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. Ã 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