Systemic Bud Induction and Retinoic Acid Signaling Underlie Whole Body Regeneration in the Urochordate Botrylloides leachi

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi in which an entire adult zooid is restored from a miniscule blood vessel fragment. In contrast to all other documented cases, regeneration is induced systemically in blood vessels. Multiple buds appear simultaneously in newly established regeneration niches within vasculature fragments, stemming from composites of pluripotent blood cells and terminating in one functional zooid. We found that retinoic acid (RA) regulates diverse developmental aspects in WBR. The homologue of the RA receptor and a retinaldehyde dehydrogenase-related gene were expressed specifically in blood cells within regeneration niches and throughout bud development. The addition of RA inhibitors as well as RNA interference knockdown experiments resulted in WBR arrest and bud malformations. The administration of all-trans RA to blood vessel fragments resulted in doubly accelerated regeneration and multibud formation, leading to restored colonies with multiple zooids. The Botrylloides system differs from known regeneration model systems by several fundamental criteria, including epimorphosis without the formation of blastema and the induction of a “multifocal regeneration niche” system. This is also to our knowledge the first documented case of WBR from circulating blood cells that restores not only the soma, but also the germ line. This unique Botrylloides WBR process could serve as a new in vivo model system for regeneration, suggesting that RA signaling may have had ancestral roles in body restoration events.     

Piwi positive cells that line the vasculature epithelium, underlie whole body regeneration in a basal chordate

The colonial tunicate Botrylloides leachi can regenerate functional adults from minute vasculature fragments, in a poorly understood phenomenon termed Whole Body Regeneration (WBR). Using Piwi expression (Bl-Piwi), blood cell labeling and electron microscopy, we show that WBR develops through activation, mobilization and expansion of ‘dormant’ cells which normally line the internal vasculature epithelium of blood vessels. Following a mechanical insult, these cells express Bl-Piwi de novo, change morphology and invade niches of the vasculature lumen, where they proliferate and differentiate, regenerating a functional organism. Mitomycin C treatments and siRNA knockdown of Bl-Piwi result in deficient cells incapable of expanding or differentiating and to subsequent regeneration arrest. Last, we find similar transient mobilization of Piwi⁺ cells recurring every week, as part of normal colony development, and also during acute environmental stress. This recurrent activation of Piwi⁺ cells in response to developmental, physiological and environmental insults may have enabled the adaptation of colonial tunicates to the imposed varied conditions in the marine, shallow water environment.     

Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate

Background  

The restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR]) represents an inimitable event in the chordates, which is poorly understood on the mechanistic level.     

The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian,Polyzoa vesiculiphora

The blood cells in the bud and the zooid of the polystyelid ascidian, Polyzoa vesiculiphora, were examined by means of light and electron microscopy to identify the cells that have been named trophocytes. The large blood cells were abundant in the mesenchymal space of the bud, but not in that of the functional zooid. They contained glycogen particles, lipid droplets, large protein granules and autophagosomes in their cytoplasm and were identified as granular amoebocytes. The majority of these cells were specifically phagocytized by phagocytes during bud development and disappeared. These results indicate that the granular amoebocytes virtually represent trophocytes in Polyzoa and may participate in bud development via nutrient supply to the developing tissues.     

Msx-1 acts as a regulator for blastema growth

Msx-1 is known to regulate ectoderm-mesenchyme and mesenchyme-mesenchyme tissue interactions in the developing vertebrate limb bud. In this study, the spatial and temporal expression pattern ofMsx-1 in the regenerating limbs of axolotls (Ambystoma mexicanum) was examined by whole mountin situ hybridization and RT-PCR. In addition, the effects of retinoic acid (RA) and denervation on theMsx-1 expression were examined as well. In the regenerating normal limbs, the weak expression ofMsx-1 was detected in the mesenchymal cells under the apical ectodermal cap. From the early bud stage, the elevated level ofMsx-1 expression was maintained until the early digit stage. However,Msx-1 expression was rapidly declined by the completion of regeneration. Compared to the normal limb regeneration,Msx-1 expression in RA treated limb regenerates was low until 9 days after RA treatment but increased during blastema growth period both m the lower and the upper arm regenerates. These results suggest that theMsx-1 regulates the proliferation and growth of blastema cells, and its expression may not be responsible for the dedifferentiation process. On the other hand, high level ofMsx-1 expression was noted in the limb stump tissue after denervation. This result indicates thatMsx-1 is also related to the regression of denervated mature tissue.     

Stromal Protein Ecm1 Regulates Ureteric Bud Patterning and Branching

The interactions between the nephrogenic mesenchyme and the ureteric bud during kidney development are well documented. While recent studies have shed some light on the importance of the stroma during renal development, many of the signals generated in the stroma, the genetic pathways and interaction networks involving the stroma are yet to be identified. Our previous studies demonstrate that retinoids are crucial for branching of the ureteric bud and for patterning of the cortical stroma. In the present study we demonstrate that autocrine retinoic acid (RA) signaling in stromal cells is critical for their survival and patterning, and show that Extracellular matrix 1, Ecm1, a gene that in humans causes irritable bowel syndrome and lipoid proteinosis, is a novel RA-regulated target in the developing kidney, which is secreted from the cortical stromal cells surrounding the cap mesenchyme and ureteric bud. Our studies suggest that Ecm1 is required in the ureteric bud for regulating the distribution of Ret which is normally restricted to the tips, as inhibition of Ecm1 results in an expanded domain of Ret expression and reduced numbers of branches. We propose a model in which retinoid signaling in the stroma activates expression of Ecm1, which in turn down-regulates Ret expression in the ureteric bud cleft, where bifurcation normally occurs and normal branching progresses.     

Comparative studies on the circulatory system of the compound ascidians,Botryllus, Botrylloides and Symplegma

The circulatory systems of four polystyelids, Botryllus schlosseri, B. primigenus, Botrylloides violaceus and Symplegma reptans, were compared. The palleal buds are connected to the parent zooid by a peduncle and to the colonial vascular system by connecting vessels. The peduncle of S. reptans disappears at an earlier stage of bud development than in B. primigenus; it survives the dissolution of the parent zooid in B. schlosseri and B. violaceus. The connecting vessel is formed by anastomosis between an epidermal outgrowth from the bud and a neighboring colonial vessel, and is characterized by the presence of a sphincter. The number of connecting vessels formed in a palleal bud is three in S. reptans, two in B. primigenus and one each in B. schlosseri and B. violaceus. In each species, the larva has eight rudiments of ampullae. In B. primigenus, the original ampullae degenerate soon after metamorphosis and new ampullae extend from the ventral epidermis of the oozooid. In the other species, the colonial vascular system is derived from the original ampullae. The whole colonial vascular system contracts and expands periodically, with regionally different phases. During each expansion cycle, the sphincter contracts once in B. primigenus and twice in S. reptans. The correlation may be due to blood pressure and the propagation of excitation through the colonial vascular system.     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

Multicellular cuddling in a stem cell niche

Haematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate in any blood cell type throughout life and thereby sustain the entire blood system. To do so, HSPCs had been shown to seed, in a multi-step process, intermediate haematopoietic niches before colonizing the adult marrow. While HSPC birth had been thoroughly characterized in the past, both in mammals and in zebrafish, how perivascular niches could host HSPCs and sustain their expansion was poorly understood. In an article published in the last issue of Cell, Tamplin et al.¹ elegantly exploited the many advantages provided by the zebrafish embryo to describe how endothelium remodeling in the perivascular niche, referred to as “cuddling,” favors HSPCs colonization and expansion.     

On Leukocytes in Mammary Development and Cancer

During metastasis, tumor cells may be copying a program that is executed by hematopoietic stem cells during development.That cancer is development gone awry is not a new concept. Most of the “hallmarks” ascribed to cancer—proliferation, invasion and induction of blood vessel growth—also occur during organogenesis and development. Therefore, tumors are not necessarily learning new tricks during their development, but how about when they metastasize? In colonizing a new organ, often with some degree of specificity, tumor cells may simply be copying a program that is executed during development by hematopoietic stem cells (HSCs)—the stem cells that ultimately generate all of the cells in our blood and maintain its homeostasis. One family of cells generated by HSCs—leukocytes—is the focus of the work by Coussens and Pollard (2012). These two scientists have woven together several studies that revolutionized the way we think of immune cells. As pointed out by the investigators (whose respective laboratories are responsible for much of the seminal work on this subject), immune cells also have a variety of trophic functions, and it is these functions that are used rationally during development, and recklessly during tumor growth.This leads us back to metastasis. There is so much to learn about why a tumor travels from one organ to another, how it does so, and the manner by which it adapts to and ultimately flourishes (or fails) in a foreign microenvironment. And as stated above, immune cell precursors, HSCs, do the same. In the mouse, HSCs have originated in one tissue (the dorsal aorta), traveled to another (the placenta) via the circulation, and matured somewhere else (the liver)—all before birth. Finally, HSCs make their way to the bone marrow, where they reside postnatally. Specialized niches in the bone marrow are thought to mediate HSC dormancy as a means to preserve the “stemness” of this population, and there are mechanisms in place that allow these cells to rapidly exit these environs and proliferate in response to injury. Therefore, it should not come as a surprise that a common site where micrometastases are found is the bone marrow for many cancers (including that of the breast).Uncovering whether the same niches that control HSC expansion in the bone marrow are also responsible for maintaining quiescence of tumor cell populations is an exciting prospect, as is deciphering the precise components of these niches. Such work could explain the seemingly incongruous observation that despite an absence of clinically detectable disease, circulating tumor cells are present in the blood of post-treatment cancer patients sometimes even decades later! Perhaps the niches that regulate prolonged dormancy of tumors are dynamic and inhibit tumor proliferation while allowing them to mobilize periodically, much like for HSCs. It also stands to reason that loss of the same controls that prevent HSC expansion until systemic damage occurs could awaken dormant tumors.Shiozawa et al. (2011) have demonstrated that prostate cancer cells do in fact compete with HSCs for niches within the bone marrow, and that tumor cells are mobilized from HSC niches by similar mechanisms as for HSCs. Whether this is the case for other cancers and whether these similarities can be exploited therapeutically remain to be seen.So what more is there to be learned about immune cells? By furthering our understanding of how solid cancers mimic and hijack components of our immune system, we may not “cure” cancer, but we very well may uncover a means to suppress some cancers into a state of permanent dormancy.     

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian Botryllus schlosseri.

Reversal of the bilateral asymmetry of the zooids was induced in a series of colonies of Botryllus schlosseri. Palleal buds from colonies with normal or reversed bilateral asymmetry were isolated in the early stages from the parental zooids and cultured in the vascularized tunic of the same colony or of another colony with opposite asymmetry. Vascular budding was induced in colonies with either type of asymmetry.The bud polarity was shown to depend on the vascularization; the test vessel entering the isolated palleal bud always causes the entrance point to become the posterior end of the developing zooid. On the contrary, the bilateral asymmetric type is predetermined in the bud primordium; the isolated palleal buds develop the type of asymmetry of their parents, even when grafted in the test of a colony with opposite asymmetry. Since the same was also true of the vascular buds, it is concluded that the information for the kind of bilateral asymmetry to be developed is conveyed by the epidermal envelope of the bud. The epidermis of the parental zooids influences the palleal buds, whereas the wall of the test vessels, epidermal extrusions of the zooids, influences the vascular buds.     

Studies on the asexual reproduction in the polystyelid ascidian,Polyzoa vesiculiphora Tokioka

New blastozooids of Polyzoa vesiculiphora, the polysytelid ascidian are produced by pallial budding of three types depending on the method of “isolated bud” formation; stolonic, planktonic and intermediate types. Differences among each type of bud are attributed to behavior of test-vessels composing a part of the bud. Isolated buds produced by each type are essentially equal in terms of their internal structures and their subsequent fate, and develop independently of their parent zooids. New test-vessels originate directly from the epidermis of a “prefunctional zooid,” while the test-vessels derived from the parent zooid finally disintegrate. The new test-vessels extended with branching under the ventral side of a “functional zooid,” ascend to the lateral side of it and participate in bud formation. Budding regions exist in three dimensions on the lateral wall of the mantle of the functional zooid, especially the right posterior part. During the life cycle of one functional zooid, the stolonic type buds appear at early and/or aged stages. Appearances of the stolonic type buds in early stages tend to repress those of the planktonic types. The number of planktonic type buds formed on a functional zooid at the same time is many more than that of the stolonic type. Such budding features are discussed from the viewpoint of behavior of the test-vessel system.     

Temporal dynamics of plant community regeneration sources during tallgrass prairie restoration

Ecological restoration aims to augment and steer the composition and contribution of propagules for community regeneration in degraded environments. We quantified patterns in the abundance, richness, and diversity of seed and bud banks across an 11-year chronosequence of restored prairies and in prairie remnants to elucidate the degree to which the germinable seed bank, emerged seedlings, belowground buds, and emerged ramets were related to community regeneration. There were no directional patterns in the abundance, richness, or diversity of the germinable seed bank across the chronosequence. Emerged seedling abundance of sown species decreased during restoration. Richness and diversity of all emerged seedlings and non-sown emerged seedling species decreased across the chronosequence. Conversely, abundance and richness of belowground buds increased with restoration age and belowground bud diversity of sown species increased across the chronosequence. Numbers of emerged ramets also increased across the chronosequence and was driven primarily by the number of graminoid ramets. There were no temporal changes in abundance and richness of sown and non-sown emerged ramets, but diversity of sown emerged ramets increased across the chronosequence. This study demonstrates that after initial seeding, plant community structure in restored prairies increasingly reflects the composition of the bud bank.     

Seagrass Restoration Enhances “Blue Carbon” Sequestration in Coastal Waters

Seagrass meadows are highly productive habitats that provide important ecosystem services in the coastal zone, including carbon and nutrient sequestration. Organic carbon in seagrass sediment, known as “blue carbon,” accumulates from both in situ production and sedimentation of particulate carbon from the water column. Using a large-scale restoration (>1700 ha) in the Virginia coastal bays as a model system, we evaluated the role of seagrass, Zostera marina , restoration in carbon storage in sediments of shallow coastal ecosystems. Sediments of replicate seagrass meadows representing different age treatments (as time since seeding: 0, 4, and 10 years), were analyzed for % carbon, % nitrogen, bulk density, organic matter content, and ²¹⁰Pb for dating at 1-cm increments to a depth of 10 cm. Sediment nutrient and organic content, and carbon accumulation rates were higher in 10-year seagrass meadows relative to 4-year and bare sediment. These differences were consistent with higher shoot density in the older meadow. Carbon accumulation rates determined for the 10-year restored seagrass meadows were 36.68 g C m^-2 yr^-1. Within 12 years of seeding, the restored seagrass meadows are expected to accumulate carbon at a rate that is comparable to measured ranges in natural seagrass meadows. This the first study to provide evidence of the potential of seagrass habitat restoration to enhance carbon sequestration in the coastal zone.     

Stage-dependent effects of retinoic acid on regenerating urodele limbs

Summary Following amputation through the distal zeugopodium, regenerating limbs of larvalAmbystoma mexicanum and pre and post-metamorphicPleurodeles waltlii were treated with 150 g of retinoic acid (RA) per gram of body weight, at the dedifferentiation, early bud, medium bud, late bud or early redifferentiation stages of regeneration. The effect of RA on regenerate morphogenesis differed as a function of the stage at which it was administered. When given during dedifferentiation or at early bud stages, RA evoked proximodistal duplications of stump segments in the regenerates. The maximum duplication index (DI) inAbystoma was achieved when RA was injected at 4 days post-amputation, which corresponds to the stage of dedifferentiation; and inPleurodeles at 10 days post-amputation, which corresponds to a stage midway between early bud and medium bud. When RA was administered at later stages, the DI declined progressively to zero or nearly zero by the stage of early redifferentiation in both species. The decline in DI was due to a decreased frequency of duplication, not to a decrease in the magnitude of duplication in individual regenerates. At the same time, there was an increase in hypomorphism and aberrant morphogenesis of both duplicating and non-duplicating regenerates. These results indicate that regenerative cells are differentially sensitive to RA in a stage-dependent way.     

Role of the Y organ and the effect of ecdysterone during the regeneration of an appendage in the oniscoid Porcellio dilatatus

The rôle of the moulting hormone in regeneration was studied in Porcellio dilatatus by destroying the Y-organs (moulting glands) and by injecting synthetic ecdysterone. The destruction of the Y-organs prevents the formation of the regeneration bud of the limb. Several successive injections of a very weak solution of ecdysterone allow the formation of a regeneration bud in animals without Y-organs. A single injection of ecdysterone at a dose which induces apolysis blocks regeneration by making all the epidermal cells secrete cuticle, including those of the regeneration bud.