Abstract
Mutations in the DMD gene cause Duchenne Muscular Dystrophy, which is characterised by muscle wastage that progresses to a life limiting phenotype. Emerging evidence suggests that the onset of disease happens in early development which challenges the current understanding of the pathogenesis. The established animal models are either not suitable for developmental studies or do not fully recapitulate the human disease phenotype. Therefore, this project aimed to develop a novel model in Xenopus tropicalis, which has proved to be valuable in disease modelling and understanding developmental mechanisms.The DMD gene is 2.5 Mb long and encodes multiple isoforms, whose expression pattern is tightly regulated. Using CRISPR/Cas9 technology, isoform-specific knockout models were developed to discover which of these had early developmental roles. Mosaic crispant frogs were able to be grown with mutations in all isoforms (dystrophin-null), the full-length isoform (FL20) or the shortest isoform (Dp71-null). Adult juveniles of FL20 and dystrophin-null showed muscle wastage, a tissue phenotype with the typical characteristics of dystrophic muscle in DMD patients and a poor fertility rate.
The progeny of Dp71-null animals did not show a significant phenotype, whereas dystrophin- null embryos died early on in development and the progeny of FL20 animals showed a more progressive phenotype. These tadpoles had shorter anterior-posterior axes, craniofacial defects, kinked tails and delayed migration of melanophores, which were more marked in the homozygotes. There was clear evidence for loss of collagen fibre structure in their tails. As they grew into juvenile frogs, they showed signs of weight loss and kyphosis. Histological analysis of their skeletal muscle showed severe fibrosis, invasion of inflammatory cells and regenerating myofibres. This dystrophic phenotype was confirmed by the absence of dystrophin protein expression.
This shows that the disease initiates during embryonic stages and that the full-length isoform plays a role in early development.
| Date of Award | 1 Jul 2025 |
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| Original language | English |
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| Supervisor | Darek Gorecki (Supervisor) & Matt Guille (Supervisor) |