RESEARCH ARTICLE Sponge Cell Reaggregation: Cellular Structure and Morphogenetic Potencies of Multicellular Aggregates ANDREY I. LAVROV* AND IGOR A. KOSEVICH Department of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia

ABSTRACT 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. J. Exp. Zool. 325A:158–177, 2016. © 2016 Wiley Periodicals, Inc. J. Exp. Zool. 325A:158–177, How to cite this article: Lavrov AI, Kosevich IA. 2016. Sponge Cell Reaggregation: Cellular 2016 Structure and Morphogenetic Potencies of Multicellular Aggregates. J. Exp. Zool. 325A:158–177.

The origin and early evolution of multicellular animals is one of the most intriguing problems in modern biology. Understanding of these evolutionary events requires reconstruction of the last Grant sponsor: Russian Foundation of Basic Research; grant number: common ancestor of Metazoa. Such reconstruction should 16-34-00145. embrace all aspects of animal biology—anatomy, reproduction, Conflicts of interest: None. ecology, physiology, and of course mechanisms of cell coordina- Correspondence to: Andrey I. Lavrov, Department of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, Leninskie tion and tissue integration. Obviously, transition from unicellular gory 1-12, 119234, Moscow, Russia state to multicellularity is impossible without emergence of new E-mail: [email protected] mechanisms of cell communication and integration consolidat- Received 17 June 2015; Revised 13 January 2016; Accepted 13 January ing individual cells into tissues. To date, significant data were 2016 collected on cell behavior, mechanisms of cell-cell communica- DOI: 10.1002/jez.2006 Published online 8 February 2016 in Wiley Online Library tion and integration in intact tissue during embryogenesis and (wileyonlinelibrary.com). different regeneration processes in diverse vertebrates and

© 2016 WILEY PERIODICALS, INC. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1593 invertebrates. These data allow to find out certain patterns in Korotkova, '72, '97; Nikitin, '73; Volkova and Zolotareva, '81). functioning of metazoan tissues and to make assumptions on Thus cell reaggregation is a favorable model for analysis of events in early evolution of Metazoa (Tyler, 2003; Srivastava detailed sponge tissue functioning—behavior and interactions of et al., 2010; Adamska et al., 2011). However precise investigations different cell types, their abilities for dedifferentiations and of ancient Metazoa, in particular representatives of phylum transdifferentiations, mechanisms of cell interaction restoring, etc. Porifera, are essential to get more elaborated knowledge on —in controlled laboratory conditions. mechanisms of tissue functioning and integration in multicellu- The present study deals with analysis of cell reaggregation lar animals. process in three species from class Demospongiae: Halichondria Sponges (phylum Porifera) are the most ancient extant panicea (Pallas, 1766), Haliclona (Reniera) aquaeductus (Schmidt, multicellular animals. In adult stage these animals lack any 1862), and Halisarca dujardinii Johnston, 1842. Dynamic of cell organs, digestive, nervous and muscle systems. Aquiferous reaggregation and structure of multicellular aggregates are system is the only distinct structure in the body of adult sponge. characterized. Obtained data confirm the existence of a great Through this system sponges pump huge volumes of water variability in cell reaggregation process among various sponge obtaining oxygen and nutrition. High efficiency of water species, in particular belonging to the class Demospongiae. pumping in continuously changing hydrodynamic conditions is vital for sponges. These ancient animals found elegant solution for this problem—they permanently rearrange their aquiferous MATERIALS AND METHODS system to make it most effective in particular hydrodynamic Material Collection and Laboratory Maintenance conditions. Reversible differentiation of cells and their motility Three demosponge species were used in the present study: are at the basis for anatomical plasticity of sponges. Virtually H. panicea, H. aquaeductus (Schmidt, 1862), and H. dujardinii. all cells in the sponge body permanently move and many of Sponges were collected in the vicinity of N.A. Pertsov White Sea them are capable for transdifferentiation (Harris, '87; Bond, '92; Biological Station of Lomonosov Moscow State University Ereskovsky, 2010). (Kandalaksha Bay) (66°340 N, 33°080 E). Specimens of H. dujardinii The most outstanding manifestation of sponge anatomical were collected in August of 2013 and 2014 from the upper subtidal plasticity is the ability of their cells to reaggregate after tissue zone at 0–2 m depth at low-tide. All specimens were at the stage of dissociation. Multicellular aggregates of different structure are the postreproduction growth and did not contain any reproductive formed through cell reaggregation and in certain cases full elements. Specimens of H. panicea and H. aquaeductus collected in reconstruction of functional and viable sponge occurs (Wilson, early-June of 2012 and 2013 by SCUBA diving at 10–15 m depth '07; Huxley, '11; Galtsoff, '25b; Korotkova, '72). The peculiar stage were at the stage of growth preceding sexual reproduction and did of this process is multicellular aggregates termed primmorphs. not contain any reproductive elements. They are characterized by continuous exopinacoderm which Prior to experiments sponges were maintained in the aquarium separates internal mass of unspecialized cells from external with biological filters and fresh seawater (80 l, 6–10°C) no longer environment. Primmorphs represents intermediate stage between than 24 hr. Fifteen individuals of H. panicea, eight individuals of multicellular aggregates and initial stages of sponge reconstruc- H. aquaeductus, and seventeen individuals of H. dujardinii were tion (Custodio et al., '98; Muller€ et al., '99; Sipkema et al., 2003). used in the study (Table 1). Cell cultures from three H. panicea Only after complete primmorph formation the process of individuals and from one H. aquaeductus individual died during functional sponge reconstruction can begin. This reconstruction early stages of reaggregation. happens not due to the sorting out of already differentiated cells but involves extensive dedifferentiations and transdifferentiations Tissue Dissociation of certain cell types. The fate of transdifferentiating cells depends Sponge tissues were dissociated by mechanical squeezing in non- on cell position inside multicellular aggregate (Efremova, '69, '72; sterile conditions. Portion of the sponge tissues were cut in small

Table 1. Characteristics of reaggregation experiments of studied sponge species.

Number of individuals Overall number of Species used in experiments studied cell cultures Culturing time, dpd Final developmental stage Haliclona aquaeductus 8477–24 Early-stage primmorphs Halichondria panicea 15 75 5–29 Primmoprhs Halisarca dujardinii 17 46 5–24 Reconstructed sponge

dpd, days post-dissociation.

J. Exp. Zool. 4160 IGOREVICHLAVROV AND AND ARNOLDOVICH KOSEVICH pieces by scalpel and tweezers in sterile seawater (FSW). phosphate buffer (pH 7.4) isotonic with White Sea water Afterwards these small pieces of sponge tissue were squeezed (osmolarity 830 mOsm) (Millonig, '64), then post-fixed for 2 hr through 50 mm nylon mesh into vessels with fresh FSW to obtain with 1% OsO4 on the same buffer at room temperature (RT). cell suspensions. Between fixation and post-fixation aggregates were twice rinsed FSW was used in dissociation procedure and during subse- with Millonig's PB for 30 min. After post-fixation multicellular quent cell cultivation to avoid additional contamination. Water aggregates of H. panicea and H. aquaeductus were subjected to was sterilized with Millex-GP syringe filter units 0.22 mm (Merck spicule dissolution in 10% fluoride acid for 3 hr at RT. Finally Millipore, Billerica, Massachusetts, USA). aggregates of all species were dehydrated in ethanol series at RT and stored in 70% ethanol at 4°C. Sponge Cell Cultures Aliquot (2 mL) from each cell suspension was taken to Histological Studies of Multicellular Aggregates determine cell concentration in hemocytometer. Accordingly For histological and ultrastructural investigations fixed samples cell suspensions were diluted with FSW up to concentrations of were embedded in Araldite502/Embed-812 embedding media 1 107 3 107 cells/mL and cultured in 30 mm Petri dishes (Electron Microscopy Sciences) according to manufacturer (5 mL of suspension in each dish). Threefold variations in cell instructions. Semi-thin sections 1–2 mm thick made by LKB concentration in suspensions do not affect the course and rate Ultratome 3 (LKB, Sweden) were stained with toluidine blue— of cell reaggregation in studied species. During preliminary methylene blue mixture and studied under Leica DM5000B studies we evaluated viability of cell in suspension of each microscope (Leica, Wetzlar, Germany). species using standard Propidium Iodide staining. In all cases only few cells were stained. During main studies we supposed Transmission and Scanning Electron Microscopy that viability of cells in suspension was the same. Five to ten For scanning electron microscopy (SEM) fixed samples were Petri dishes with cell suspension were obtained from each dehydrated in ethanol/acetone series, critical point dried using individual of H. panicea and H. aquaeductus, and two to four Hitachi Critical Point Dryer HCP-2 (Hitachi, Tokyo, Japan), Petri dishes from each individual of H. dujardinii.EachPetri mounted on aluminum stabs and gold/platinum coated. For dish with cell suspension was considered as cell culture. In sum, internal structure investigation aggregates were fractured in a we studied 75 cell cultures of H. panicea (obtained from 12 liquid nitrogen bath prior to critical point drying. Investigation individuals), 47 cell cultures of H. aquaeductus (obtained from was done under CamScan S-2 and Jeol JSM6380-LA (Jeol, Tokyo, 7 individuals), and 46 cell cultures of H. dujardinii (obtained Japan) microscopes. from 17 individuals) (Table 1). For transmission electron microscopy (TEM) ultrathin section Cell cultures were maintained in FSW under temperature of of embedded samples were stained with 4% aqueous uranyl 8–10°C. To intensify the cell reaggregation Petri dishes were acetate and 0.4% lead citrate according to Reynolds ('63). Stained placed on orbital shaker (70 rotations per minute). Cell cultures ultrathin sections were studied under Jeol JEM-100B and Jeol of H. panicea and H. aquaeductus were maintained on shaker JEM-1011 (Jeol, Tokyo, Japan) microscopes. during first 24 hr after tissue dissociation, H. dujardinii cell cultures—during only 1 hr after tissue dissociation. Every 48 hr Measurements half of the culture medium was changed with fresh FSW. Each Aggregates were measured on photographs of cultures prior to cell culture was inspected and photographed daily over the fixation with the help of Leica LAS Store and Recall v.3.6. The whole period of cultivation (Table 1) with the help of measured size of the aggregate corresponded to its maximum stereomicroscope Leica M165FC (Leica, Wetzlar, Germany) dimension. The measurements were done two times during equipped with digital camera Leica DFC 320 and application cultivation period: 1) at the end of orbital shaker cultivation (size Leica LAS Store and Recall v.3.6. Viability of aggregates was of initial multicellular aggregates), 2) after formation of evaluated by their general morphology: dead aggregates primmorphs (H. panicea and H. dujardinii) or early-stage quickly disintegrated in the cultures. primmorphs (H. aquaeductus). Objects measuring on TEM micrographs were done with JR Fixation of Aggregates Screen Ruller v. 1.5. Preliminary observations allowed defining the main stages of cell For all measurements mean value and standard deviation were reaggregation for each studied species. Further on the samples of calculated. these main stages and in some cases of the intermediate ones (exact time of these samples are indicated in the Results) were RESULTS fixed for histological and ultrastructural studies. The initial phase of reaggregation is common for all investigated Specimens were fixed overnight at 4°C by 2.5% glutaraldehyde species. Further reaggregation process appeared species-specific (Ted Pella, Inc., Redding, California, USA) on 0.2 M Millonig's and described for each species.

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Cell Behavior in Suspension and Initial Phase of Reaggregation Primary multicellular aggregates arise in cultures at 24 hpd Cells of all species remain viable after the procedure of tissue (Table 2) with round or oval shape and size of 265 135 mm dissociation actively forming pseudopodia of different shape and (n ¼ 150) (Fig. 2A; Table 3). Histological studies reveal that cells number (Fig. 1A and B). The majority of cells bear filopodia, but of two dimensional classes participate in H. aquaeductus occasionally cells with lobobodia are found. Choanocytes retain aggregates formation: minute cells about 2–5 mm in diameter flagellum and move in suspension with help of it. Even within the and large cells about 8–20 mm in diameter. Minute cells aggregates choanocytes preserve their actively beating flagella. constitute majority of the cells of primary aggregates. Large Choanocytes are the only motile cell type in suspension. Other cells are present in small number. Cell packaging is very loose and cells do not move despite active formation of pseudopodia. These most of cells have round shape. There are no specialized cell-cell cells use pseudopodia for searching surroundingsand in the cases of contacts in primary aggregates of H. aquaeductus. Moreover, touching pseudopodia of neighboring cells they slowly move closer within these aggregates the cells in most cases even do not up each other forming an aggregate. Cells already incorporated in contact each other being separated by wide free spaces (Fig. 3A the multicellular aggregates continue formation of pseudopodia and C). (Fig. 1C). Interestingly that H. dujardinii cells incorporated into an In TEM investigations four cell types can be identified among aggregate switch to formation of lobopodia instead of filopodia. large cells of the H. aquaeductus primary aggregates: nucleolar The size of multicellular aggregates increases due to incorpo- amoebocytes and three types of cells with inclusions. Nucleolar ration of cells from suspension and fusion with other aggregates amoebocytes are characterized by large central nucleus with on the bases of described above peculiarities of cell behavior. The prominent nucleolus and homogeneous karyoplasm. Their first multicellular aggregates have diverse shapes, but later on the cytoplasm contains rough endoplasmic reticulum (RER) cisterns, developing aggregates become more or less spherical. small homogenous inclusions, phagosomes and one or two Golgi To start the cell reaggregation the suspensions were cultured on apparatus (Fig. 3D). the orbital shaker to intensify cell contacts and increase the size Cell with inclusions are abundant in the H. aquaeductus of primary multicellular aggregates. Preliminary studies eluci- primary multicellular aggregates. Considering their ultrastruc- dated that the optimal cultivation period on the orbital shaker for tural differences three cell types can be distinguished: H. panicea and H. aquaeductus cells was 24 hr post-dissociation (hpd) and for H. dujardinii—1 hpd. During this cultivation period 1. Granular cells. The cytoplasm is filled with 15–40 homoge- primary multicellular aggregates of similar size forms in cell neous spherical or seed-like shaped membranous inclusions cultures of each species (Table 3). Longer time lead to formation of (1.1 0.3 mm). No other organelles are present in the a single big aggregate with the size up to 10 mm in each Petri dish. cytoplasm. Nucleus lacking nucleolus (confirmed by series Such aggregates are unviable and degenerate within few days of semi-thin sections) with small amount of condensed after formation. chromatin distributed along the nuclear membrane is located at the periphery of cell (Fig. 3E). Cell Reaggregation of H. Aquaeductus 2. Spherulous cells. The cytoplasm include 5–15 large spherical Cell cultures from seven out of eight individuals of H. membranous inclusions (2.12 0.44 mm) with homogenous aquaeductus remained viable and were cultured for more than or fine-grained contents. Cisterns of RER, several Golgi 7 days (Table 1). apparatus, electron-transparent vesicles and few phagosomes

Figure 1. Cells of sponges after tissue dissociation. (A, B) H. panicea cells with pseudopodia in suspension. (C) surface of H. dujardinii multicellular aggregate with cells actively forming different types of pseudopodia. Black arrowhead, lobopodia; white arrowhead, filopodia; asterisk, nucleus with nucleolus.

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Table 2. Time of the main stages development in cell reaggregation of studied sponge species.

Species Primary multicellular aggregates Early-stage primmorphs Primmorphs Reconstructed sponges Haliclona aquaeductus 24 hpd 6–7 dpd - - Halichondria panicea 24 hpd 5–8 dpd 7–9 dpd - Halisarca dujardinii 1 hpd 3 hpd 1–3 dpd 10–12 dpd

Dash means that stage was not reached in the experiments; dpd, days post-dissociation, hpd, hours post-dissociation.

are present in the cytoplasm of spherulous cells. The nucleus is contains condensed chromatin associated with nuclear mem- nucleated and has homogenous karyoplasm (Fig. 3F). brane. Basal body of the flagellum and nucleus always lie at the 3. Presumable spongocytes. The cytoplasm contains 7–10 opposite sides of the cell. We have never observed rootlets of membranous spherical inclusions (1.08 0.24 mm) with basal body (Fig. 3H). At 24 hpd the number of choanocytes within homogenous content, multiple cisterns of RER, Golgi primary aggregates is high, but visibly declines at 72 hpd. At apparatus, electron-transparent vesicles and phagosomes. 72 hpd only few choanocytes can be found in multicellular Cell nucleus is nucleated and karyoplasm contains some aggregates. During further H. aquaeductus aggregate develop- condensed chromatin (Fig. 3G). However, these cells lack some ment the choanocytes completely disappear. features characteristic for intact spongocytes (for example, At 3 days post-dissociation (dpd) transformation of primary polarization, and certain position in sponge body). multicellular aggregates begins. Loose substance consisting of Most of the minute cells lost their typical features and look dead cells and cell debris is expelled from aggregates and forms dedifferentiated at 24 hpd. These cells have granular and an envelope around them (Figs. 2B and 3B). At 6–7 dpd sometimes dark cytoplasm with some RER cisterns, electron- aggregates free from debris envelope, become denser and almost dense and electron-transparent vesicles. Nucleus of these cells in spherical in shape (Fig. 2C). During this transformation two most cases is unnucleated. important changes in the histological structure of H. aquaeductus Choanocytes are the only cell type that can be identified among aggregates occur. Firstly, only two cell types constitute minute cells. They have rounded shape and bear flagellum with aggregates after transformation—nucleolar amoebocytes and collar of microvilli. Few homogenous inclusions, 1–2 phag- granular cells. Other cell types disappear during transformation. osomes and electron-transparent vesicles are present in the Cell packaging becomes denser and the shape of the cells shifts to cytoplasm. Choanocyte nucleus is round, unnucleated and polygonal. There are no free spaces between cells like in primary

Figure 2. Stages of H. aquaeductus cell reaggregation. (A) general view of initial multicellular aggregates in the culture (24 hpd); (B) multicellular aggregates within the envelope of cell debris during transformation; (C) general view of early-stage primmorph in the culture (11dpd). White arrowhead, the envelope of cell debris. bb, flagellum basal body; bexp, cell body of exopinacocyte; c, cavity; cc, choanocyte chambers; ccr, choanocyte chamber rudiment; cd, cell debris; cel, collagen-reinforced layer; ch, choanocytes; cl, collagen layer; cn, canals of aquiferous system; d, cell debris; ec, expelled cells; enp, endopinacocytes; ep, external parts of exopincocytes; exp, exopinacocytes; f, flagellum; fc, fibrillar collagen; GA, Golgi apparatus; gc, granular cell; l, lumen of choanocyte chamber; ME, mesohyl; mgc, microgranular cell; mt, mitochondria; mv, microvilli; in, specific inclusion; in1, homogeneous inclusion; in2, fine-grained inclusion; n, nucleus; Ph, phagosome; r, rootlet of basal body; RER, rough endoplasmatic reticulum; sc, spherulous cell; spc, presumable spongocyte; v, vesicle.

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Table 3. Size (mean value SD) of primary multicellular aggregates and final stage of reaggregation (true primmorphs of H. panicea and H. dujardinii; early-stage primmorphs of H. aquaeductus) of studied sponge species.

Species Size of primary multicellular aggregates, mm Size of final stage of reaggregation, mm Haliclona aquaeductus 265 135 1055 410 Halichondria panicea 222 78 700 376 Halisarca dujardinii 90 30 333 176 multicellular aggregates. Nevertheless, there are no specific cell- exopinacoderm. The majority of cells at the surface retain their cell contacts—outer membranes of neighbor cells just lie parallel amoeboid shape with multiple branching pseudopodia (Fig. 4F). to each other separated by narrow space. Thus, cell reaggregation of H. aquaeductus comes to the end at Secondly, three concentric cell zones emerge inside H. aqua- the stage of early primmorphs and true primmorphs with eductus aggregates (Fig. 4A and B). TEM and SEM investigations continuous exopinacoderm never form in cell cultures of this show that the surface zone of aggregates consists of cells of species (Table 2). different shapes with several pseudopodia. Sometimes these cells At 8 dpd the size of H. aquaeductus early-stage primmorphs is are slightly flattened. There is no fibrillar extracellular matrix 1055 410 mm(n¼ 47) (Table 3). They can remain viable in (ECM) in this cell zone (Fig. 4C and F). The intermediate zone cultures without any sign of further development for 9–17 days. contains densely packed cells with some fibrils of ECM between During this time they merge with each other visually increasing them (Fig. 4D). In the central zone the cell packing is loose, cells are in size. round shaped and surrounded by considerable amount of fibrillar ECM (Fig. 4E). This stage of H. aquaeductus cell reaggregation can Cell reaggregation of H. panicea be considered as early-stage primmorph (Table 2). Cell cultures from twelve individuals of H. panicea were viable Histological and SEM investigations show emergence of few and cultured for more than 5 days (Table 1). exopinocytes on the surface of early-stage primmorphs during In H. panicea cell cultures primary multicellular aggregates their further development. However, they never form continuous form at 24 hpd having size of 222 78 mm(n¼ 118) and different

Figure 3. Ultrastructure and cell types of H. aquaeductus primary multicellular aggregates. (A) semithin section of initial multicellular aggregate (24 hpd); (B) primary multicellular aggregate (3 dpd) (1) surrounded by the envelope of dead cells and cell debris (2); (C) ultrathin section showing loose packaging of cells within initial multicellular aggregate (24 hpd) (TEM); (D) nucleolar amoebocyte (TEM); (E) granular cell (TEM); (F) spherulous cell (TEM), inset shows area of cell cytoplasm with higher magnification; (G) presumable spongocyte (TEM), inset shows cisterns of RER from another presumable spongocyte with higher magnification; (H) choanocyte (TEM). bb, flagellum basal body; d, cell debris; ec, expelled cells; gc, granular cell; f, flagellum; mv, microvilli; in1, homogeneous inclusion; in2, fine-grained inclusion, n, nucleus; Ph, phagosome; RER, rough endoplasmatic reticulum; sc, spherulous cell; spc, presumable spongocyte; v, vesicle.

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Figure 4. Structure of H. aquaeductus early-stage primmorphs. (A, B) concentric cell zones within early-stage primmorph (7 dpd); (A) semithin section; (B) fractured early-stage primmorph (SEM); 1, surface cell zone; 2, intermediate cell zone; 3, central cell zone; (C) surface cell zone of the fractured early-stage primmorph (SEM); (D) intermediate cell zone of the fractured early-stage primmorph (SEM); (E) central cell zone of the fractured early-stage primmorph (SEM); (F) surface of early-stage primmorph (14 dpd) (SEM). n, nucleus.

shapes—from round to complex branching (Fig. 5A) (Tables 2 and microvilli. Despite strong structural similarity with H. aqua- 3). Most likely branching aggregates arise by merging of several eductus choanocytes, there is one clear-cut distinction between round aggregates. choanocytes of these two species: relative position of flagellum Histological investigations show two dimensional classes of basal body and nucleus. In H. panicea choanocytes nucleus is cells constitute the primary multicellular aggregates of H. located close to basal body that affect nucleus shape (Fig. 6F). panicea: minute (2–5 mm) and large (8–20 mm) cells. Majority At 3 dpd the transformation of H. panicea primary multicellu- of cells belong to the cells of minute class, large cells are rare. lar aggregates starts and thin envelope of debris appears around There are clusters of debris inside these aggregates which them (Fig. 5B). During next few days the envelope of debris probably appear due to cell disintegration after the procedure of gradually increases and can become larger than aggregate itself. sponge tissue dissociation (Fig. 6A and B). Cell packaging in H. Sometimes during this process the branching aggregates can fall panicea primary multicellular aggregates is loose and cells are into several round aggregates surrounded by common envelope separated by wide free spaces. There are no specific cell-cell of debris. On the whole the transformation of H. panicea primary contacts in H. panicea aggregates. multicellular aggregates at histological level resembles the same In TEM investigations two cell types can be distinguished of H. aquaeductus primary multicellular aggregates: certain cell among large cells of multicellular aggregates: nucleolar types disappear and packaging of residual cells becomes denser amoebocytes and granular cells. Halichondria panicea nucleolar (Fig. 7A). amoebocytes structurally are very similar to those of H. At 5–8 dpd the transformation process ends and aggregates aquaeductus (Figs. 3D and 6C). During H. panicea aggregate become free from envelopes of debris. At this time they are very development the relative number of phagosomes per amoebo- dense and spherically shaped (Fig. 5C). During histological and cytes increases. TEM studies of aggregates after the transformation we were able Cytoplasm of granular cells contains 10–20 membranous to find only one cell type constituting inner parts of these inclusions (0.57 0.10 mm) with homogenous content and large aggregates—the nucleolar amoebocytes polygonal in shape due to cisterns of RER. Round nucleus with homogenous karyoplasm the dense cell packaging and with cytoplasm filled with bears nucleolus (Fig. 6D). numerous phagosomes (Fig. 7A). Some flattened cells appear Most of the minute cells within primary multicellular on the surface of such aggregates. This stage of H. panicea cell aggregates of H. panicea look dedifferentiated losing their reaggregation can be considered as early-stage primmorph typical characters just as in the case of H. aquaeductus aggregates (Table 2). Similarly to the early-stage primmorphs of H. (Fig. 6E). Only choanocytes remain recognizable at this time aquaeductus those of H. panicea have three concentric cell among minute cells as they still possess flagellum and collar of zones with similar structure (Fig. 7B).

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Figure 5. Stages of H. panicea cell reaggregation. (A) general view of initial multicellular aggregates in the culture (24 hpd); (B) multicellular aggregates within the envelope of cell debris during transformation; (C) general view of early-stage primmorphs in the culture (9 dpd); (D) general view of primmorphs in the culture (13 dpd). White arrowhead, the envelope of cell debris.

During next 2 days H. panicea early-stage primmorphs Several cell types can be distinguished during TEM inves- transform into primmorphs (Table 2). At histological level this tigations of primary multicellular aggregates of H. dujardinii: transformation is accompanied by emergence of continuous exopinacoderm at the surface of aggregates (Figs. 5D and 7C). 1. Nucleolar amoebocytes—cells with central round nucleated Since H. panicea early-staged primmorphs are composed of only nucleus and phagosomes, cell size is 7–8 mm (Fig. 10A); nucleolar amoebocytes new exopinacocytes apparently differen- 2. choanocytes—cells with irregular shape and size of 5–7 mm, tiate from this cell type. flagellum surrounded with collar of microvilli and pyriform At 9 dpd primmorphs of H. panicea have size of 700 376 mm nucleated nucleus (Fig. 10B); (n ¼ 69) (Table 3). They stay viable in the cultures for 10–20 days 3. spherulous cells—large cells (8–12 mm) with cytoplasm almost showing no further development. Primmorphs can merge with completely filled with 3–5 large membranous inclusions each other visually increasing in size. (3.14 0.64 mm) and nucleated nucleolus lying at the periphery of the cell (Fig. 10C); Cell Reaggregation of Halisarca dujardinii 4. granular cells—cells with cytoplasm containing 5–10 round or Cell cultures from all individuals of H. dujardinii remained viable seed-like membranous inclusions (1.05 0.44 mm) and nu- and were cultured for more than 5 days long (Table 1). Cell cleated nucleus bearing some condensed chromatin associated reaggregation of H. dujardinii proceeds very rapidly compared to with nucleolar membrane, cell size is 6–10 mm (Fig. 10D); other species. As early as at 1 hpd primary multicellular 5. microgranular cells—cells with cytoplasm containing large aggregates emerge in cultures (Table 2) having size of amounts of small round or oval membranous inclusions 90 30 mm(n¼ 46) and round shape (Table 3). They actively (0.33 0.04 mm) and central nucleated nucleus, cell size is 7– merge with each other forming larger aggregates of complex 8 mm (Fig. 10E). shapes (Fig. 8A). Histological studies show that primary Ultrastructure of all mentioned cells is the same as in intact multicellular aggregates of H. dujardinii consist of loosely tissue of H. dujardinii and was described earlier (Gonobobleva packed cells about 5–12 mm in size. Within the primary and Maldonado, 2009; Ereskovsky et al., 2011). aggregates all cells form pseudopodia and probably retain active Most of the cells constituting the aggregates are choanocytes and locomotion (Fig. 9A). nucleolar amoebocytes. Choanocytes retain their characteristic

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Figure 6. Ultrastructure and cell types of H. panicea primary multicellular aggregates. (A) semithin section of initial multicellular aggregate (24 hpd); (B) ultrathin section showing loose packaging of cells within the initial multicellular aggregate (24 hpd) (TEM, montage); (C) nucleolar amoebocyte (TEM); (D) granular cell, inset shows part of the cell with higher magnification (TEM); (E) dedifferentiated minute cell (TEM); (F) choanocyte (TEM). bb, flagellum basal body; cd, cell debris; f, flagellum; GA, Golgi apparatus; in, specific inclusion; n, nucleus; Ph, phagosome; RER, rough endoplasmatic reticulum; v, vesicle. features up to 3–4 dpd. During this time they can be easily dedifferentiation this inclusions coexist with typical choanocyte distinguished from other cell types even on semi-thin sections of characters like pyrifom nucleus. Later on electron-dense aggregates. However, in the course of further aggregate inclusions become the only sign of choanocyte nature of the development choanocytes gradually dedifferentiate losing their cell allowing tracing the choanocytes up to 6–7 dpd but only at typical features and becoming indistinguishable from other the ultrastructural level. cells. During their dedifferentiation choanocytes pass through As early as 3 hpd primary multicellular aggregates of H. the stage when small electron-dense inclusions appear in their dujardinii transform into early-stage primmorphs (Fig. 8B; cytoplasm. These inclusions are few in number and uniformly Table 2). Histological and ultrastructural studies show that by distributed in the cytoplasm (Fig. 10F). At early stages of this time first exopinacocytes appear and at 5–7 hpd almost

Figure 7. Ultrastructure of H. panicea early-stage primmorphs and primmorphs. (A) ultrathin section of the early-stage primmorph (7 dpd) showing dense packaging of cell within it (TEM, montage); (B) semithin section showing three cell zones within the early-stage primmorph (7 dpd); 1, surface cell zone; 2, intermediate cell zone; 3, central cell zone; (C) surface of primmorph (17 dpd) with continuous exopinacoderm layer (ruptures in the exopinacoderm layer are artifacts of fixation process) (SEM). n, nucleus; Ph, phagosome.

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1673

Figure 8. Stages of H. dujardinii cell reaggregation. (A) general view of initial multicellular aggregates in the culture (2 hpd); (B) general view of the early-stage primmorph in the culture (7 hpd); (C) general view of the primmorph in the culture (24 hpd); (D) primmorphs with developing cavities of aquiferous system in the culture, black arrowheads—cavities of aquiferous system; (E) attached primmorph with developing cavities of aquiferous system, white arrowheads—cavities of aquiferous system; (F) fully developed sponges, asterisk—osculums; (G) unattached sponges with multiple osculum rudiments, asterisk—osculum rudiments.

Figure 9. Structure of H. dujardinii primary multicellular aggregates, early-stage primmorphs and primmorphs. (A) semithin section of the initial multicellular aggregate (3 hpd); (B) surface of the early-stage primmorph (7 hpd) covered with unstable layer of exopinacocytes (SEM), white arrowheads—pseudopodia of exopinacocytes; (C) surface of the primmorph (24 hpd) covered with layer of exopinacocytes (SEM); (D) semithin section of interior part of the primmorph (24 hpd), mgc—microgranular cell.

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Figure 10. Cell types of H. dujardinii primary multicellular aggregates, early-stage primmorphs and primmorphs. (А) nucleolar amoebocyte (TEM, montage); (B) choanocyte from primary multicellular aggregate, inset shows nucleus with more typical form of another choanocyte (TEM); (C) spherulous cell (TEM); (D) granular cell (TEM); (E) microgranular cell (TEM); (F) dedifferentiating choanocyte (TEM); (G) exopinacocyte of the early-stage primmorph (7 hpd) (TEM). bb, flagellum basal body; f, flagellum; GA, Golgi apparatus; in, specific inclusion; in1, homogenous inclusion; mt, mitochondria; mv, microvilli; n, nucleus; Ph, phagosome; RER, rough endoplasmatic reticulum; v, vesicle. entirely covering the surface of the aggregate. But this At 5–7 dpd at histological sections of developing primmorphs exopinacocyte layer is unstable as majority of cells actively numerous choanocyte chamber rudiments looking like rosettes of produce pseudopodia (Fig. 9B). Moreover, at this moment cells can be found. Cells of these rudiments are columnar shape exopinacocytes are fusiform, not T-shaped like exopinacocytes with long axis oriented radially (Fig. 11B and C). TEM of intact H. dujardinii (Fig. 10G). investigations show that these cells bear nucleated nucleolus Final epithelization and transformation of primary aggregates and numerous phagosomes in the cytoplasm. Considering to primmorphs occurs at 1–3 dpd (Table 2). In H. dujardinii this structure of these cells they can originate either from amoebo- process is not accompanied by formation of the envelope of cell cytes or choanocytes (at this stage choanocytes lose their typical debris and disappearance of certain cell types. Primmorphs of H. features and become indistinguishable from amoebocytes) dujardinii have spherical shape and size of 333 176 mm (Fig. 11D). Latter on some cells start to differentiate into (n ¼ 134) (Figs. 8C and 9C; Table 3). Histological investigations choanocytes acquiring typical features of this cell type reveal that by this time some exopinacocytes acquire T-shape like (Fig. 11E and F). exopinacocytes of intact sponge. Cell packaging inside the Simultaneously rudiments of aquiferous system canals become primmorphs becomes denser: some cells acquire polygonal shape larger obtaining endopinacocyte lining (Fig. 11C). Histological but free spaces still separate most of them. Majority of cells have and TEM studies show that endopinacocytes are either fusiform numerous pseudopodia (Fig. 9D). or their central part with nucleus is submersed into the mesohyl. Primmorphs of H. dujardinii are capable of further progressive These cells have nucleated nucleus, Golgi apparatus, cisterns of development leading to the functional sponge reconstruction. At RER, vesicles, few inclusions with homogenous contents and histological level different structures of intact sponge gradually form short pseudopodia at the side of mesohyl (Fig. 11G). emerge—canals and choanocyte chambers, mesohyl and exopi- Exopinacoderm of developing primmorphs also changes— nacoderm. The progressive decrease of cell packaging density is an majority of exopinacocytes become T-shaped. Moreover, a important peculiarity of H. dujardinii primmorph development. supporting fibrillar layer arises under exopinacoderm (Fig. 11H). Simultaneously with final epithelization of the aggregates’ The rest volume of developing primmorphs is occupied by the surface the first rudiments of aquiferous system canals emerge. At mesohyl of the future sponge. At histological level of the mesohyl histological sections they look like small cavities between cells undergo further structural changes connected with decreasing of without endopinacocyte lining (Fig. 8D). Many cavities contain the cell density: at this time only few cells can be found in the some cell material (Fig. 11А). Most likely these are pseudopodia of mesohyl while most of its volume is composed of fibrillar ECM. cells surrounding cavity. Active formation of pseudopodia by This ECM is probably synthesized by the lophocytes (Fig. 11I). We most of the cells is characteristic feature of this stage of H. were able to find only few of these cells in developing dujardinii primmorph development. primmorphs. The relative amount of cells with inclusions

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1693

Figure 11. Ultrastructure and cell types of developing H. dujardinii primmorphs. (A) semithin section of internal part of primmorph (5 dpd) with developing cavities; (B, C) semithin sections of primmorph (7 dpd) with developing parts of aquiferous system; (D) cell from developing choanocyte chamber (TEM); (E) developing choanocyte chamber cell with choanocyte traits (TEM); (F) small central cavity of developing choanocyte chamber (TEM); (G) endopinacocyte (TEM); (H) collagen layer beneath exopinacoderm of the developing primmorph (7 dpd) (SEM), white arrowheads—exopinacocytes pseudopodia with which they contact collagen layer; (I) lophocyte of developing primmorph (TEM); (J) semithin section of reconstructed sponge (15 dpd); (K) semithin section of choanocyte chamber of the reconstructed sponge; (L) choanocyte of the reconstructed sponge (TEM); (M) exopinacoderm of reconstructed sponge, white arrowheads—ostia (SEM); (N) ectosomal region of the reconstructed sponge. bb, flagellum basal body; bexp, body of exopinacocyte; c, cavity; cc, choanocyte chambers; ccr, choanocyte chamber rudiment; cel, collagen-reinforced layer; ch, choanocytes; cl, collagen layer; cn, canals of aquiferous system; enp, endopinacocytes; ep, external parts of exopincocytes; exp, exopinacocytes; f, flagellum; fc, fibrillar collagen; GA, Golgi apparatus; in, specific inclusion; l, lumen of choanocyte chamber; ME, mesohyl; mgc, microgranular cell; mt, mitochondria; mv, microvilli; n, nucleus; Ph, phagosome; r, rootlet of basal body; RER, rough endoplasmatic reticulum; sc, spherulous cell; v, vesicle.

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(especially, microgranular cells) increases within mesohyl Eerkes-Medrano et al., 2015), physiological properties of (Fig. 11C). multicellular aggregates (Custodio et al., '98; Krasko et al., At 9–10 dpd the histological structure of H. dujardinii 2002; Le Pennec et al., 2003; Zhang et al., 2003a; Muller€ et al., developing primmorphs show some features of intact sponge 2004b; Cao et al., 2007a,b; Valisano et al., 2007; Chernogor et al., structure: 2011b), and multicellular aggregate potential for obtaining sponge cell cultures for biotechnological purposes (Muller€ 1. Aquiferous system is developed to a considerable degree— et al., 2000, 2004a; Sipkema, 2004). Nevertheless, many details canals with endopinacocyte lining pass through the entire of sponge cell reaggregation still remain unclear (Lavrov and body of developing primmorph, first fully developed choano- Kosevich, 2014) including questions of cell behavior and cyte chambers with wide lumen and consisting of differenti- aggregates structure changes in the course of reaggregation. ated choanocytes occur (Fig. 11J–L); 2. the amount of ECM increase further within the mesohyl and Cell Behavior in Suspension and Initial Phase of Reaggregation the number of wandering cells decreases; Many sponge species were investigated during the century and 3. dense fibrillar layer develops under exopinacoderm, all nearly every species showed unique pattern of reaggregation. The exopinacocytes are T-shaped and first ostia appear differences among species may be observed at any step of the (Fig. 11M and N); reaggregation process. Behavior of cells in suspension is the first 4. osculum rudiments emerge, but they are still closed and do not point. For majority of studied species the ability for amoeboid function. movement of all cell types or certain cell types after tissue Osculums of reconstructed sponges open at 10–12 dpd, and dissociation was shown. Moving cells can meet each other forming sponges begin to pump water (Fig. 8F; Table 2). small multicellular aggregates which in some case remain motile Both attached (Fig. 8E) and unattached primmorphs of H. (Wilson, '07; Galtsoff, '23, '25a; Efremova and Drozdov, '70; dujardinii can pass through described developmental stages and Efremova, '72; Gaino et al., '85). The careful studies of single cell reconstruct functional and viable sponge. In attached prim- migrations of Clathria prolifera (Ellis and Solander, 1786) (Galtsoff, morphs the process of attachment to the substrate takes place '23) and Clathrina sp. (Gaino et al., '85) indicate random nature of immediately after continuous exopinacoderm formation. Devel- cell movements in suspension. During movement the cells opment of two kinds of primmorphs occurs similarly concerning frequently change the direction and velocity of movement. The both structure and emergence time of developmental stages. The reaggregation in such sponge species proceeds apparently due to only difference is the number of osculum rudiments formed at the occasional contacts of cells and small groups of cells in suspension. end of primmorph development. Attached primmorphs always However, our observations on H. panicea, H. aquaeductus,and have single osculum rudiment, unattached primmorphs—several H. dujardinii showed quite distinct behavior of cells in suspension. rudiments (Fig. 8G). Nevertheless only one osculum rudiment in All cells produce pseudopodia using them for searching activity. unattached primmorph henceforth opens and functions. Other Similar cell behavior was reported by Volkova and Zolotareva ('81) osculum rudiments gradually disappear from reconstructed for all cell types of H. dujardinii and by Efremova ('72) for certain sponges. These sponges stay unattached during further obser- cell types of Ephydatia fluviatilis (Linnaeus, 1759). vations (Fig. 8F). In such a case cells must be located close enough allowing them approaching each other only due to the contact between their DISCUSSION pseudopodia. At the same time the size of multicellular Since the pioneer work of Wilson ('07) many studies dealt with aggregates is a very important parameter because small sponge cell reaggregation. These studies had different aims and aggregates are incapable of progressive development as it was investigated diverse aspects of reaggregation process: molecular shown for E. fluviatilis (Muller,€ '11; Brien, '37), C. prolifera basis of cell reaggregation (Spiegel, '55; Humphreys, '63, '70; (Galtsoff, '25b; Bagby, '72), and Sycon lingua (Haeckel, 1870) Muller€ and Muller,€ '80; Fernandez-Busquets, 2008), self/non-self (Korotkova, '72). That is why we had to start our sponge cells recognition (Wilson, '10; Galtsoff, '23, '25a; Spiegel, '54; Curtis, cultures on orbital shaker increasing probability of cell contacts '62; Humphreys, '63, '70; Van de Vyver, '75; Leith, '79; Custodio and the size of primary multicellular aggregates. et al., 2004), cell dedifferentiation/transdifferentiation (Efre- Despite practically equal initial cells concentrations in the mova, '68; Bagby, '72; Korotkova and Sokolova, '73; Nikitin, '73; suspensions the cells of H. dujardinii reaggregate much quicker Buscema et al., '80; Volkova and Zolotareva, '81), cell behavior in than these of H. panicea and H. aquaeductus. Significant suspension and during early stages of reaggregation (Galtsoff, difference in optimal time of cultivation on the orbital shaker '23, '25a; Efremova and Drozdov, '70; Noble and Peterson, '72; revealed in present study may reflect differences in certain Gaino et al., '85; Gaino and Magnino, '99), intact sponge processes affecting cell aggregation. At the moment it is difficult regeneration from multicellular aggregates (Wilson, '07, '10; to state the precise mechanisms intensifying cell aggregation. Huxley, '11; Galtsoff, '25b; Efremova, '69, '72; Korotkova, '72; Possible mechanisms may be: 1) formation of higher number of

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1713 pseudopodia, 2) formation of longer pseudopodia, 3) motility of after tissue dissociation and become indistinguishable from all or certain cell types. The latter can explain the high rate of cell nucleolar amoebocytes. During further development of H. reaggregation in H. dujardinii. The choanocytes are the only dujardinii aggregates choanocytes lose their typical characters motile cell type in suspensions of studied species. Presumably the and also become indistinguishable from amoebocytes. On the sponge cells retain their differentiated state during tissue contrary the identified cells with inclusions do not change dissociation so the cell suspension of H. dujardinii may contain significantly their structure in the course of reaggregation high number of choanocytes (Ereskovsky et al., 2011). Visually process. Similar fate was earlier shown for spherulous cells of H. the fraction of choanocytes in cell suspension of H. dujardinii dujardinii (Volkova and Zolotareva, '81) and grey cells of C. was higher compared to cell suspensions of H. panicea and H. prolifera (Wilson and Penney '30; Bagby, '72). Interestingly, we aquaeductus. The random movement of these choanocytes can never saw another cell type with inclusion in the primary intensifying the reaggregation process as a whole. But further multicellular aggregates of H. dujardinii—vacuolar cells, al- studies are needed to clarify this problem. though this cell type is always presented in the mesohyl of intact sponge (Ereskovsky et al., 2011). Primary Multicellular Aggregates: Formation and Structure In most cases the primary multicellular aggregates of Difference in cell reaggregation rates of investigated species investigated species demonstrated loose cell packaging. We appears again after the initial stage of reaggregation—primary never observed any sign of ECM in these spaces. They look multicellular aggregates and primmorphs of H. dujardinii transparent both under light and transmission electron micros- develop in the cultures much quicker compared to H. panicea copy. The same results were obtained by Eerkes-Medrano et al. and H. aquaeductus. Such significant difference in time of (2015) in their ultrastructural investigations of primary multicel- primary multicellular aggregates and primmorphs formation was lular aggregates of seven marine and freshwater species. Most also shown for other sponge species. On the basis of large-scale likely primary multicellular aggregates are filled with non- screening of reaggregation dynamic among Mediterranean fibrillar electron-transparent ECM which is hard to visualize with sponges Valisano and colleagues (Valisano et al., 2006a) develop conventional methods. a general scheme of cell reaggregation dynamics. According to Primary multicellular aggregates transforms to early-stage this scheme all species studied in present work are characterized primmorphs. This stage resembles primmorphs in many charac- by quick primmorph formation, long-time survival of the ters, but lacks continuous exopinacoderm. The process of primmorphs and fusion during further primmorph development. transformation of primary multicellular aggregates and resulting Structure of primary multicellular aggregates is very similar in structure of early-stage primmorphs differs greatly among all sponge species studied to date, including the primary studied species. multicellular aggregates of H. panicea, H. aquaeductus, and H. dujardinii studied in the present work. Mostly aggregates have Possible Reasons of Arrested Development of H. panicea irregular shape and consist of loosely packed cells. Primary Primmorphs and H. aquaeductus Early-Stage Primmorphs multicellular aggregates should comprise all cell types of intact Transformation of H. panicea and H. aquaeductus primary sponge. But not all the cells in the aggregates are readily multicellular aggregates into early-stage primmorphs is accom- recognizable as they lose typical characters some time after tissue panied with alteration in their cell type composition: many cell dissociation. That may be considered as dedifferentiation, at least types (e.g., minute cells including dedifferentiated choanocytes) partial (Efremova, '69, '72; Bagby, '72; Korotkova, '72; Efremova disappear during this transformation. One possible way of such and Nikitin, '73; Volkova and Zolotareva, '81; Sipkema et al., disappearance is expelling from aggregates or phagocytation by 2003; Chernogor et al., 2011a; Eerkes-Medrano et al., 2015). We nucleolar amoebocytes. Two facts support this assumption: 1) could definitely identify choanocytes, certain cells with in- during transformation the envelope consisting of dead cells clusions, nucleolar amoebocytes constituting majority of cell forms around all aggregates and 2) number of phagosomes in the inside aggregates and minute cells. Researchers who worked nucleolar amoebocytes visibly increases meaning these cells with H. panicea (Korotkova, '72; Eerkes-Medrano et al., 2015) and actively phagocytized some other cells. Similar process was H. dujardinii (Volkova and Zolotareva, '81) obtained similar observed by Eerkes-Medrano et al. (2015) during cell reaggrega- results. tion of Haliclona cf. permollis and Spongilla lacustris (Linnaeus, Unfortunately, precise cell type composition of intact tissues in 1759) immediately before primmorph formation at 24–48 hpd. our investigated species is known only for H. dujardinii The other possibility is transdifferentiation of cells into nucleolar (Ereskovsky et al., 2011). In primary aggregates of H. dujardinii amoebocytes. However, we never observed intermediated stages we identified only choanocytes, nucleolar amoebocytes, granular of such transdifferentiation in the multicellular aggregates of H. cells, microgranular cells and spherulous cells. Cell types that we panicea and H. aquaeductus. were unable to find in the aggregates (like exo-, endo- and Eerkes-Medrano et al. (2015) declared that cells of H. panicea basopinacocytes, lophocytes, etc.) may dedifferentiate some time did not form even primmorphs and reaggregation ceased at the

J. Exp. Zool. 4172 IGOREVICHLAVROV AND AND ARNOLDOVICH KOSEVICH stage of primary multicellular aggregates. In our experiments system and even whole sponge movement (Harris, '87; Bond, '92; development of this species stopped after primmorph formation, Gorin and Kosevich, 2009; Ereskovsky, 2010; Borisenko et al., while Korotkova ('72) described the reconstruction of functional 2015; Ereskovsky et al., 2015a). In several studies it was shown and viable sponge within 1 month. that cells of certain sponges can form two types of aggregates: Our results showed that reaggregation of H. aquaeductus cells with dense or loose cell packaging. While primmorphs with loose is arrested at the stage of early primmorphs. There are no other cell packaging showed ability for progressive development and data on reaggregation experiments with H. aquaeductus cells. reconstructed sponges, primmorphs with dense cell packaging in Cells of Haliclona cf. permollis, the most closely related species, most of cases did not develop further (Huxley, '11, '21; Korotkova, were able to reconstruct sponge in the course of reaggregation '72; Volkova and Zolotareva, '81). but with low probability (2 out of 10 experiments) (Eerkes- Cell morphology and ultrastructure in the concentric cell zones Medrano et al., 2015). of H. panicea and H. aquaeductus primmorphs and early-stage Several reasons could cause arrested development of H. primmorphs indicate different conditions within these zones. The panicea and H. aquaeductus. One is the loss of majority of cell narrow peripheral zone provides the most favorable conditions types during transformation of primary aggregates to early-stage for cell movement. Intermediate zone is characterized by very primmorphs in both species. We suppose that after this process dense cell packaging restricting movement of cells. Central zone primmorphs of H. panicea consist virtually only of nucleolar comprises loosely packed and possibly disintegrating round cells amoebocytes and early-stage primmorphs of H. aquaeductus—of due to the oxygen deficiency. Hence one of the possible reasons of nucleolar amoebocytes and few granular cells. One can assume H. panicea primmorphs and H. aquaeductus early-stage that the presence of only mentioned cell types is insufficient for primmorphs inability for further development is limited motility sponge reconstruction. Inability of different cells with inclusions of cells in the intermediate zone accompanied with disintegration to transdifferentiate during reaggregation was shown for some of cells in the central zone. The only progressive development in species (Wilson and Penney, '30; Bagby, '72; Volkova and these aggregates is differentiation of exopinacocytes taking place Zolotareva, '81), including our data on H. dujardinii (see below). in the peripheral zone where active cell locomotion and shape However, nucleolar amoebocytes display the ability for trans- changing are possible. differentiation into: 1) exo- and endopinacocytes (Efremova, '69, '72; Bagby, '72; Volkova and Zolotareva, '81), 2) choanocytes Reconstruction of Functional Sponge During Development of (Efremova, '69, '72; Volkova and Zolotareva, '81), 3) collencytes H. dujardinii Aggregates: Mechanism and Cell Fates and unnucleolar amoebocytes (Efremova, '69, '72). Moreover E. H. dujardinii is the only of investigated species that showed fluviatilis multicellular aggregates consisting only of nucleolar ability to reconstruct functional sponge in the course of cell amoebocytes were capable to reconstruct functional and viable reaggregation. Primmorphs of H. dujardinii include all cell types sponge meaning that nucleolar amoebocytes (at least of this from primary multicellular aggregates which are not tightly species) can transdifferentiate into all cell types (Nikitin, '73). So packed allowing cell locomotion and shape changing. different factor caused the arrested development of H. panicea The principal processes of sponge reconstruction from and H. aquaeductus aggregates. multicellular aggregate are epithelization and construction of Some researchers considered that transdifferentiation of cell aquiferous system including formation of canals and choano- and hence progressive development of aggregates depends on the cyte chambers. According to our observations formation of establishment of certain physiological gradient. This gradient is both these structures in H. dujardinii relies on cell locomotion oriented from the periphery to the center of aggregate and which is confirmed by the increase of pseudopodial activity controls (trans) differentiation of the cells. In the case of small of all cells at the very beginning of aquiferous system aggregates the gradient is not so pronounced and development development. ceases (Nikitin, '73; Volkova and Zolotareva, '81). Moreover the So we suppose that during canal formation cells move away size of aggregates less than 100–200 mm was reported as possible from each other leaving free space in-between them. Choano- reason of arrested development in some sponge species (Muller,€ cyte chambers can develop by the opposite process—cells inside '11; Galtsoff, '25b; Brien, '37; Bagby, '72; Korotkova, '72). But the primmorphs move to each other forming spherical groups— size and existence of three concentric cell zones in early-stage choanocyte chamber rudiments looking like rosettes on the primmorphs and primmorphs of H. panicea and H. aquaeductus histological sections. Similar rosettes-like cell groups were clearly indicate the presence of a certain physiological gradient. shown during aquiferous system development in the prim- Embryonic development, regeneration and continuous main- morphs of H.cf.permollis, S. lacustris,andL. baiсolensis tenance of normal structure of any multicellular organism (Eerkes-Medrano et al., 2015; Ereskovsky et al., 2015b). Further depend on controlled cell locomotion. Coordinated locomotion of development of choanocyte chamber rudiments includes individual cells participates in sponge growth, asexual reproduc- expansion of their internal lumen, differentiation of cells tion, continuous reconstruction of highly effective aquiferous into the choanocytes and establishing connections with other

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1733 choanocyte chambers and/or canals. Similar steps of choano- in absence of vacuolar cells. Moreover, we never observed this cyte chamber development occur during normal and experi- cell type in the reconstructed sponges. The probable explanation mental metamorphosis (when only larval flagellated cells is that vacuolar cells disintegrate during sponge tissue dissocia- participate in the process) of H. dujardinii larva (Gonobobleva tion not participating in aggregate formation, and their and Ereskovsky, 2004; Ereskovsky et al., 2007), and during subsequent restoration occurs after the latest investigated stage choanosome regeneration in the adult individuals of H. of reaggregation. dujardinii (Borisenko et al., 2015). Unfortunately the present data do not allow determining the Formation of aquiferous system parts during reconstruction exact source of exo- and endopinacocytes, choanocytes and of sponges by cell reaggregation based on active cell lophocytes emergence during H. dujardinii reconstruction. On locomotion was also described for H. dujardinii, C. prolifera, time of aquiferous system and mesohyl development these cell and H. panicea (Galtsoff, '25b; Korotkova, '72; Volkova and types lose their typical characters and look like nucleolar Zolotareva, '81). However, other mechanisms of aquiferous amoebocytes, while maternal granular, microgranular, and system development are known. In the study of H. dujardinii spherulous cells obviously do not dedifferentiate during whole cell reaggregation Volkova and Zolotareva ('81) showed that process of cell reaggregation. We never observed intermediate within primmorphs with loose cell packaging the canals and stages between cell with inclusions and other cell types. Later on choanocyte chambers of aquiferous system developed by active the lacking cell types of reconstructed sponge probably originate cell locomotion, while in primmorphs with dense cell packaging from this pool of nucleolar amoebocytes. canals developed through disintegration of cell groups. Indications of the certain cell types’ origin during sponge Disintegration of cells was recognized as the main mechanism reconstruction were found by several researchers through of aquiferous system development in the course of cell morphological analysis of developing aggregates of different reaggregation for all calcarean sponges studied to date sponge species. According to these data both exo- and (Leucosolenia complicata (Montagu, 1814), Sycon raphanus endopinacocytes in Demospongiae and Calcarea can arise from Schmidt, 1862, and S. lingua) (Huxley, '11, '21; Korotkova, '72). nucleolar amoebocytes or choanocytes. For H. dujardinii it was In freshwater demosponges (E. fluviatilis and S. lacustris) also shown that maternal endopinacocytes do not transdiffer- choanocytes constituting the chambers emerged as conse- entiate during reaggregation and always become a part of canal quence of cell divisions of nucleolar amoebocytes while the lining of newly formed sponge (Efremova, '69, '72; Bagby, '72; canals of aquiferous system formed by active cell locomotion Korotkova, '72; Volkova and Zolotareva, '81; Ereskovsky et al., like in other demosponges (Efremova, '69, '72). 2015b). In Demospongiae choanocytes originate from maternal Simultaneously with aquiferous system formation progressive choanocytes and nucleolar amoebocytes, and in Calcarea— development of mesohyl occurs in H. dujardinii primmorphs. maternal choanocytes are the only source of choanocytes of Since majority of cells take part in the formation of choanocyte reconstructed sponge (Efremova, '69, '72; Korotkova, '72; chambers amount of free mesohyl cells not participating in Volkova and Zolotareva, '81). In demosponges both lophocytes aquiferous system formation decreases. The space filled with and sclerocytes originate from maternal nucleolar amoebocytes ECM between mesohyl cells becomes pronounced. Granular, (Efremova, '68, '69, '72; Bagby, '72). microgranular and spherulous cells become the main cell types Hence, we suppose that all maternal cells with inclusions in the mesohyl of developing primmorphs and newly formed become a part of mesohyl of reconstructed sponge and never give sponge while nucleolar amoebocytes are present in low amount. rise to another cell type. At the same time restoration of exo- and This can indicate that numerous nucleolar amoebocytes trans- endopinacocytes, choanocytes and lophocytes occurs through differentiate into choanocytes, endopinacocytes and cells with transdifferentiation of nucleolar amoebocytes. inclusions. Composition of mesohyl ECM also changes. While the ECM of CONCLUSION primary multicellular aggregates is probably composed of non- Data collected by many researchers so far assume different modes fibrillar material, during primmorph development a lot of fibrils of cell reaggregation among sponge species. Variations include appear in the mesohyl especially under exopinacoderm. Obvi- both rate of reaggregation and final stage of reaggregation. Not ously these are collagen fibrils produced by lophocytes. all studied species display ability to reconstruct functional and Considering amount of collagen fibrils and short period of their viable individuals during cell reaggregation. In many species emergence (between 5 and 7 dpd) great number of lophocytes reaggregation stops at primmorphs stage, but some species even should arise at this developmental stage, although we found only fail to develop continuous exopinacoderm and stop at the stage of few cells of this type. The other possibility is the very high primary multicellular aggregates (Custodio et al., '98; Zhang secretion activity of lophocytes. et al., 2003a; Valisano et al., 2006a; Eerkes-Medrano et al., 2015). The structure of mesohyl in developing primmorphs resembles Our study is in agreement with this body of data: H. dujardinii that in intact H. dujardinii (Ereskovsky et al., 2011) differing only showed ability to reconstruct functional and viable sponge, while

J. Exp. Zool. 4174 IGOREVICHLAVROV AND AND ARNOLDOVICH KOSEVICH both H. aquaeductus and H. panicea stopped their development at certain cell type. Such experiments were performed on certain stages of early primmorphs and primmorphs, respectively, due to sponge species by several investigators (Korotkova and immobility of cell inside their aggregates. Sokolova, '73; Nikitin, '73; Buscema et al., '80; Zhang et al., However, the inability of certain sponge species to recon- 2003b), but their results are yet insufficient to make struct functional and viable individuals during cell reaggrega- unequivocal conclusions about cell fates. Future elucidation tion may be not the fixed characteristic of the species. First of of morphogenetic potencies of cell types should be done on all, it is well known that physiological state and anatomical broad variety of sponges from different taxonomic groups and structure of tissue varies greatly during sponge life cycle and at at different stages of their life cycles. Such body of data will different seasons during the year (Korotkova, '79; Ereskovsky, provide our better understanding on how sponges combine 2000). That means that for a certain species the ability of cells tissue integration with magnificent plasticity of their cells. to reaggregate can vary at different stages of their life cycle and during the year. Dependence of competence to cell reaggrega- ACKNOWLEDGMENTS tion upon the stage of sponge life cycle was shown for some The authors thank SCUBA divers from N.A. Pertsov White Sea species (Efremova, '69, '72; Korotkova, '79, '97) and variations Biological Station of Lomonosov Moscow State University for the of cell reaggregation intensity during the annual cycle were help with specimens’ collection and the Electron Microscopy reported for Petrosia ficiformis (Poiret, 1789) (Valisano et al., Laboratory of the Shared Facilities Center of Lomonosov Moscow 2006b). Moreover, H. panicea showed significant response even State University sponsored by the RF Ministry of Education and on tiny variations in experimental conditions and successfully Science. The authors also thank three anonymous reviewers for regenerated only within narrow range of conditions (Korotkova helpful comments and suggestions for improving the manuscript. and Nikitin, '69a,b). Hence ability to cell reaggregation of Both authors conceived the study, read, and approved the final certain sponge species also can vary greatly depending on version of the manuscript. experimental conditions. These facts explain why many researchers obtained so differing results in experiments on LITERATURE CITED cell reaggregation working with the same sponge species (for Adamska M, Degnan BM, Green K, et al. 2011. What sponges can tell example, results on H. panicea cell reaggregation reported in us about the evolution of developmental processes. Zoology this article and results of Korotkova ('72) and Eerkes-Medrano 144:1–10. et al. (2015)). Bagby RM. 1972. Formation and differentiation of the upper That means that aiming to reveal full morphogenetic potential pinacoderm in reaggregation masses of the sponge Microciona of multicellular aggregates of certain sponge species during cell prolifera (Ellis and Solander). J Exp Zool 180:217–225. reaggregation we have to investigate this process at the different Bond C. 1992. Continuous cell movements rearrange anatomical stages of live cycle of this species related to the seasonality and structures in intact sponges. J Exp Zool 263:284–302. sexual reproduction. What is more, we have to elucidate and Borisenko IE, Adamska M, Tokina DB, Ereskovsky AV. 2015. Trans- maintain the optimal experimental conditions which will allow differentiation is a driving force of regeneration in Halisarca certain species to uncover its full potential. Only such approach dujardini (Demospongiae, Porifera). PeerJ 3:e1211. can guarantee the unambiguous conception of sponge cell Brien P. 1937. La reorganization d’eponge apres dissotiation par reaggregation process. filtration et phenomens d’involution chez Ephydatia fluviatilis. Arch Another important task is the elucidation of morphogenetic Biol Liege 48:185–268. potencies of certain sponge cell types for precise understanding Buscema M, De Sutter D, Van de Vyver G. 1980. Ultrastructural of tissue functioning and coordination in these ancient study of differentiation processes during aggregation of metazoans. Cell reaggregation is very promising model for purified sponge archaeocytes. Wilhelm Roux Arch Dev Biol solving this problem. During reaggregation cells are exposed to 188:45–53. the extreme conditions and can reveal all their potencies for Cao X, Fu W, Yu X, Zhang W. 2007a. Dynamics of spicule production in dedifferentiations/transdifferentiations. Moreover it allows us the marine sponge Hymeniacidon perlevis during in vitro cell to set experiments under fully controlled laboratory conditions. culture and seasonal development in the field. Cell Tissue Res Unfortunately dedifferentiation of majority of cells at the 329:595–608. beginning of reaggregation makes it highly difficult to trace Cao X, Yu X, Zhang W. 2007b. Comparison of spiculogenesis in in vitro cell types resting only on cell morphology and ultrastructure. ADCP-primmorph and explants culture of marine sponge Hyme- During morphological analysis certain mistakes and inaccura- niacidon perleve with 3-TMOSPU supplementation. Biotechnol cies are possible. Hence precise tracing of dedifferentiating cell Prog 23:707–714. fate in the course of reaggregation needs elaboration of specific Chernogor LI, Denikina NN, Belikov SI, Ereskovsky AV. 2011a. Long- methods of each cell type labeling. Another promising term cultivation of primmorphs from freshwater Baikal sponge approach is reaggregation experiments with pure fraction of Lubomirskia baikalensis. Mar Biotechnol 13:782–792.

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1753

Chernogor LI, Denikina NN, Belikov SI, Ereskovsky AV. 2011b. Ereskovsky AV, Borisenko IE, Lapebie P, et al. 2015a. Oscarella Formation of spicules during the long-term cultivation of lobularis (Homoscleromorpha, Porifera) regeneration: epithelial primmorphs from the freshwater Baikal sponge Lubomirskia morphogenesis and metaplasia. Plos One 10:e0134566. baikalensis. Organic Chem Curr Res S2:001. Ereskovsky AV, Chernogor LI, Belikov SI. 2015b. Ultrastructural Curtis ASG. 1962. Pattern and mechanism in the reaggregation of description of development and cell composition of primmorphs in sponges. Nature 196:245–248. the endemic Baikal sponge Lubomirskia baicalensis. Zoomorphol- Custodio MR, Prokic I, Steffen R, et al. 1998. Primmorphs generated ogy 11:1–17. from dissociated cells of the sponge Suberites domuncula: a model Fernandez-Busquets X. 2008. The sponge as a model of system for studies of cell proliferation and cell death. Mech Ageing cellular recognition. In: Conn M, editor. Sourcebook of Dev 105:45–59. models for biomedical research. Totowa, NJ: Humana Press Inc. Custodio MR, Hajdu E, Muricy G. 2004. Cellular dynamics of in vitro p75–83. allogeneic reactions of Hymeniacidon heliophila (Demospongiae: Gaino E, Zunino L, Burlando B, Sara M. 1985. The locomotion of Halichondrida). Mar Biol 144:999–1010. dissociated sponge cells: a cell-by-cell, time-lapse film analysis. Eerkes-Medrano D, Feehan CJ, Leys S. 2015. Sponge cell aggregation: Cell Motil 5:463–473. checkpoints in development indicate a high level of organismal Gaino E, Magnino G. 1999. Dissociated cells of the calcareous sponge complexity. Invetbr Biol 134:1–18. clathrina: a model for investigating cell adhesion and cell motility Efremova SM. 1968. Character of proliferative activity of cells of in vitro. Microsc Res Tech 44:279–292. various types in Ephydatia fluviatilis in the course of development Galtsoff PS. 1923. The amoeboid movement of dissociated sponge after dissociation. Archive AGE 54:96–100 (in Russian with English cells. Biol Bull 45:153–161. summary). Galtsoff PS. 1925a. Regeneration after dissociation (an experimental Efremova SM. 1969. Morphologichesky analiz razvitia gubki study on sponges). I. Behavior of dissociated cells of Microciona Ephydatia fluviatilis iz dissotziirovannyh kletok. Vestnik LGU prolifera under normal and altered conditions. J Exp Zool 42: 9:39–53 (in Russian with English summary). 183–221. Efremova SM. 1972. Morphophiziologichesky analiz razvitia presno- Galtsoff PS. 1925b. Regeneration after dissociation (an experimental vodnyh gubok Ephydatia fluviatilis i Spongilla lacustris iz study on sponges). II. Histogenesis of microciona prolifera. J Exp dissotziirovannyh kletok. In: Tokin BP, editor. Asexual reproduction, Zool 42:223–255. somatic embrogenesis and regeneration. Leningrad, Leningrad: Gonobobleva EL, Ereskovsky AV. 2004. Metamorphosis of the larva of University Press. p 110–154. (in Russian with English summary). Halisarca dujardini (Demospongiae, Halisarcida). Bull inst Roy Sci Efremova SM, Drozdov AS. 1970. Prizhiznenye nabludenia nad nat Belgique Biol 74:10–115. rannimi etapamy agregatzii izolirovannyh kletok gubki Ephydatia Gonobobleva EL, Maldonado M. 2009. Choanocyte ultrastructure in fluviatilis. Vestnik LGU 15:18–23. (in Russian with English Halisarca dujardini (Demospongiae, Halisarcida). J Morphol summary). 270:615–627. Efremova SM, Nikitin NS. 1973. Formoobrazovatelnye potencii Gorin S, Kosevich I. 2009. How the sponge can travel. International razlichnogo razmera konglomeratov somaticheskih kletok pre- workshop “The evolution of multicellularity: insights from hydra snovodnoj gubki Ephydatia fluviatilis. In: Tokin BP, editor. and other basal metazoans.” Evangelische Akademie Tutzing: Morphogenetic processes during asexual reproduction, somatic Germany. p 77. embryogenesis and regeneration. Leningrad, Leningrad: University Haeckel E. 1870. Prodromus of a system of the Calcareous sponges. Press. p 97–105 (in Russian). Ann. Mag. Nat. Hist. 5:176–191. Ereskovsky AV. 2000. Reproduction cycles and strategies of the cold- Harris AK. 1987. Cell motility and the problem of anatomical water sponges Halisarca dujardini (Demospongiae, Halisarcida), homeostasis. J Cell Sci 8:121–140. Myxilla incrustans and Iophon piceus (Demospongiae, Poecilo- Humphreys T. 1963. Chemical dissolution and in vitro reconstruction sclerida) from the White Sea. Biol Bull 198:77–87. of sponge cell adhesion. I. Isolation and functional demonstration Ereskovsky AV. 2010. The comparative embryology of sponges. of components involved. Dev Biol 8:27–47. Netherlands: Springer. Humphreys T. 1970. Biochemical analysis of sponge cell aggregation. Ereskovsky AV, Konjukov P, Willenz P. 2007. Experimental metamor- Symp Zool Soc Lond 25:325–334. phosis of Halisarca dujardini larvae (Demospongiae, Halisarcida): Huxley JS. 1911. Some phenomena of regeneration in Sycon; with a evidence of flagellated cell totipotentiality. J Morph 268:529–536. note on the structure of its collar-cells. Phil Trans R Soc B Ereskovsky AV, Lavrov DV, Boury-Esnault N, Vacelet J. 2011. Molecular 202:165–189. and morphological description of a new species of Halisarca Huxley JS. 1921. Further studies on restitution-bodies and free tissue (Demospongiae: Halisarcida) from Mediterranean Sea and a culture in Sycon. Quart J Micr Sci 65:293–322. redescription of the type species Halisarca dujardini. Zootaxa Johnston G. 1842. A history of British sponges and lithophytes. 2768:5–31. Edinburgh: W.H. Lizars.

J. Exp. Zool. 4176 IGOREVICHLAVROV AND AND ARNOLDOVICH KOSEVICH

Korotkova GP, Nikitin NS. 1969a. Sravnitel’no-morphologicheskiy of avarol by primmorphs from Dysidea avara. J Nat Prod analiz regeneratzii i somaticheskogo embriogeneza u cremner- 63:1077–1081. ogovoj gubki Halichondria panicea. Tr Murman Morsk Boil In-Ta AN Muller€ WEG, Grebenjuk VA, Le Pennec G, et al. 2004a. Sustainable SSSR 16:9–16 (in Russian). production of bioactive compounds by sponges—cell cultures Korotkova GP, Nikitin NS. 1969b. Osobennosti morphogeneza pri and gene cluster approach: a review. Mar Biotechnol 6: razvitii kremnerogovoj gubki Halichondria panicea iz nebol'shogo 105–117. fragmenta tela. Tr Murman Morsk Boil In-Ta AN SSSR 16:17–26. (in Muller€ WEG, Thakur NL, Ushijima H, et al. 2004b. Matrix-mediated Russian). canal formation in primmorphs from the sponge Suberites Korotkova GP. 1972. Sravnitelno-morphologicheskie issledovania domuncula involves the expression of CD36 receptor-ligand razvitia gubok iz dissotziirovannyh kletok. In: Tokin BP, editor. system. J Cell Sci 117:2579–2590. Asexual reproduction, somatic embrogenesis and regeneration. Nikitin NS. 1973. Formoobrazovatel’nye potentzii konglomeratov Leningrad, Leningrad: University Press. p 74–109 (in Russian with iadryshkovyh amebocitov presnovodnoj gubki Ephydatia fluviatilis English summary). v zavisimosti ot ih razmera. In: Tokin BP, editor. Morphogenetic Korotkova G. 1979. Pecularities of somatic embriogenesis in sponges. processes during asexual reproduction, somatic embryogenesis In: Levi C, Boury-Esnault N, editors. Biologie des spongiaures. Paris: and regeneration. Leningrad: Leningrad University Press. p 134–142 Coll. Internat. p 53–58. (in Russian). Korotkova GP. 1997. Regeneracia zhivotnyh. Saint-Petersburg: Saint- Noble PB, Peterson SC. 1972. A two-dimensional random-walk Petersburg University Press. (In Russian). analysis of aggregating sponge cells prior to cell contact. Exp Cell Korotkova GP, Sokolova ED. 1973. Zavisimost’ formoobrazovatel’nyh Res 2:288–290. potencij choanocitov izvestkovoj gubki ot kletochnogo sostava Pallas PA. 1766. Elenchus zoophytorum sistens generum adumbra- konglomeratov. In: Tokin BP, editor. Morphogenetic processes tiones generaliores et specierum cognitarium succinctas descrip- during asexual reproduction, somatic embryogenesis and regener- tiones cum selectis auctorum synonymis. Fransiscum Varrentrapp, ation. Leningrad: Leningrad University Press. p 118–133 (in Hagae. Russian). Reynolds ES. 1963. The use of lead citrate at high pH as an electron- Krasko A, Schr€oder HC, Batel R, et al. 2002. Iron induces poliferation opaque stain in electron microscopy. J Cell Biol 17:208–212. and morphogenesis in primmorphs from the marine sponge Schmidt O. 1862. Die Spongien des adriatischen Meeres. Wilhelm Suberites domuncula. DNA Cell Biol 21:67–80. Engelmann, Leipzig. Lavrov AI, Kosevich IA. 2014. Sponge cell reaggregation: mechanisms Sipkema D, van Wielink R, van Lammeren AAM, et al. 2003. and dynamics of the process. Russ J Dev Biol 45:205–223. Primmorphs from seven marine sponges: formation and structure. Le Pennec G, Perovic S, Ammar MSA, et al. 2003. Cultivation of J Biotechnol 100:127–139. primmorphs from the marine sponge Suberites domuncula: Sipkema D. 2004. Cultivation of marine sponges: from sea to cell. PhD morphogenetic potential of silicon and iron. J Biotech 100:93–108. thesis, The Netherlands: Wageningen University. Leith A. 1979. Role of aggregation factor and cell type in sponge cell Spiegel M. 1954. The Role of specific antigens in cell adhesion. adhesion. Biol Bull 156:212–223. Part I. The reaggregation of sponge cells. Biol Bull 107:130– Linnaeus C. 1759. Systema naturæ per regna tria naturæ, secundum 148. classes, ordines, genera, species, cum characteribus, differentiis, Spiegel M. 1955. The Reaggregation of dissociated sponge cells. Ann synonymis, locis. Tomus II. Editio decima, reformata. Holmiæ. NY Acad Sci 60:1056–1078. Millonig G. 1964. Study on the factors which influence preservation of Srivastava M, Simakov O, Chapman J, et al. 2010. The Amphimedon fine structure. Sympos. on electron microscopy. Rome, Italy: queenslandica genome and the evolution of animal complexity. Consiglio Nazionale delle Ricerche. p 347. Nature 466:720–726. Montagu G. 1814. An Essay on Sponges, with Descriptions of all the Tyler S. 2003. Epithelium—the primary building block for metazoan Species that have been discovered on the Coast of Great Britain. complexity. Integr Comp Biol 43:55–63. Mem. Wernerian Nat. Hist. Soc. 2:67–122. Valisano L, Bavestrello G, Giovine M, Cerrano C. 2006a. Primmorphs Muller€ H. 1911.Das regenerationsverm€ogen der susswasserschw€ €amme. formation dynamics: a screening among Mediterranean sponges. Roux’Arch Entwickl Mech 32:397–446. Mar Biol 149:1037–1046. Muller€ WEG, Muller€ IM. 1980. Sponge cell aggregation. Mol Cell Valisano L, Bavestrello G, Giovine M, Arillo A, Cerrano C. 2006b. Biochem 29:131–143. Seasonal production of primmorphs from the marine sponge Muller€ WEG, Wiens M, Batel R, et al. 1999. Establishment of a primary Petrosia ficiformis (Poiret, 1789) and new culturing approaches. cell culture from a sponge: primmorphs from Suberites domuncula. Exp Mar Biol Ecol 337:171–177. Mar Ecol Proc Ser 178:205–219. Valisano L, Bavestrello G, Giovines M, Arillo A, Cerrano C. 2007. Effect Muller€ WEG, Bohm M, Batel R, et al. 2000. Application of cell culture of iron and dissolved silica on primmorphs of Petrosia ficiformis for the production of bioactive compounds from sponges: systhesis (Poiret, 1789). Chem Ecol 23:233–241.

J. Exp. Zool. SPONGE CELL REAGGREGATION: CELLULAR STRUCTURE 1773

Van de Vyver G. 1975. Phenomena of cellular recognition in sponges. Wilson HV, Penney JT. 1930. The regeneration of sponges (Microciona) Curr Top Dev Biol 10:123–140. from dissociated cells. J Exp Zool 56:73–147. Volkova MA, Zolotareva GA. 1981. The development of Halisarca Zhang W, Zhang X, Cao X, et al. 2003a. Optimizing the formation of in dujardin i Johnston from conglomerates of somatic cells. In: vitro sponge primmorph from Chinese sponge Stylotella agminata. Korotkova GP, editor. Morphogenesis in sponges. Leningrad: J Biotech 100:161–168. Leningrad University Press. p 74–93 (in Russian). Zhang X, Cao X, Zhang W, Yu X, Jin M. 2003b. Primmorphs from Wilson HV. 1907. On some phenomena of coalescence and archaeocytes-dominant cell population of the sponge hymeniaci- regeneration in sponges. J Exp Zool 5:245–258. don perleve: improved cell proliferation and spiculogenesis. Wilson HV. 1910. Development of sponges from dissociated tissue Biotechnol Bioeng 84:583–590. cells. Bull Bureau Fisheries 30:1–30.

J. Exp. Zool.