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

Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ^(ct90a)). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and “flip” in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation

Dmd-citrine expression

<p>Wide-field fluorescent images of Dmd-Citrine expression in Gt(dmd-citrine)ct90a embryo in the trunk skeletal muscles at 24hpf</p

Dmd-citrine expression

<p>Wide-field fluorescent images of Dmd-Citrine expression in Gt(dmd-citrine)ct90a embryo in the trunk skeletal muscles at 24hpf</p

Dmd-citrine localize to the myosepta

<p>Antibody staining for Dmd-Citrine(green) and Tropomyosin(red) in ct90aGT embryo showing localization of Dmd-citrine to the myosepta.</p

Gt(dmd-citrine)ct90a line enable visualization of differential expression in heterozygous and homozygous embryo via in situ HCR

<p>In situ HCR staining of tpm3 (red), dmd (blue) and dmd-citrine (green) transcript in heterozygous ct90aGT embryo.</p