Characterization of Morphological and Cellular Events Underlying Oral
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Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis Aldine Amiel, Hereroa Johnston, Karine Nedoncelle, Jacob Warner, Solène Ferreira, Eric Röttinger To cite this version: Aldine Amiel, Hereroa Johnston, Karine Nedoncelle, Jacob Warner, Solène Ferreira, et al.. Charac- terization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis. International Journal of Molecular Sciences, MDPI, 2015, 16 (12), pp.28449- 28471. 10.3390/ijms161226100. hal-02411397 HAL Id: hal-02411397 https://hal.archives-ouvertes.fr/hal-02411397 Submitted on 24 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Article Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis Aldine R. Amiel, Hereroa T. Johnston, Karine Nedoncelle, Jacob F. Warner, Solène Ferreira and Eric Röttinger * Received: 8 September 2015; Accepted: 6 November 2015; Published: 1 December 2015 Academic Editor: Francesc Cebrià Institute for Research on Cancer and Aging, Université de Nice Sophia-Antipolis UMR 7284, INSERM U1081, CNRS UMR 7284, Nice 06107 Cedex 02, France; [email protected] (A.R.A.); [email protected] (H.T.J.); [email protected] (K.N.); [email protected] (J.F.W.); [email protected] (S.F.) * Correspondence: [email protected]; Tel.: +33-493-377-791 Abstract: Cnidarians, the extant sister group to bilateria, are well known for their impressive regenerative capacity. The sea anemone Nematostella vectensis is a well-established system for the study of development and evolution that is receiving increased attention for its regenerative capacity. Nematostella is able to regrow missing body parts within five to six days after its bisection, yet studies describing the morphological, cellular, and molecular events underlying this process are sparse and very heterogeneous in their experimental approaches. In this study, we lay down the basic framework to study oral regeneration in Nematostella vectensis. Using various imaging and staining techniques we characterize in detail the morphological, cellular, and global molecular events that define specific landmarks of this process. Furthermore, we describe in vivo assays to evaluate wound healing success and the initiation of pharynx reformation. Using our described landmarks for regeneration and in vivo assays, we analyze the effects of perturbing either transcription or cellular proliferation on the regenerative process. Interestingly, neither one of these experimental perturbations has major effects on wound closure, although they slightly delay or partially block it. We further show that while the inhibition of transcription blocks regeneration in a very early step, inhibiting cellular proliferation only affects later events such as pharynx reformation and tentacle elongation. Keywords: regeneration; wound healing; tissue tracking; marine invertebrate; sea anemone; Nematostella vectensis 1. Introduction Regeneration is the biological process that enables organisms to regrow missing body parts after amputation. This fascinating phenomenon has intrigued naturalists and scientists for more than 300 years. Among the first animals in which regeneration has been described was the freshwater polyp Hydra, a cnidarian that belongs to the group of hydrozoans. While regenerative potential has since then been described in other groups of cnidarians (Anthozoa [1,2], Cubozoa [3], and Scyphozoa [4]), the majority and most detailed cellular and molecular regeneration studies in this phylum have been carried out using Hydrozoa (reviewed in [5,6]). These studies highlight variations in the cellular mechanisms (e.g., cellular proliferation) underlying regeneration in different groups of cnidarians (the hydrozoan Hydra vs. the anthozoan Nematostella [7]). They also demonstrate variations within Hydrozoa (Hydra vs. Hydractinia [8]) and even within the same species; in Hydra, for example, the regenerative mechanism varies depending on the amputation site [9]. Therefore, Int. J. Mol. Sci. 2015, 16, 28449–28471; doi:10.3390/ijms161226100 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2015, 16, 28449–28471 Int. J. Mol. Sci. 2015, 16, page–page additional systems are required to determine whether common mechanisms govern regeneration throughoutTherefore, additional Cnidaria andsystems to understandare required to the determ evolutionine whether of this common process mechanisms within the animalgovern regeneration kingdom. throughout Cnidaria and to understand the evolution of this process within the animal kingdom. The majority of cnidarian species belong to the Anthozoa class (sea anemones and corals) The majority of cnidarian species belong to the Anthozoa class (sea anemones and corals) which which diverges basally to their other cnidarian sister groups [10–12]. This places anthozoans at a diverges basally to their other cnidarian sister groups [10–12]. This places anthozoans at a key key phylogenetic position to understand the evolution of the regeneration process among Cnidaria. phylogenetic position to understand the evolution of the regeneration process among Cnidaria. Several anthozoans have been shown to display high regenerative capacities [1,2,13]. However, very Several anthozoans have been shown to display high regenerative capacities [1,2,13]. However, very littlelittle is is known known about about the the cellular/molecular cellular/molecular mechanis mechanismsms that that underlie underlie this this process process in inthese these animals. animals. TheThe anthozoan anthozoan sea sea anemone anemoneNematostella Nematostella vectensisvectensis ((Nv)) is is a a well-established well-established system system for for the the study study of of embryonicembryonic and and larval larval development development [[13–18].13–18]. Interestingly, it it is is also also able able to to regrow regrow half half of ofitsits oral oral or or aboralaboral body body within within five five to to seven seven days daysafter after bisectionbisection (Figure 11))[ [12,13].12,13]. FigureFigure 1. 1.General General anatomy anatomyof of adultadult andand juvenilejuvenile Nematostella vectensis vectensis. .Photographs Photographs illustrating illustrating the the adultadult (left (left) and) and juvenile juvenile ( right(right)) morphologymorphology ofof Nematostella;;( (aa) )Polyps Polyps are are oriented oriented toward toward the the oral oral region to the top and aboral region to the bottom. Red dotted lines indicate the future amputation region to the top and aboral region to the bottom. Red dotted lines indicate the future amputation site site for the following experiments of this study. (b–e) Close-up confocal stack images of the tentacle for the following experiments of this study. (b–e) Close-up confocal stack images of the tentacle (b); (b); pharyngeal (c); mesenterial (along the body column) (d); or the physal (e) regions. Juvenile pharyngeal (c); mesenterial (along the body column) (d); or the physal (e) regions. Juvenile polyps polyps were stained with DAPI to label the nuclei (white staining). An asterisk (*) indicates the were stained with DAPI to label the nuclei (white staining). An asterisk (*) indicates the position of the position of the mouth. Orange and blue arrowheads indicate the gastrodermal and ectodermal mouth. Orange and blue arrowheads indicate the gastrodermal and ectodermal epithelia, respectively. epithelia, respectively. ten, tentacles; pha, pharynx; m, mesenteries. ten, tentacles; pha, pharynx; m, mesenteries. Nematostella is a diploblastic animal comprised of an outer ectodermal epithelium and an inner gastrodermisNematostella thatis aare diploblastic separated by animal muscle comprised fibers and of the an mesoglea outer ectodermal (Supplementary epithelium Figure and S1A,B). an inner gastrodermisThe oral region that areconsists separated of a pharynx by muscle and fibers tentacles and thethat mesoglea surround (Supplementarythe mouth opening. Figure Freshly S1A,B). Themetamorphosed oral region consists juveniles of possess a pharynx four andtentacles tentacles that are that used surround to catch the food mouth that opening.floats by, while Freshly metamorphosedadult polyps have juveniles up to 16 possess [19]. The four body tentacles column that (or arescapus) used ends to catch in its foodaboral-most that floats region by, in while a adultstructure polyps that have is called up to 16the [19physa]. The (Figure body 1; column [20–22]). (or Inside scapus) the ends body in cavity, its aboral-most internal structures region in a structuretermed thatmesenteries is called stretch the physa from (Figure the pharynx1;[20–22 to]). the Inside physa the bodyand correspond cavity, internal to the structures digestive termed and mesenteriesreproductive stretch organs from of thethe pharynxanimal (Figure to thephysa 1). Two and primary correspond mesenteries to the digestive are distinctly and reproductive visible in