RESUMEN
Many animals share a lifelong capacity to adapt their growth rates and body sizes to changing environmental food supplies. However, the cellular and molecular basis underlying this plasticity remains only poorly understood. We therefore studied how the sea anemones Nematostella vectensis and Aiptasia (Exaiptasia pallida) respond to feeding and starvation. Combining quantifications of body size and cell numbers with mathematical modelling, we observed that growth and shrinkage rates in Nematostella are exponential, stereotypic and accompanied by dramatic changes in cell numbers. Notably, shrinkage rates, but not growth rates, are independent of body size. In the facultatively symbiotic Aiptasia, we show that growth and cell proliferation rates are dependent on the symbiotic state. On a cellular level, we found that >7% of all cells in Nematostella juveniles reversibly shift between S/G2/M and G1/G0 cell cycle phases when fed or starved, respectively. Furthermore, we demonstrate that polyp growth and cell proliferation are dependent on TOR signalling during feeding. Altogether, we provide a benchmark and resource for further investigating the nutritional regulation of body plasticity on multiple scales using the genetic toolkit available for Nematostella.
Asunto(s)
Tamaño Corporal , Proliferación Celular , Anémonas de Mar , Animales , Anémonas de Mar/citología , Anémonas de Mar/fisiología , Ciclo Celular/fisiología , Conducta Alimentaria/fisiología , Transducción de Señal , Simbiosis , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.
Asunto(s)
Quinasas Similares a Doblecortina , Neuronas , Anémonas de Mar , Animales , Anémonas de Mar/embriología , Anémonas de Mar/citología , Anémonas de Mar/genética , Neuronas/metabolismo , Neuronas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Microtúbulos/metabolismo , Neurogénesis/fisiología , Animales Modificados Genéticamente , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
The capacity to regenerate lost or injured body parts is a widespread feature within metazoans and has intrigued scientists for centuries. One of the most extreme types of regeneration is the so-called whole body regenerative capacity, which enables regeneration of fully functional organisms from isolated body parts. While not exclusive to this habitat, whole body regeneration is widespread in aquatic/marine invertebrates. Over the past decade, new whole-body research models have emerged that complement the historical models Hydra and planarians. Among these, the sea anemone Nematostella vectensis has attracted increasing interest in regard to deciphering the cellular and molecular mechanisms underlying the whole-body regeneration process. This manuscript will present an overview of the biological features of this anthozoan cnidarian as well as the available tools and resources that have been developed by the scientific community studying Nematostella. I will further review our current understanding of the cellular and molecular mechanisms underlying whole-body regeneration in this marine organism, with emphasis on how comparing embryonic development and regeneration in the same organism provides insight into regeneration specific elements.
Asunto(s)
Modelos Animales , Regeneración/fisiología , Anémonas de Mar/citología , Anémonas de Mar/fisiología , Animales , Hemostasis , Filogenia , Reproducción , Anémonas de Mar/genéticaRESUMEN
Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance.
Asunto(s)
Ganglios Espinales/ultraestructura , Microtúbulos/ultraestructura , Células Receptoras Sensoriales/ultraestructura , Piel/inervación , Animales , Animales Modificados Genéticamente , Axones/fisiología , Axones/ultraestructura , Cuerpo Celular/ultraestructura , Polaridad Celular , Dendritas/fisiología , Drosophila/citología , Drosophila/crecimiento & desarrollo , Ganglios Espinales/fisiología , Centro Organizador de los Microtúbulos/ultraestructura , Anémonas de Mar/citología , Anémonas de Mar/crecimiento & desarrollo , Anémonas de Mar/ultraestructura , Células Receptoras Sensoriales/fisiología , Pez CebraRESUMEN
Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis. In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12-13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.
Asunto(s)
Antozoos/citología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas/metabolismo , Anémonas de Mar/citología , Animales , Supervivencia CelularRESUMEN
Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.
Asunto(s)
Células Endoteliales/metabolismo , Leucocitos/metabolismo , Proteínas/genética , Migración Transcelular de la Célula/genética , Transcriptoma , Migración Transendotelial y Transepitelial/genética , Animales , Evolución Biológica , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Caenorhabditis elegans/clasificación , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Pollos/clasificación , Pollos/genética , Pollos/metabolismo , Ciona intestinalis/clasificación , Ciona intestinalis/citología , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Drosophila melanogaster/clasificación , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Endoteliales/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Leucocitos/citología , Ratones , Pan troglodytes/clasificación , Pan troglodytes/genética , Pan troglodytes/metabolismo , Petromyzon/clasificación , Petromyzon/genética , Petromyzon/metabolismo , Filogenia , Placozoa/clasificación , Placozoa/citología , Placozoa/genética , Placozoa/metabolismo , Proteínas/clasificación , Proteínas/metabolismo , Anémonas de Mar/clasificación , Anémonas de Mar/citología , Anémonas de Mar/genética , Anémonas de Mar/metabolismo , Tiburones/clasificación , Tiburones/genética , Tiburones/metabolismo , Pez Cebra/clasificación , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Animal regeneration is a biological process leading to the reformation of injured or lost tissues/body parts. One of the most fascinating regenerative phenomena is the so-called whole-body regeneration, leading to the reformation of fully functional organisms within days after bisection. The sea anemone Nematostella vectensis is currently emerging as novel whole-body regeneration model. Here we describe the methods of inducing the regenerative process in this cnidarian as well as the fixation and staining protocols for morphological, molecular, and cellular analysis.
Asunto(s)
Anémonas de Mar/fisiología , Anémonas de Mar/ultraestructura , Animales , Proliferación Celular , Inmunohistoquímica/métodos , Regeneración , Anémonas de Mar/citología , Coloración y Etiquetado/métodos , Fijación del Tejido/métodos , Cicatrización de HeridasRESUMEN
Two distinct mechanisms for primordial germ cell (PGC) specification are observed within Bilatera: early determination by maternal factors or late induction by zygotic cues. Here we investigate the molecular basis for PGC specification in Nematostella, a representative pre-bilaterian animal where PGCs arise as paired endomesodermal cell clusters during early development. We first present evidence that the putative PGCs delaminate from the endomesoderm upon feeding, migrate into the gonad primordia, and mature into germ cells. We then show that the PGC clusters arise at the interface between hedgehog1 and patched domains in the developing mesenteries and use gene knockdown, knockout and inhibitor experiments to demonstrate that Hh signaling is required for both PGC specification and general endomesodermal patterning. These results provide evidence that the Nematostella germline is specified by inductive signals rather than maternal factors, and support the existence of zygotically-induced PGCs in the eumetazoan common ancestor.
Asunto(s)
Tipificación del Cuerpo/genética , Estratos Germinativos , Proteínas Hedgehog , Anémonas de Mar , Transducción de Señal/genética , Animales , Femenino , Técnicas de Silenciamiento del Gen , Células Germinativas/citología , Células Germinativas/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/crecimiento & desarrollo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Estadios del Ciclo de Vida/genética , Masculino , Anémonas de Mar/citología , Anémonas de Mar/genética , Anémonas de Mar/crecimiento & desarrolloRESUMEN
Terminal selectors are transcription factors that control the morphological, physiological, and molecular features that characterize distinct cell types. Here, we show that, in the sea anemone Nematostella vectensis, NvPOU4 is expressed in post-mitotic cells that give rise to a diverse set of neural cell types, including cnidocytes and NvElav1-expressing neurons. Morphological analyses of NvPOU4 mutants crossed to transgenic reporter lines show that the loss of NvPOU4 does not affect the initial specification of neural cells. Transcriptomes derived from the mutants and from different neural cell populations reveal that NvPOU4 is required for the execution of the terminal differentiation program of these neural cells. These findings suggest that POU4 genes have ancient functions as terminal selectors for morphologically and functionally disparate types of neurons and they provide experimental support for the relevance of terminal selectors for understanding the evolution of cell types.
Asunto(s)
Sistema Nervioso/metabolismo , Anémonas de Mar/genética , Factores de Transcripción/genética , Animales , Blástula/metabolismo , Diferenciación Celular/genética , Proliferación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Glutamatos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Anémonas de Mar/citología , Factores de Transcripción/metabolismo , Transcriptoma/genética , TransgenesRESUMEN
The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G2/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.
Asunto(s)
Ciclo Celular , Dinoflagelados/fisiología , Nutrientes/metabolismo , Anémonas de Mar/fisiología , Simbiosis/fisiología , Animales , Dinoflagelados/citología , Anémonas de Mar/citología , Especificidad de la EspecieRESUMEN
The cadherin-catenin complex is a conserved, calcium-dependent cell-cell adhesion module that is necessary for normal development and the maintenance of tissue integrity in bilaterian animals. Despite longstanding evidence of a deep ancestry of calcium-dependent cell adhesion in animals, the requirement of the cadherin-catenin complex to coordinate cell-cell adhesion has not been tested directly in a non-bilaterian organism. Here, we provide the first analysis of classical cadherins and catenins in the Starlet Sea Anemone, Nematostella vectensis. Gene expression, protein localization, siRNA-mediated knockdown of α-catenin, and calcium-dependent cell aggregation assays provide evidence that a bonafide cadherin-catenin complex is present in the early embryo, and that α-catenin is required for normal embryonic development and the formation of cell-cell adhesions between cells dissociated from whole embryos. Together these results support the hypothesis that the cadherin-catenin complex was likely a complete and functional cell-cell adhesion module in the last common cnidarian-bilaterian ancestor. SUMMARY STATEMENT: Embryonic manipulations and ex vivo adhesion assays in the sea anemone, Nematostella vectensis, indicate that the necessity of the cadherin-catenin complex for mediating cell-cell adhesion is deeply conserved in animal evolution.
Asunto(s)
Cadherinas/metabolismo , Cateninas/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Anémonas de Mar/embriología , Animales , Adhesión Celular/fisiología , Embrión no Mamífero/citología , Anémonas de Mar/citologíaRESUMEN
Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry. Although Hox genes are also present within Cnidaria, these simple animals lack a definitive A-P axis, leaving it unclear how and when a functionally integrated Hox code arose during evolution. We used short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9 mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larval endoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbx activity also elicits marked defects in tentacle patterning along the directive (orthogonal) axis of primary polyps. On the basis of our results, we propose that an axial Hox code may have controlled body patterning and tissue segmentation before the evolution of the bilaterian A-P axis.
Asunto(s)
Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/fisiología , Anémonas de Mar/crecimiento & desarrollo , Factores de Transcripción/fisiología , Animales , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Endodermo/citología , Endodermo/crecimiento & desarrollo , Endonucleasas , Técnicas de Silenciamiento del Gen/métodos , Genes Homeobox/genética , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Mutagénesis , ARN Interferente Pequeño/genética , Anémonas de Mar/citología , Anémonas de Mar/genética , Factores de Transcripción/genéticaRESUMEN
Hox gene transcription factors are important regulators of positional identity along the anterior-posterior axis in bilaterian animals. Cnidarians (e.g., sea anemones, corals, and hydroids) are the sister group to the Bilateria and possess genes related to both anterior and central/posterior class Hox genes. Here we report a previously unrecognized domain of Hox expression in the starlet sea anemone, Nematostella vectensis, beginning at early blastula stages. We explore the relationship of two opposing Hox genes (NvAx6/NvAx1) expressed on each side of the blastula during early development. Functional perturbation reveals that NvAx6 and NvAx1 not only regulate their respective expression domains, but also interact with Wnt genes to pattern the entire oral-aboral axis. These findings suggest an ancient link between Hox/Wnt patterning during axis formation and indicate that oral-aboral domains are likely established during blastula formation in anthozoan cnidarians.
Asunto(s)
Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Anémonas de Mar/genética , Proteínas Wnt/genética , Animales , Blástula/citología , Blástula/crecimiento & desarrollo , Blástula/metabolismo , Gastrulación/genética , Anémonas de Mar/citología , Anémonas de Mar/crecimiento & desarrollo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Robust morphogenetic events are pivotal for animal embryogenesis. However, comparison of the modes of development of different members of a phylum suggests that the spectrum of developmental trajectories accessible for a species might be far broader than can be concluded from the observation of normal development. Here, by using a combination of microsurgery and transgenic reporter gene expression, we show that, facing a new developmental context, the aggregates of dissociated embryonic cells of the sea anemone Nematostella vectensis take an alternative developmental trajectory. The self-organizing aggregates rely on Wnt signals produced by the cells of the original blastopore lip organizer to form body axes but employ morphogenetic events typical for normal development of distantly related cnidarians to re-establish the germ layers. The reaggregated cells show enormous plasticity including the capacity of the ectodermal cells to convert into endoderm. Our results suggest that new developmental trajectories may evolve relatively easily when highly plastic embryonic cells face new constraints.
Asunto(s)
Estratos Germinativos/citología , Anémonas de Mar/embriología , Animales , Evolución Biológica , Agregación Celular , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Anémonas de Mar/citología , Anémonas de Mar/genética , Anémonas de Mar/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.
Asunto(s)
Cultivo Primario de Células/métodos , Anémonas de Mar/citología , Animales , Proliferación Celular/fisiología , Calor , Anémonas de Mar/fisiología , Estrés FisiológicoRESUMEN
BACKGROUND: Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians. METHODS: Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. RESULTS: We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. CONCLUSIONS: In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.
Asunto(s)
Antozoos/citología , Biomarcadores/análisis , Separación Celular/métodos , Citometría de Flujo , Animales , Reproducibilidad de los Resultados , Anémonas de Mar/citología , Coloración y EtiquetadoRESUMEN
Nervous systems often consist of a large number of different types of neurons which are generated from neural stem and progenitor cells by a series of symmetric and asymmetric divisions. The origin and early evolution of these neural progenitor systems is not well understood. Here we use a cnidarian model organism, Nematostella vectensis, to gain insight into the generation of neural cell type diversity in a non-bilaterian animal. We identify NvFoxQ2d as a transcription factor that is expressed in a population of spatially restricted, proliferating ectodermal cells that are derived from NvSoxB(2)-expressing neural progenitor cells. Using a transgenic reporter line we show that the NvFoxQ2d cells undergo a terminal, symmetric division to generate a morphologically homogeneous population of putative sensory cells. The abundance of these cells, but not their proliferation status is affected by treatment with the γ-secretase inhibitor DAPT, suggesting regulation by Notch signalling. Our data suggest that intermediate progenitor cells and symmetric divisions contribute to the formation of the seemingly simple nervous system of a sea anemone.
Asunto(s)
Células-Madre Neurales/citología , Neurogénesis , Anémonas de Mar/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neurogénesis/fisiología , Filogenia , Receptores Notch/genética , Receptores Notch/metabolismo , Anémonas de Mar/citología , Anémonas de Mar/genética , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Transducción de SeñalRESUMEN
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 2 = 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.
Asunto(s)
Ectodermo/citología , Modelos Biológicos , Anémonas de Mar/citología , Animales , Calcio/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocalasina D/farmacología , Ectodermo/efectos de los fármacos , Femenino , Histonas/metabolismo , Magnesio/farmacología , Masculino , Mitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Regeneración/efectos de los fármacos , Anémonas de Mar/efectos de los fármacosRESUMEN
BACKGROUND: The evolution of novel genes is thought to be a critical component of morphological innovation but few studies have explicitly examined the contribution of novel genes to the evolution of novel tissues. Nematosomes, the free-floating cellular masses that circulate through the body cavity of the sea anemone Nematostella vectensis, are the defining apomorphy of the genus Nematostella and are a useful model for understanding the evolution of novel tissues. Although many hypotheses have been proposed, the function of nematosomes is unknown. To gain insight into their putative function and to test hypotheses about the role of lineage-specific genes in the evolution of novel structures, we have re-examined the cellular and molecular biology of nematosomes. RESULTS: Using behavioral assays, we demonstrate that nematosomes are capable of immobilizing live brine shrimp (Artemia salina) by discharging their abundant cnidocytes. Additionally, the ability of nematosomes to engulf fluorescently labeled bacteria (E. coli) reveals the presence of phagocytes in this tissue. Using RNA-Seq, we show that the gene expression profile of nematosomes is distinct from that of the tentacles and the mesenteries (their tissue of origin) and, further, that nematosomes (a Nematostella-specific tissue) are enriched in Nematostella-specific genes. CONCLUSIONS: Despite the small number of cell types they contain, nematosomes are distinct among tissues, both functionally and molecularly. We provide the first evidence that nematosomes comprise part of the innate immune system in N. vectensis, and suggest that this tissue is potentially an important place to look for genes associated with pathogen stress. Finally, we demonstrate that Nematostella-specific genes comprise a significant proportion of the differentially expressed genes in all three of the tissues we examined and may play an important role in novel cell functions.
Asunto(s)
Evolución Biológica , Anémonas de Mar/genética , Animales , Conducta Animal , Escherichia coli , Regulación de la Expresión Génica , Fagocitos/fisiología , Fagocitosis/genética , Conducta Predatoria , Anémonas de Mar/citología , TranscriptomaRESUMEN
The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one ß-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and ß-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/ß-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans.