Cell cycle dynamics of food-entrapping cells of sponges: an experimental approach.

Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase.

Regeneration in calcareous sponge relies on 'purse-string' mechanism and the rearrangements of actin cytoskeleton.

The crucial step in any regeneration process is epithelization, i.e. the restoration of an epithelium structural and functional integrity. Epithelization requires cytoskeletal rearrangements, primarily of actin filaments and microtubules. Sponges (phylum Porifera) are early branching metazoans with pronounced regenerative abilities. Calcareous sponges have a unique step during regeneration: the formation of a temporary structure, called regenerative membrane which initially covers a wound. It forms due to the morphallactic rearrangements of exopinaco- and choanoderm epithelial-like layers. The current study quantitatively evaluates morphological changes and characterises underlying actin cytoskeleton rearrangements during regenerative membrane formation in asconoid calcareous sponge Leucosolenia variabilis through a combination of time-lapse imaging, immunocytochemistry, and confocal laser scanning microscopy. Regenerative membrane formation has non-linear stochastic dynamics with numerous fluctuations. The pinacocytes at the leading edge of regenerative membrane form a contractile actomyosin cable. Regenerative membrane formation either depends on its contraction or being coordinated through it. The cell morphology changes significantly during regenerative membrane formation. Exopinacocytes flatten, their area increases, while circularity decreases. Choanocytes transdifferentiate into endopinacocytes, losing microvillar collar and flagellum. Their area increases and circularity decreases. Subsequent redifferentiation of endopinacocytes into choanocytes is accompanied by inverse changes in cell morphology. All transformations rely on actin filament rearrangements similar to those characteristic of bilaterian animals. Altogether, we provide here a qualitative and quantitative description of cell transformations during reparative epithelial morphogenesis in a calcareous sponge.

Tissue homeostasis in sponges: Quantitative analysis of cell proliferation and apoptosis.

Tissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Sponges are early branching metazoans essential to understanding the key mechanisms of tissue homeostasis. This article is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea). Labeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. Quantitative DNA staining reveals the classic cell cycle distribution curve. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant throughout various areas of sponge bodies that contain choanocytes. The EdU tracking experiments conducted in H. dujardinii indicate that choanocytes may give rise to mesohyl cells through migration. The number of apoptotic cells in tissues of both species is insignificant, although being comparable to the renewing tissues of other animals. In vivo studies with tetramethylrhodamine ethyl ester and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization. Altogether, a combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell proliferation and apoptosis in sponges displaying either rapid growth or cell turnover.

Studying Porifera WBR Using the Calcerous Sponges Leucosolenia.

Sponges (Porifera), basal nonbilaterian metazoans, are well known for their high regenerative capacities ranging from reparation of a lost body wall to whole-body regeneration from a small piece of tissues or even from dissociated cells. Sponges from different clades utilize different cell sources and various morphological processes to complete the regeneration. This variety makes these animals promising models for studying the evolution of regeneration in Metazoa. However, there are few publications concerning the regenerative mechanisms in sponges. This could be partially explained by the delicacy of sponge tissues, which requires modifying and fine adjusting of common research protocols. The current chapter describes various methods for studying regeneration processes in the marine calcareous sponge, Leucosolenia. Provided protocols span all significant research steps: from sponge collection and surgical operations to various types of microscopy and immunohistochemical studies.

Fine details of the choanocyte filter apparatus in asconoid calcareous sponges (Porifera: Calcarea) revealed by ruthenium red fixation.

Sponges (phylum Porifera) are highly specialized filter-feeding metazoans, pumping and filtering water with a network of canals and chambers, the aquiferous system. Most sponges have a leuconoid aquiferous system, characterized by choanocytes organized in small spherical chambers connected with ambient water by a complex net of canals. Such organization requires substantial pressure difference to drive water through an elaborate system of canals, so the choanocytes in leuconoid sponges have several structural features to generate pressure difference. In contrast, it is generally accepted that asconoid and syconoid sponges with long choanocyte tubes or large choanocyte chambers have no similar structures in their choanocytes. The present study is devoted to the detailed ultrastructural analysis of the choanocytes and their filter apparatus in the asconoid calcareous sponge Leucosolenia variabilis. The general structure of L. variabilis choanocytes is similar to that described for other sponge species. However, the fixation with 0.1% ruthenium red allowed us to reveal for the first time a complex of glycocalyx structures (vanes on the flagella, a fine glycocalyx sealing microvilli in the collar, and a glycocalyx strainer, embedding the apical parts of neighboring collars) in the choanocytes of L. variabilis, which are traditionally associated with the pumping and filtration process in leuconoid demosponges. All revealed glycocalyx structures have dimensions and locations similar to those found in the choanocyte chambers of some demosponges. The data suggest that L. variabilis utilizes the principles of water pumping and filtration similar to those in demosponges and revealed glycocalyx structures are potentially crucial for these processes. It seems that sponges from distant phylogenetic lineages and with different body plans rely on common principles of choanoderm organization for effective pumping and filtration of water. However, while some adaptation for effective pumping and filtration of water have possibly arisen before the diversification of Porifera, others have appeared independently in different lineages.

Expression of Wnt and TGF-Beta Pathway Components during Whole-Body Regeneration from Cell Aggregates in Demosponge Halisarca dujardinii.

The phenomenon of whole-body regeneration means rebuilding of the whole body of an animal from a small fragment or even a group of cells. In this process, the old axial relationships are often lost, and new ones are established. An amazing model for studying this process is sponges, some of which are able to regenerate into a definitive organism after dissociation into cells. We hypothesized that during the development of cell aggregates, primmorphs, new axes are established due to the activation of the Wnt and TGF-beta signaling pathways. Using in silico analysis, RNA-seq, and whole-mount in situ hybridization, we identified the participants in these signaling pathways and determined the spatiotemporal changes in their expression in demosponge Halisarca dujardinii. It was shown that Wnt and TGF-beta ligands are differentially expressed during primmorph development, and transcripts of several genes are localized at the poles of primmorphs, in the form of a gradient. We suppose that the Wnt and TGF-beta signaling cascades are involved in the initial axial patterning of the sponge body, which develops from cells after dissociation.

Whole-Body Regeneration in Sponges: Diversity, Fine Mechanisms, and Future Prospects.

While virtually all animals show certain abilities for regeneration after an injury, these abilities vary greatly among metazoans. Porifera (Sponges) is basal metazoans characterized by a wide variety of different regenerative processes, including whole-body regeneration (WBR). Considering phylogenetic position and unique body organization, sponges are highly promising models, as they can shed light on the origin and early evolution of regeneration in general and WBR in particular. The present review summarizes available data on the morphogenetic and cellular mechanisms accompanying different types of WBR in sponges. Sponges show a high diversity of WBR, which principally could be divided into (1) WBR from a body fragment and (2) WBR by aggregation of dissociated cells. Sponges belonging to different phylogenetic clades and even to different species and/or differing in the anatomical structure undergo different morphogeneses after similar operations. A common characteristic feature of WBR in sponges is the instability of the main body axis: a change of the organism polarity is described during all types of WBR. The cellular mechanisms of WBR are different across sponge classes, while cell dedifferentiations and transdifferentiations are involved in regeneration processes in all sponges. Data considering molecular regulation of WBR in sponges are extremely scarce. However, the possibility to achieve various types of WBR ensured by common morphogenetic and cellular basis in a single species makes sponges highly accessible for future comprehensive physiological, biochemical, and molecular studies of regeneration processes.

Intraspecific variability of cell reaggregation during reproduction cycle in sponges.

The ability of sponge cells to reaggregate and reconstruct intact functional organism is known for more than 100 years. This process was studied in numerous species of sponges, and its interspecific variability is well described. However, some data also indicate the existence of a certain intraspecific variability of the cell reaggregation. The present study deals with the cell reaggregation in two demosponges, Halichondria panicea and Halisarca dujardinii, during different periods of their sexual reproduction. In both species, cell reaggregation shows a common pattern at all studied periods of reproduction. However, the course of the reaggregation process significantly depends on the reproduction period of an individual used in the experiment, thus demonstrating pronounced intraspecific variability, which concerns the rate of the cell reaggregation and the final stage of the process. This variability occurs due to tissue rearrangements that accompany reproduction and changes cell composition and amount of available somatic stem cells in sponge tissues, and consequently alters morphogenetic potencies of a cell suspension and multicellular aggregates. In both Halichondria panicea and Halisarca dujardinii, the growth period is the most favorable for the reaggregation process, while the cell reaggregation is depressed during periods of embryogenesis and restoration of somatic tissues after the reproduction. At the same time, the structure of a particular stage of reaggregation and morphogenetic processes underlying the development of multicellular aggregates are always identical, independently from the period of the reproductive cycle.

Transdifferentiation and mesenchymal-to-epithelial transition during regeneration in Demospongiae (Porifera).

Origin and early evolution of regeneration mechanisms remain among the most pressing questions in animal regeneration biology. Porifera have exceptional regenerative capacities and, as early Metazoan lineage, are a promising model for studying evolutionary aspects of regeneration. Here, we focus on reparative regeneration of the body wall in the Mediterranean demosponge Aplysina cavernicola. The epithelialization of the wound surface is completed within 2 days, and the wound is completely healed within 2 weeks. The regeneration is accompanied with the formation of a mass of undifferentiated cells (blastema), which consists of archaeocytes, dedifferentiated choanocytes, anucleated amoebocytes, and differentiated spherulous cells. The main mechanisms of A. cavernicola regeneration are cell dedifferentiation with active migration and subsequent redifferentiation or transdifferentiation of polypotent cells through the mesenchymal-to-epithelial transformation. The main cell sources of the regeneration are archaeocytes and choanocytes. At early stages of the regeneration, the blastema almost devoid of cell proliferation, but after 24 hr postoperation (hpo) and up to 72 hpo numerous DNA-synthesizing cells appear there. In contrast to intact tissues, where vast majority of DNA-synthesizing cells are choanocytes, all 5-ethynyl-2'-deoxyuridine-labeled cells in the blastema are mesohyl cells. Intact tissues, distant from the wound, retains intact level of cell proliferation during whole regeneration process. For the first time, the apoptosis was studied during the regeneration of sponges. Two waves of apoptosis were detected during A. cavernicola regeneration: The first wave at 6-12 hpo and the second wave at 48-72 hpo.

Sewing up the wounds : The epithelial morphogenesis as a central mechanism of calcaronean sponge regeneration.

Sponges (Porifera) demonstrate prominent regeneration abilities and possess a wide variety of mechanisms, used during this process. In the current study, we combined in vivo observations with histological, immunohistochemical, and ultrastructural technics to elucidate the fine cellular mechanisms of the regeneration in the calcareous sponge Leucosolenia cf. variabilis. The regeneration of Leucosolenia cf. variabilis ends within 4-6 days. The crucial step of the process is the formation of the transient regenerative membrane, formed by the epithelial morphogenesis-spreading of the intact exopinacoderm and choanoderm. The spreading of the choanoderm is accompanied by the transdifferentiation of the choanocytes. The regenerative membrane develops without any contribution of the mesohyl cells. Subsequently, the membrane gradually transforms into the body wall. The cell proliferation is neither affected nor contributes to the regeneration at any stage. Thus, Leucosolenia cf. variabilis regeneration relies on the remodeling of the intact tissues through the epithelial morphogenesis, accompanied by the transdifferentiation of some differentiated cell types, which makes it similar to the regeneration in homoscleromorphs and eumetazoans.

Stolonial Movement: A New Type of Whole-Organism Behavior in Porifera.

Sponges (phylum Porifera) traditionally are represented as inactive, sessile filter-feeding animals devoid of any behavior except filtering activity. However, different time-lapse techniques demonstrate that sponges are able to show a wide range of coordinated but slow whole-organism behavior. The present study concerns a peculiar type of such behavior in the psychrophilic demosponge Amphilectus lobatus: stolonial movement. During stolonial movement, sponges produce outgrowths (stolons) that crawl along a substrate with a speed of 4.4 ± 2.2 µm min-1 and branch, thus forming a complex net covering a considerable area of a substrate. This net is used by sponges to search for new points with appropriate environmental conditions for individual relocation. After such points are found, all cells of the parental sponge migrate through stolons, leaving a naked parental skeleton, forming one or several filial sponges in the new location. Thus, stolonial movement combines traits of crawling along the substrate and asexual reproduction. This behavior relies on massive cell dedifferentiation followed by coordinated cell migration to the point of new sponge body formation and their subsequent differentiation into specialized cell types.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 ß subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions.