In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis

Claudette Rabinowitz, Elisabeth Moiseeva, Baruch Rinkevich

Research output: Contribution to journalArticlepeer-review

Abstract

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; R2 = 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.

Original languageEnglish
Pages (from-to)693-705
Number of pages13
JournalCell and Tissue Research
Volume366
Issue number3
DOIs
StatePublished - 1 Dec 2016
Externally publishedYes

Bibliographical note

Funding Information:
We thank Guy Paz for figure preparations and Elad Rachmilovitz for statistical advice. This study was supported by a grant from the Ministry of National Infrastructures, Energy and Water Resources in Israel.

Funding Information:
We thank Guy Paz for figure preparations and Elad Rachmilovitz for statistical advice. This study was supported by a grant from the Ministry of National Infrastructures, Energy and Water Resources in Israel.

Publisher Copyright:
© 2016, Springer-Verlag Berlin Heidelberg.

Keywords

  • Ectodermis
  • Monolayers
  • Mucolytic agent
  • Nematostella vectensis
  • Regeneration

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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