The role of Delta-like protein 1 (DLK1) has been implicated in the muscle hypertrophy observed in DLK1 transgenic mice, callipyge sheep, mouse paternal uniparental disomy (pUPD) 12 and human pUPD14 syndromes. However, no study on DLK1 has been conducted in the avian species. The ultimate goal of this dissertation is to identify the role of chicken delta-like protein 1 (gDLK1) in skelelal muscle development, growth, and regeneration in poultry.
The objectives of the first study were to clone and sequence a full length of chicken DLK1 cDNA, and to investigate the developmental regulation of gDLK1 during embryonic muscle development and postnatal muscle growth. The cloning and sequencing data revealed that gDLK1 contains a total of 1,161 base pairs, encoding 386 amino acids. The similarities of gDLK1 nucleotide and protein sequences were over 50% compared to mammalian species. In addition, chickens only express a full-length gDLK1 in various tissues at different ages without the alternative splicing variants. The expression of gDLK1 gene in pectoralis major muscle was significantly higher in embryonic stages rather than posthatching stages during muscle development (P < 0.05). The expression pattern of gDLK1 was very similar to the expression patterns of myogenin and Pax7 genes, suggesting a close association with myogenic activities.
The objectives of the second study were to determine gDLK1 mRNA expression during primary muscle cell differentiation and during muscle regeneration after cold injury, as well as to compare gDLK1mRNA expression during skeletal muscle development in layers and broilers. In the chicken primary muscle cell culture, the peak expression of gDLK1 mRNA was highly associated with late stages of myogenesis after primary myoblast differentiation. During muscle regeneration, the induction of the gDLK1 gene consistently appeared at late stages of regenerative myogenesis. The expression data of gDLK1 associated with myogenic markers (Pax7, MyoD, and myogenin) and morphological data suggest that gDLK1 may be involved in the late myogenic stages of primary muscle cell differentiation and muscle regeneration. The gDLK1 mRNA in the muscle tissues was very abundant at embryonic ages, but decreased after hatching in both broiler and layer chickens. In addition, the relatively greater expression of the gDLK1 gene in the muscles of broilers compared to layers suggests that gDLK1 may serve as a new selection marker for high muscle growth in chickens.
Muscular dystrophic (MD) and Low Score Normal (LSN) chickens have a genetic muscle weakness, losing pectoralis major (PM) muscle function during muscle development and growth. Therefore, the objectives for the third study were to compare: 1) the morphological changes, and 2) the expression level and pattern of myogenic regulatory genes (MyoD, Pax7, myogenin, and MRF4) and the gDLK1 gene during muscle regeneration of control, LSN, and MD chickens with those of the control chickens. Both histological analysis and quantitative real-time PCR were used for this comparison and association studies. Histological section showed the delayed and impaired myofiber formation in LSN and MD chickens. A significantly high basal level of MyoD, Pax7, myogenin, and gDLK1 mRNA expression (P < 0.05) in LSN chickens suggests that muscle degeneration may have already progressed. LSN chickens had a delayed pattern of myogenin mRNA expression and the maintained expression of Pax7 and MyoD mRNA from D4 to D7 (P < 0.05); whereas, MD chickens had a significantly high peak expression of myogenin mRNA, a low peak expression of MRF4 mRNA, and a high peak expression of gDLK1 mRNA (P < 0.05). Although both LSN and MD chickens have delayed muscle regeneration, however, distinctive expression of stage-specific myogenic markers suggests that the regenerative mechanisms for their impaired muscles might be different.