Identification of genes directly responding to <italic>DLK1</italic> signaling in Callipyge sheep.

Background: In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of <italic>DLK1</italic> and <italic>RTL1</italic> results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to <italic>DLK1</italic> and <italic>RTL1</italic> signaling from the genes that change as the result of muscle specific effects. Results: The quantitative PCR results indicated that <italic>DLK1</italic> expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, <italic>RTL1</italic> was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including <italic>PARK7</italic>, <italic>DNTTIP1, SLC22A3, METTL21E</italic> and <italic>PDE4D,</italic> were consistently co-expressed with <italic>DLK1</italic>, and therefore were possible transcriptional target genes responding to <italic>DLK1</italic> signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in <italic>Dnttip1</italic> and <italic>Pde4d</italic> expression respectively. <italic>Myh4</italic> expression was significantly elevated in DLK1-treated myotubes, whereas the expression of <italic>Mettl21e</italic> was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on <italic>Park7</italic> expression. In addition, <italic>Park7</italic> and <italic>Dnttip1</italic> increased <italic>Myh4</italic> and decreased <italic>Myh7</italic> promoter activity, resemble to the effects of <italic>Dlk1</italic>. In contrast, expression of <italic>Mettl21e</italic> increased <italic>Myh7</italic> and decreased <italic>Myh4</italic> luciferase activity. Conclusion: The study provided additional supports that <italic>RTL1</italic> alone was insufficient to induce muscle hypertrophy and concluded that <italic>DLK1</italic> was likely the primary effector of the hypertrophy phenotype. The results also suggested that <italic>DNTTIP1</italic> and <italic>PDE4D</italic> were secondary effector genes responding to <italic>DLK1</italic> signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb. [ABSTRACT FROM AUTHOR]