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We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

A new progressive, fatal disease called 'cup cell disease' was characterized in ex situ cultures of Botryllus schlosseri, a colonial tunicate. The disease originated as a few dark spots growing within zooids. The infected colonies then started to deteriorate, morphologically diagnosed by ampullar retraction, lethargic blood circulation and by a swollen and soft tunic matrix. In later stages of the disease, developed buds were also affected. Many large black dots were scattered within the tunic matrix, and zooids were transformed to opaque, dilated, sac-like structures, signaling impending death. Colonies were infected periodically, even without direct tissue contact. The time course from first appearance to colony death ranged between 30 and 45 d. Histological studies, in vitro culturing of blood cells and blood smears revealed the existence of numerous cup-like cells (up to 4.8 µm diameter on average) with a yellowish cell wall and transparent cytoplasm that was not stained by various dyes (except azocarmine-G). Cells were refractive under bright-field illumination and revealed a flattened wall with flanges, characteristic of species of the phylum Haplosporidia. Cup cells aggregated in blood vessels and in internal parts of zooids and buds and were phagocytosed by blood cells. In a single case, plasmodia-like structures were found only in the tunic matrix of an infected colony. This is the first record in botryllid ascidians of an infectious lethal disease associated with haplosporidian protists.KEY WORDS: Ascidians · Botryllus · Disease · Haplosporidia · Mariculture Resale or republication not permitted without written consent of the publisherDis Aquat Org 60: [77][78][79][80][81][82][83][84] 2004 Using the laboratory facilities at Haifa, we maintain and culture Botryllus schlosseri colonies first established in 1988. The culture conditions, which have improved with time (Rinkevich & Shapira 1998), maintain the tunicates in good condition (i.e. fast growth rate), and support sexual reproduction, high survivorship of young individuals and long-term maintenance under laboratory conditions. However, occasionally, the entire stock of colonies growing in one of the culturing tanks (17 l standing-seawater tanks; Rinkevich & Shapira 1998) or the entire set of colonies growing in the culture room (20°C) dies simultaneously without the emergence of any known agent. Diseases causing mortality to urochordates have rarely been reported (Monniot 1990). Herein, we describe 'cup cell disease' in cultured Botryllus schlosseri colonies that is associated with the appearance of a haplosporidian protist. Infected colonies characteristically exhibited swollen zooids with dark pigmentation that gradually deteriorated and died. MATERIALS AND METHODSAnimal maintenance. Botryllus schlosseri colonies (originating from Monterey, Half Moon Bay and Moss Landing, Marinas, California, USA, and from the Istra Peninsula, Croatia) were kept in the laboratory at Haifa in 17 l standing-seawater, glass tanks (up to 20 colonies per t...

The original version of this article inadvertently contained a mistake.We wish to bring to your attention an inadvertent omission in Figure 1d in the above manuscript that was not depicted during proofreading. In the image of Figure 1d the annotations mg and bm lack the indicating lines pointing to their specific locations, accidentally lost during resubmission. Please find the revised Figure 1d below.The online version of the original article can be found at http://dx.doi.