GFP expression in Hydra : lessons from the particle gun

Böttger, Angelika; Alexandrova, Olga; Cikala, Mihai; Schade, Marsha; Herold, Michaela; David, Charles N.

doi:10.1007/s00427-002-0245-0

Cited by 43 publications

(32 citation statements)

References 6 publications

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“…The 3′ end of HyBcl-2-like 7 was obtained by RACE reactions using the GeneRacer Kit (Invitrogen). For the fluorescent fusion proteins GFP-HyBcl-2-like 1-7 and GFP-HyBak-like 1 and 2, the corresponding sequences were cloned into the vector hoT G [30] to the 3′ end of the sequence encoding the fluorescent protein, using the EcoRI site. For yeast two-hybrid assays, the coding sequences of the HyBcl-2 family genes were cloned into the EcoRI and XhoI sites of the bait vector pEG202 or the prey vector pJG4-5 (MoBiTec).…”

Section: Hydra Culturementioning

confidence: 99%

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The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins

et al. 2010

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The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.

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Section: Hydra Culturementioning

confidence: 99%

“…They were introduced into hydra cells with the Helios gene gun system (BioRad), as previously described [30].…”

Section: Hydra Culturementioning

confidence: 99%

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins

et al. 2010

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“…In both lines, expression of the reporter gene was under the control of an actin gene promoter (Böttger et al, 2002;Wittlieb et al, 2006). Animals from these two lines were bisected and grafted according to standard methods.…”

Section: Treatment Of Hydra With Dac-2-25 and Its Derivativesmentioning

confidence: 99%

A small molecule screen identifies a novel compound that induces a homeotic transformation inHydra

et al. 2013

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Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.

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“…In addition to looking for sequence similarities, this will allow the investigation of functional homologies of morphogenes and others in the most basal extant metazoa. Five years ago possible constructs for heterologous expression in Hydra vulgaris were reported' (Boettger et al, 2002, Miljkovic et al, 2002. We sample.…”

Section: Dsred2 In Porifera 1115mentioning

confidence: 99%

Heterologous expression of DsRed2 in young sponges (Porifera)

Pfannkuchen¹,

Brümmer²

2009

Int. J. Dev. Biol.

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Sponges (Porifera) are currently considered to be the first branch off the Urmetazoa, common ancestors of all multicellular animals or metazoa. Research in the field of the developmental biology of sponges was restricted to morphological observations. Nowadays, research is mainly concentrated on larval development, primarily dealing with tissue formation. Already since 1907, methods for developing functional sponges from stem cells have been at hand. Functional freshwater sponges can be grown from stem cell populations originating from gemmulae. A number of poriferan sequences with high similarity to regulative genes in higher metazoa have already been found. We have now succeeded in heterologously expressing the red fluorescent protein DsRedN1 under the control of the cytomegalovirus promoter in young specimens of the freshwater sponge Spongilla lacustris. The protein folded correctly, polymerized and subsequently was detected by fluorescence microscopy. Reporting this expression system, we now consider this appealing system for early meatazoan development to be ready for molecular developmental biology and functional genetics research.

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GFP expression in Hydra : lessons from the particle gun

Cited by 43 publications

References 6 publications

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins

A small molecule screen identifies a novel compound that induces a homeotic transformation inHydra

Heterologous expression of DsRed2 in young sponges (Porifera)

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