression in neuroblasts, suggesting that Hb might activate a negative feedback loop to limit its own expression. We conclude that Dan/Danr and Svp act in parallel pathways to limit Hb expression and allow neuroblasts to transition from making early-born neurons to late-born neurons at the proper time.