Myogenic Progenitors in the Rat Hindlimb: Role of Innervation

Development of a functional relationship between nerve and muscle is important both embryonically and throughout postnatal life. A breakdown of this relationship can lead to muscle wasting and dysfunction, to motor neuron death and may be involved in diseases such as the muscular dystrophies. In order to better understand the relationship between developing muscle and nerve, the rat was used as an animal model to investigate the timing of entry into the limb bud of muscle progenitor cells expressing the paired homeobox genes Pax3 and Pax7 - which are early markers of the limb myogenic lineage. Additionally, the timing of nerve outgrowth into the hindlimb bud was also assessed, in order to ascertain the spatial and temporal relationships between early muscle progenitors and limb innervation. Following this analysis of the normal dynamics of nerve and muscle progenitors within the hindlimb bud, the effect of an in utero denervation, using β-bungarotoxin (β-BTX), on the development of muscle progenitors in the tibialis anterior (TA) and extensor digitorum longus (EDL) was also carried out. Behaviour of the progenitors were first examined qualitatively and quantitatively, using immunohistochemistry. Finally, qPCR analysis was conducted on denervated and control muscles, to ascertain the more global effects of the loss of innervation on the TA and EDL muscles. In normal embryos, immunohistochemical examination showed that Pax3+ve progenitors were distributed throughout the proximal margins of the hindlimb from embryonic day (E) E12.5, whereas Pax7+ve progenitors appeared at E13.5, concurrent with the entry of nerve. Pax7+ve cells were first seen in the central region of the limb, surrounding nerve as it entered the limb. Pax7 immunoreactivity then rapidly appeared within the Pax3+ve muscle masses. Pax3+ve cells proliferated at a steady rate (approximately 30% of cells labeled by a 1 hr BrDU pulse) until Pax3 expression was lost by E16.0. In contrast, proliferation of Pax7+ve cells was initially low (approximately 30%), then accelerated to over 50% at E16.0 & E16.5. Denervation in utero by injection of β-BTX into the embryonic peritoneum at either E15.5 or E16.5, followed by immunohistochemical examination of extensor digitorum longus (EDL) and tibialis anterior (TA) muscles either 24 hrs or 48 hrs later, revealed a significant decrease in the number of Pax7+ve progenitors compared to controls at all time points assessed. Concurrently, after denervation there was a significant increase in the number of cells expressing myogenin (a marker of muscle differentiation) and a slight but significantly increased apoptosis of cells at various stages of myogenic development (assessed by active caspase-3 labeling). Quantitative PCR analysis corroborated these findings, with many genes associated with differentiation and apoptosis being upregulated following denervation, while genes associated with proliferation and cell cycle regulation being downregulated. In this analysis, the most profound effects on gene expression were seen after 48 hrs of denervation from E15.5, with tissues then analysed at E17.5, a time corresponding to the commencement of secondary myogenesis in the rat. From these results it can be concluded that the balance between proliferation, differentiation and survival of Pax7+ve myogenic progenitors is critically dependent on developmental interactions with the muscle nerve