Date of Award

2015

Document Type

Thesis - ECU Access Only

Publisher

Edith Cowan University

Degree Name

Doctor of Philosophy

School

School of Natural Sciences

Faculty

Faculty of Health, Engineering and Science

First Supervisor

Dr Annette Koenders

Second Supervisor

Dr Angus Stewart

Abstract

The development and regeneration of skeletal muscle is fuelled by muscle stem/progenitor cell biogenesis. The ability of these cells to survive and contribute to muscle growth and regeneration is dependent on master regulator genes, Pax3 and Pax7. These genes control key aspects of cell behaviour by their regulation of, and interactions with, other genes and proteins.

Exactly how the Pax7 protein recognises and regulates specific genes to effect appropriate muscle progenitor behaviour is not fully understood. However as for other Pax genes, which similarly regulate the development and maintenance of different tissue lineages, this is likely to be linked to the production of multiple splice forms.

Surprisingly little is known about 1) the contribution of Pax7 at the later, foetal, stages of muscle development and, 2) Pax7 splice forms to the regulation of muscle stem cell biogenesis in both developing and adult muscles. This gap in knowledge was the impetus for this PhD research.

As the roles of many Pax genes are specific to the stage of cell biogenesis as well as the status of the tissue in which the cell is found, an in vivo approach was taken to characterise the natural history of Pax7 transcript expression and investigate the relationship of Pax7 with the powerful myogenic inducer, MyoD.

Using mouse models of development and muscle maintenance and regeneration, this work suggests that the functional diversity of Pax7 in normal foetal and adult myogenesis in vivo is due to a factor other than changes in the relative proportions of alternate paired box transcripts.

A second key finding of this work was the demonstrated expression of an alternate transcript, Pax7A, in the early mouse embryo as well as foetal muscle and brain, arising from proximal polyadenylation and splicing. Dynamic expression during foetal myogenesis suggests this mechanism may be functionally important during this time. Computational analyses predicted that Pax7A lacks miRNA control elements in the 3’UTR, leading to the hypothesis that changes in the ratios of Pax7A to Pax7B could affect the known reciprocal inhibition between Pax7 and MyoD.

Endogenous levels of Pax7 and MyoD proteins were investigated to explore the spatio-temporal contribution of Pax7 to foetal limb myogenesis. This work showed that Pax7 and MyoD are co-expressed in foetal limb muscles and that the Pax7 and MyoD expression profile is highly likely to play a role in myogenic progression of foetal precursors in vivo, as reported for adult satellite cells. Further, temporal expression patterns suggest that MyoD protein can be downregulated in Pax7 precursors in foetal muscles. This could be related to the specification of satellite cells to the sublaminar position that occurs under the guidance of Notch, which is known to inhibit MyoD-mediated differentiation. It could be important that these cells do not differentiate during this time.

Lastly, to investigate if Pax3 cells may also regulate muscle progenitor biogenesis during normal foetal myogenesis, spatio-temporal expression of endogenous Pax3 protein in situ was qualitatively analysed. Whilst Pax3 is mostly absent, a novel finding is that endogenous Pax3 protein may be re-expressed in some foetal limb muscles.

In conclusion, this thesis reveals new insights into the contributions of Pax7 and its transcripts to adult and foetal myogenesis in vivo, particularly of the foetal limb.

LCSH Subject Headings

Muscle cells.

Myogenesis.

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