PMID- 32724863 OWN - NLM STAT- PubMed-not-MEDLINE VI - 5 TI - Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo. PG - 76 CI - Copyright: © 2020 Hildyard JCW et al. LA - eng PT - Journal Article PL - England TA - Wellcome Open Res JT - Wellcome open research JID - 101696457 IS - 2398-502X (Print) LID - 10.12688/wellcomeopenres.15762.1 [doi] FAU - Hildyard, John C W AU - Hildyard JCW AUID- ORCID: https://orcid.org/0000-0003-2283-2118 AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. FAU - Crawford, Abbe H AU - Crawford AH AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. FAU - Rawson, Faye AU - Rawson F AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. FAU - Riddell, Dominique O AU - Riddell DO AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. FAU - Harron, Rachel C M AU - Harron RCM AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. FAU - Piercy, Richard J AU - Piercy RJ AUID- ORCID: https://orcid.org/0000-0002-4344-6438 AD - Department of Clinical Science and Services, Royal Veterinary College, London, Camden, London, NW1 0TU, UK. IS - 2398-502X (Linking) OTO - NOTNLM OT - Brain OT - DMD OT - DeltaE50-MD OT - Development OT - Dp140 OT - Dp427 OT - Dp71 OT - Dystrophin OT - Embryogenesis OT - Expression OT - In-situ hybridisation OT - Muscle OT - Nerve OT - RNAscope PMC - PMC7372313 LR - 20200928 DP - 2020 DEP - 20200423 AB - 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.