Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

Abstract

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

“…Supporting this, labelling of dystrophic mdx muscle with our probes ( Fig 4 ) revealed key differences to healthy muscle: prominent 5’ nuclear foci were readily detected, including within centrally-located nuclei characteristic of regenerated mdx muscle (see left side of Fig 4B ), but smaller punctate foci from both probe sets appeared greatly reduced in number, and were almost exclusively found within (or adjacent to) nuclei. Additionally, dystrophic muscle also exhibited rare nuclei associated with multiple 3’ foci alone ( Fig 4B , Arrows), a labelling pattern consistent with expression of the short dystrophin isoform dp71 (as we have shown previously [ 26 ]).…”

“…Interestingly, counts of 3’ foci were typically higher than 5’, by a small but consistent amount (50–100). The consistent size and intensity of 3’ foci allows this signal to be collected with greater ease than 5’ (where overexposure of the strong nuclear foci must be balanced against underexposure of the weaker sarcoplasmic foci, and 5’ foci in mature dp427 molecules close to nuclei may be lost amidst strong nascent 5’ staining), however 3’ foci without adjacent 5’ might also represent labelling of dp71 [ 26 ]. While not reported to be expressed in muscle fibres specifically, this isoform is expressed in blood vessel endothelia [ 49 ] and proliferating myoblasts [ 50 ], and is detected at low levels within whole muscle tissue homogenates, particularly those from young dystrophic muscle undergoing acute degeneration/regeneration [ 51 ].…”

“…Subcellular resolution of individual transcripts -and moreover discrete regions of those transcripts- has wider applications. Use of a third ‘middle’ probe alongside the 5’ and 3’ probe sets described here allows the full-length dp427 isoform to be distinguished from dp260/dp140, and in turn from dp116/dp71: we have used this ISH method to study expression of dystrophin isoforms during embryonic development [ 26 ], something historically precluded by shared sequence identity at both protein and mRNA level. A logical target for further investigations would be the brain, a complex tissue known to express a similarly complex combination of dystrophin isoforms: our ISH approach would allow these isoforms to be mapped to subcellular levels and might allow us to determine whether brain dp427 isoforms (dp427c, dp427p) are as short-lived as dp427m.…”

“…We reasoned that this approach might be further refined: at 14kb, a full-length dystrophin transcript should permit multiple 20ZZ probe sets to bind simultaneously (a hypothesis supported by Smith et al , using conventional fluorescent probes [ 25 ] to resolve nascent transcriptional loci within cultured myonuclei). We have previously confirmed the validity of this approach, using a triplex strategy (targeting 5’, middle and 3’ sequence of the dp427 transcript) to reveal expression of different dystrophin isoforms in the developing embryo [ 26 ]: dp427 transcripts in nascent muscle were labelled with all three probes, dp140 transcripts in the developing brain were labelled with only middle and 3’, while cell types known to be rich in the short dp71 isoform (such as epithelia) labelled with the 3’ probe alone.…”

Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy

“…Histological detection of dystrophin protein can be limited by the typically low levels of protein, close to or below the limit of detection, and indeed we were unable to discern the smaller dystrophin isoforms by immunolabelling. RNAScope offers extremely high specificity (target sequences of ~1000 bases) and sensitivity, with single mRNA transcripts resolved as punctate fluorescent foci with high signal to noise ratio (Hildyard et al, 2020a, Hildyard et al, 2020b. Furthermore, its capacity for multiplexing enables simultaneous detection of multiple target mRNA species each labelled with a distinct fluorophore.…”