Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffinembedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

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“…3B). Putative sites of active transcription (Ruf-Zamojski et al, 2015) appear as two bright dots in some nuclei (Fig. 3A and Fig.…”

Section: Resultsmentioning

confidence: 99%

Mapping a multiplexed zoo of mRNA expression

et al. 2016

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“…We questioned whether a mobile-bound pool can also be found in endogenous zebrafish Dystrophin, where a cytoplasmic pool of the protein is not known. We have analysed Gt(dmd-Citrine) ct90a zebrafish embryos, in which Citrine was inserted by gene-trap into the endogenous Dystrophin locus, creating fluorescently tagged zfDys ( Trinh et al, 2011 ; Ruf-Zamojski et al, 2015 ).…”

Section: Resultsmentioning

confidence: 99%

“…In human embryos, Dystrophin first appears in the cytoplasm, at the tips of myotubes, then becomes widespread throughout the myofibres in foetal stages ( Wessels et al, 1991 ; Clerk et al, 1992 ; Chevron et al, 1994 ; Mora et al, 1996 ; Torelli et al, 1999 ). In embryonic zebrafish muscle, Dystrophin transcripts are reported to accumulate initially in the cytoplasm, and from 24 hr post fertilization (hpf) until early larval stages, Dystrophin protein and transcripts are primarily located at muscle fibre tips ( Bassett et al, 2003 ; Guyon et al, 2003 ; Jin et al, 2007 ; Böhm et al, 2008 ; Ruf-Zamojski et al, 2015 ). In both species, Dystrophin becomes localised under the sarcolemma in maturing and adult muscle fibres where it concentrates at costameres, neuromuscular and myotendinous junctions ( Samitt and Bonilla, 1990 ; Miyatake et al, 1991 ; Chambers et al, 2001 ; Guyon et al, 2003 ).…”

Section: Introductionmentioning

confidence: 99%

In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

Bajanca

González-Pérez

Gillespie

et al. 2015

eLife

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Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics.DOI: http://dx.doi.org/10.7554/eLife.06541.001

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“…We questioned whether a mobile-bound pool can also be found in endogenous zebrafish Dystrophin, where a cytoplasmic pool of the protein is not known. We have analysed Gt(dmd-Citrine) ct90a zebrafish embryos, in which Citrine was inserted by gene-trap into the endogenous Dystrophin locus, creating fluorescently tagged zfDys (Trinh et al, 2011;Ruf-Zamojski et al, 2015). We first searched for signs of cytoplasmic Dystrophin.…”

Section: Endogenous Zebrafish Dystrophin Diffusion and Binding Dynamicsmentioning

confidence: 99%

Decision letter: In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

2015

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Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmd ta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt (dmd-Citrine) ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics.

show abstract

Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line

Cited by 20 publications

References 58 publications

Mapping a multiplexed zoo of mRNA expression

Mapping a multiplexed zoo of mRNA expression

In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

Decision letter: In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

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