A quantitative method for separation of livingHydra cells

Greber, Martin; David, Charles N.; Holstein, Thomas W.

doi:10.1007/bf00592110

Cited by 26 publications

(15 citation statements)

References 15 publications

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“… A single polyp Hydra animal consists of ~1–2.10 5 cells (Greber et al . ) and harbours 1–2 bacterial cells per host cell (Franzenburg et al . ). …”

Section: The Coral Holobiontmentioning

confidence: 99%

Coral—the world's most diverse symbiotic ecosystem

2015

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Zooxanthellate corals (i.e. those harbouring Symbiodinium) are the main builders of the world's shallow-water marine coral reefs. They represent intimate diverse symbioses between coral animals, single-celled photosynthetic dinoflagellates (Symbiodinium spp.), other microscopic eukaryotes, prokaryotes and viruses. Crabs and other crustaceans, worms, sponges, bivalves and hydrozoans, fishes, sea urchins, octopuses and sea stars are itinerant members of these 'rainforests of the sea'. This review focuses on the biodiversity of scleractinian coral animals and their best studied microscopic epi-and endosymbionts. In relation to coral-associated species diversity, Symbiodinium internal transcribed spacer region sequence types tally 10 2 -10 3 or up tõ 15 different operational taxonomic units (OTUs, or putative species at the 97% sequence identity level; this cut-off was chosen based on intragenomic sequence diversity observed in monoclonal cultures) and prokaryotes (mostly bacterial) total 10 2 -10 4 OTUs. We analysed all publically accessible 16S rRNA gene sequence data and found Gammaproteobacteria were extremely abundant, followed by Alphaproteobacteria. Notably, Archaea were poorly represented and 'unassigned OTUs' were abundant in data generated by high-throughput DNA sequencing studies of corals. We outline and compare model systems that could be used in future studies of the coral holobiont. In our future directions, we recommend a global coral sampling effort including substantial attention being paid to method of coral tissue acquisition, which compartments (mucus, tissue, skeleton) to explore, broadening the holobiont members considered and linking biodiversity with functional investigations.

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“… A single polyp Hydra animal consists of ~1–2.10 5 cells (Greber et al . ) and harbours 1–2 bacterial cells per host cell (Franzenburg et al . ). …”

Section: The Coral Holobiontmentioning

confidence: 99%

Coral—the world's most diverse symbiotic ecosystem

2015

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“…Cell-type specificity of HySmad1 expression was analyzed by northern hybridization in a cell type northern blot using poly A + RNA from elutriated cell fractions (Greber et al 1992). A single band of about 2.7 kb was detected in all fractions (Fig.…”

Section: Expression Of Hysmad1 In Intact and Regenerating Polypsmentioning

confidence: 99%

“…Separation of wild-type Basel polyps into cell type fractions by counterflow centrifugation elutriation was performed as described by Greber et al (1992). Poly A + RNA was isolated from each fraction, and 1 µg poly A + RNA per fraction was size separated by gel electrophoresis and blotted on nylon membrane.…”

Section: Cell Type Northern Analysismentioning

confidence: 99%

Identification and expression of HySmad1, a member of the R-Smad family of TGFβ signal transducers, in the diploblastic metazoan Hydra

2001

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Members of the Smad family of TGFbeta signal transducers are important regulators of proliferation and cell fate during axis formation, organogenesis, and tumorigenesis. A canonical TGFbeta/Smad signaling pathway is conserved in nematodes, insects, and vertebrates. However, its evolutionary origin before the divergence of protostomes and deuterostomes is unclear. Here, we present the cloning and expression of a highly conserved orthologue of receptor-activated Smads in Hydra, which represents clear evidence of the presence of TGFbeta signaling in the ancient phylum Cnidaria. HySmad1 is expressed rather uniformly during asexual reproduction and regeneration, and is transcriptionally upregulated during oocyte development. This suggests that multiple functions of TGFbeta/Smad signaling might be conserved between diploblastic and triploblastic metazoans.

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“…Cellular localisation or cell-type specificity of GFP expression was analysed after dissociating live hydra 48 hours after EP. For dissociation, hydra were macerated according to David's method (David, 1973) or treated either with pronase (Greber et al, 1992) and cells were spread over gelatine-coated slides (0.5% gelation, 0.1% Figure 1. Analysis of the UV-induced GFP-like fluorescence in DAPI-stained live animals (a-d) and fixed cells (e-h).…”

Section: Cellular Analysis Of Gfp Expressionmentioning

confidence: 99%

Cnidarian and Bilaterian Promoters Can Direct GFP Expression in Transfected Hydra

Miljkovic

Mazet

Galliot

2002

Developmental Biology

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Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.

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A quantitative method for separation of livingHydra cells

Cited by 26 publications

References 15 publications

Coral—the world's most diverse symbiotic ecosystem

Coral—the world's most diverse symbiotic ecosystem

Identification and expression of HySmad1, a member of the R-Smad family of TGFβ signal transducers, in the diploblastic metazoan Hydra

Cnidarian and Bilaterian Promoters Can Direct GFP Expression in Transfected Hydra

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