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

Asthma is a common debilitating inflammatory lung disease affecting over 200 million people worldwide. Here, we investigated neurogenic components involved in asthmatic-like attacks using the ovalbumin-sensitized murine model of the disease, and identified a specific population of neurons that are required for airway hyperreactivity. We show that ablating or genetically silencing these neurons abolished the hyperreactive bronchoconstrictions, even in the presence of a fully developed lung inflammatory immune response. These neurons are found in the vagal ganglia and are characterized by the expression of the transient receptor potential vanilloid 1 (TRPV1) ion channel. However, the TRPV1 channel itself is not required for the asthmatic-like hyperreactive airway response. We also demonstrate that optogenetic stimulation of this population of TRP-expressing cells with channelrhodopsin dramatically exacerbates airway hyperreactivity of inflamed airways. Notably, these cells express the sphingosine-1-phosphate receptor 3 (S1PR3), and stimulation with a S1PR3 agonist efficiently induced broncho-constrictions, even in the absence of ovalbumin sensitization and inflammation. Our results show that the airway hyperreactivity phenotype can be physiologically dissociated from the immune component, and provide a platform for devising therapeutic approaches to asthma that target these pathways separately.bronchospasms | airway inflammation

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“…To genetically ablate selective populations of sensory neurons, we relied on cre-dependent expression of diphtheria toxin (DTX) (23). Fig.…”

Section: Significancementioning

confidence: 99%

Population of sensory neurons essential for asthmatic hyperreactivity of inflamed airways

Tränkner

Hahne

Sugino

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

193

195

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“…To do so, we created mice expressing the tamoxifen-dependent Cre recombinase (CreER T ) under the control of the proteolipid protein 1 (Plp1) promoter (PlpCreER T ) (26,27) and carrying an inducible flox-stop DTA transgene (Rosa26 DTA ) (25) together with an inducible Tomato reporter (Ai14:Rosa26 tdTom ) (28). Tamoxifen treatment of neonatal [postnatal day (P)0-1] PlpCreER T ;Ai14:Rosa26 tdTom (PlpTom) mice resulted in specific and effective gene recombination in IBCs/IPhCs in all regions of the cochlea (apex, 25.9 ± 4.1%; middle, 71.9 ± 2.3%; base, 86.1 ± 4.2%; mean ± SEM; statistical differences exist only between the apex and the other cochlear turns) ( Fig.…”

Section: Significancementioning

confidence: 99%

“…To determine the consequences of neonatal IBC and IPhC loss on the mature organ of Corti, we ablated these cells in vivo using an inducible diphtheria toxin fragment A (DTA) transgenic approach (25). Unexpectedly, we found that when these IHC supporting cells are eliminated immediately after birth, they are replaced efficiently within days.…”

mentioning

confidence: 99%

Spontaneous regeneration of cochlear supporting cells after neonatal ablation ensures hearing in the adult mouse

Lagarde

Wan

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Supporting cells in the cochlea play critical roles in the development, maintenance, and function of sensory hair cells and auditory neurons. Although the loss of hair cells or auditory neurons results in sensorineural hearing loss, the consequence of supporting cell loss on auditory function is largely unknown. In this study, we specifically ablated inner border cells (IBCs) and inner phalangeal cells (IPhCs), the two types of supporting cells surrounding inner hair cells (IHCs) in mice in vivo. We demonstrate that the organ of Corti has the intrinsic capacity to replenish IBCs/IPhCs effectively during early postnatal development. Repopulation depends on the presence of hair cells and cells within the greater epithelial ridge and is independent of cell proliferation. This plastic response in the neonatal cochlea preserves neuronal survival, afferent innervation, and hearing sensitivity in adult mice. In contrast, the capacity for IBC/IPhC regeneration is lost in the mature organ of Corti, and consequently IHC survival and hearing sensitivity are impaired significantly, demonstrating that there is a critical period for the regeneration of cochlear supporting cells. Our findings indicate that the quiescent neonatal organ of Corti can replenish specific supporting cells completely after loss in vivo to guarantee mature hearing function.deafness | cell ablation | hair cell | organ of Corti | glia

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“…The main stumbling block will be achieving a high enough level of expression in an inducible manner. To study the requirement for a certain cell type, the combination of GIFM and cellular phenotyping alleles harboring LoxP-STOP-diphtheria toxic (DT) A fragment or its receptor (e.g., Buch et al, 2005;Ivanova et al, 2005) will allow for the ablation of a genetically defined population of cells (Fig. 3D).…”

Section: Future Prospects For Using Gifm To Analyze Cell Morphology mentioning

confidence: 99%

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

Joyner

Zervas

2006

Developmental Dynamics

172

167

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A fascinating aspect of developmental biology is how organs are assembled in three dimensions over time. Fundamental to understanding organogenesis is the ability to determine when and where specific cell types are generated, the lineage of each cell, and how cells move to reside in their final position. Numerous methods have been developed to mark and follow the fate of cells in various model organisms used by developmental biologists, but most are not readily applicable to mouse embryos in utero because they involve physical marking of cells through injection of tracers. The advent of sophisticated transgenic and gene targeting techniques, combined with the use of site-specific recombinases, has revolutionized fate mapping studies in mouse. Furthermore, using genetic fate mapping to mark cells has opened up the possibility of addressing fundamental questions that cannot be studied with traditional methods of fate mapping in other organisms. Specifically, genetic fate mapping allows both the relationship between embryonic gene expression and cell fate (genetic lineage) to be determined, as well as the link between gene expression domains and anatomy (genetic anatomy) to be established. In this review, we present the ever-evolving development of genetic fate mapping techniques in mouse, especially the recent advance of Genetic Inducible Fate Mapping. We then review recent studies in the nervous system (focusing on the anterior hindbrain) as well as in the limb and with adult stem cells to highlight fundamental developmental processes that can be discovered using genetic fate mapping approaches. We end with a look toward the future at a powerful new approach that combines genetic fate mapping with cellular phenotyping alleles to study cell morphology, physiology, and function using examples from the nervous system. Developmental Dynamics 235:2376 -2385, 2006.

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

Cited by 225 publications

References 40 publications

Population of sensory neurons essential for asthmatic hyperreactivity of inflamed airways

Population of sensory neurons essential for asthmatic hyperreactivity of inflamed airways

Spontaneous regeneration of cochlear supporting cells after neonatal ablation ensures hearing in the adult mouse

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

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