Cell Depletion Due to Diphtheria Toxin Fragment A after Cre-Mediated Recombination

Brockschnieder, Damian; Lappe-Siefke, Corinna; Goebbels, Sandra; Boesl, Michael; Nave, Klaus Armin; Riethmacher, Dieter

doi:10.1128/mcb.24.17.7636-7642.2004

Cited by 104 publications

(91 citation statements)

References 42 publications

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“…To this end, we used microarray analysis to compare cDNAs from the brainstems of wild-type mice with those found in dysmyelinated mice expressing a diphtheria toxin in oligodendrocytes (Brockschnieder et al, 2004). Initial sequence analysis of one of the differentially expressed cDNAs suggested that it encodes for a novel cytoskeletal protein, herein referred to as Ermin.…”

Section: Resultsmentioning

confidence: 99%

“…Oligodendrocyte-ablated animals were obtained by crossing CNP-Cre mice (Lappe-Siefke et al, 2003) with a Rosa26-lacZ flox DTA mice, in which an additional polyadenylation stop signal was introduced upstream of the loxP-DTA gene described previously (Brockschnieder et al, 2004). Total RNA was isolated with TRIzol reagent (Invitrogen, San Diego, CA) from dissected brainstems of 11-d-old animals.…”

Section: Methodsmentioning

confidence: 99%

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Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Brockschnieder¹,

Sabanay²,

Riethmacher³

et al. 2006

J. Neurosci.

109

102

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Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actinrich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Brockschnieder¹,

Sabanay²,

Riethmacher³

et al. 2006

J. Neurosci.

109

102

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“…As of yet, efficient and rapid cell ablation has been achieved in vivo by the use of diphtheria toxin that inactivates the elongation factor 2 resulting in termination of protein synthesis and apoptosis of the targeted cells (Brockschnieder et al, 2004). By ablation of TIF-IA we do not interfere directly with protein synthesis, but with synthesis of rRNA and nucleolar integrity.…”

Section: Discussionmentioning

confidence: 99%

Activation of an Endogenous Suicide Response after Perturbation of rRNA Synthesis Leads to Neurodegeneration in Mice

Parlato¹,

Kreiner

Erdmann

et al. 2008

J. Neurosci.

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Transcription of rRNA genes is essential for maintaining nucleolar integrity, a hallmark for the healthy state and proliferation rate of a cell. Inhibition of rRNA synthesis leads to disintegration of the nucleolus, elevated levels of p53, and induction of cell suicide, identifying the nucleolus as a critical stress sensor. Whether deregulation of rRNA synthesis is causally involved in neurodegeneration by promoting cell death and/or by inhibiting cellular growth has however not been addressed. The transcription factor TIF-IA plays a central role in mammalian rRNA synthesis, regulating the transcriptional activity of RNA polymerase I. To investigate the consequences of nucleolar perturbation in the nervous system, we have chosen to specifically ablate the gene encoding the transcription factor TIF-IA in two different contexts: neural progenitors and hippocampal neurons. Here, we show that ablation of TIF-IA leads to impaired nucleolar activity and results in increased levels of the proapoptotic transcription factor p53 in both neural progenitors and hippocampal neurons but induces rapid apoptosis only in neural progenitors. Nondividing cells of the adult hippocampus are more refractory to loss of rRNA transcription and face a protracted degeneration. Our study provides an unexploited strategy to initiate neurodegeneration based on perturbation of nucleolar function and underscores a novel perspective to study the cellular and molecular changes involved in the neurodegenerative processes.

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“…4a-d and data not shown). We estimate a minimum time lapse of around 16-20 h between the onset of Cre expression in the midbrain (presomitic stages) and cell ablation (8-10 somites), which is similar to that observed in ROSA26 eGFP-DTA/+ ;Nkx2.5 Cre/+ mutants.Similar DTA mouse lines to the one described here have been generated recently (Brockschnieder et al, 2004;Matsumura et al, 2004;Sato and Tanigawa, 2005). In comparison with the mouse line of Brockschnieder et al (2004), our mouse line offers the advantage of expressing eGFP rather than lacZ ubiquitously.…”

mentioning

confidence: 94%

“…In comparison with the mouse line of Brockschnieder et al (2004), our mouse line offers the advantage of expressing eGFP rather than lacZ ubiquitously. Moreover, Cre-mediated recombination excises not only eGFP and the polyadenylation signals, but also the Neo cassette in our construct.…”

mentioning

confidence: 99%

In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

Ivanova

Signore

Caro

et al. 2005

genesis

230

236

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SummaryWe generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells. KeywordsCre; loxP; diphtheria toxin; eGFP; Nkx2.5; Wnt1; genetic ablation; heart; midbrain The role of individual cells in complex tissues or within the whole organism can be examined by specific deletion of appropriate lineages. For example, during embryogenesis specific groups of cells (signalling centres) control the fate of neighbouring cells by emanating diffusible molecules (Meinhardt, 1983;Martinez et al., 1991;Shimamura and Rubenstein, 1997;Placzek and Briscoe, 2005). Mechanical ablation of restricted embryonic regions has been useful in identifying these signalling centres and their role during development (Placzek et al., 1995;Shimamura and Rubenstein, 1997;Thomas and Beddington, 1996). However, these experiments require very precise surgery on the developing embryo, which can often be a difficult task. In adulthood the loss of small numbers of strategically placed cells may result in conditions such as Parkinson's disease (Moore, 2005), Type I diabetes (Butler et al., 2003), and Hirschsprung's disease (Amiel and Lyonnet, 2001). Thus, a versatile system to specifically ablate cells of any lineage during embryogenesis or in adulthood would be of substantial benefit for studies of development, as well as to model human diseases of various aetiologies. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author ManuscriptWith the aim of developing such a system, we generated a ROSA26-eGFP-DTA mouse line, which combines the use of enhanced green fluorescent protein (GFP), diphtheria toxin A subunit (DTA), and Cre recombinase. eGFP is a mutated version of GFP that displays enhanced fluorescence in mammalian cells in vivo and in vitro (Srinivas et al., 2001). Diphtheria toxin is secreted by pathogenic strains of Corynebacterium diphtheriae and is composed of two subunits, A and B. Subunit B is responsible for the internalisation of the toxin upon binding to its receptor. Once inside the cell, subunit A catalyses the inactivation of elongation factor 2, resulting in termination of protein synthesis and apoptosis of the target cell (Maxw...

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Cell Depletion Due to Diphtheria Toxin Fragment A after Cre-Mediated Recombination

Cited by 104 publications

References 42 publications

Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Activation of an Endogenous Suicide Response after Perturbation of rRNA Synthesis Leads to Neurodegeneration in Mice

In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

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