Ectopic ovine Dlk1 inhibits expression of endogenous Dlk1 and affects myostatin expression pattern.

<p>A. Endogenous Dlk1 expression was detected during regeneration using rabbit-anti-murine FA1 antibody and days 3–9 following stab wound are represented. Anti-human FA1 antibody cross-reacts with the ovine form of Dlk1/FA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060692#pone.0060692-Davis1" target="_blank">[8]</a>. Days 3–7 following stab wound are represented. A staining of uninjured LC control muscle confirms that the staining of the ovine form is specific. Scalebar: 50 µm. In LC control mice endogenous Dlk1 is expressed in mononuclear cells located within the injury and primarily observed along the periphery of newly formed myotubes (arrows). In Dlk1 TG mice the expression of Dlk1 is strongly inhibited within the muscle injury and only few mononuclear cells expressing Dlk1 can be observed (arrows). Ectopic ovine Dlk1 is expressed only in the TG mice and not by LC mice. The expression is under control by the myosin light chain promoter and is primarily expressed by myotubes and mature muscle fibers (arrows). B. qPCR analysis of endogenous <i>Dlk1</i> mRNA confirms the down regulation by ectopic ovine Dlk1. Dlk1 was included in the TaqMan™ Low Density Array Card set-up and calculated as described in materials and methods. Black squares: Dlk1 TG mice +/− SD. Open circles: LC control mice. C. RT-PCR analyses of the Dlk1 splice variants present during regeneration. The primers used span exon 5 which contain all splice sites of Dlk1 and theoretically yields bands corresponding to the A (824 bp), B (671 bp), C/C₂ (605/599 bp) and D/D₂ (545/539 bp) forms. Pituitary gland (PG) is used as positive control. This analysis shows that the splice variants present are full-length A, C/C₂ and confirms the down regulation of endogenous Dlk1 by ectopic Dlk, with all splice variants being equally affected. D. Immunohistochemical analysis of myostatin expression in Dlk1 TG and LC mice. Scalebar representative for all images: 20 µm. Myostatin expression is generally patchy and variable both in TG and LC mice, but TG mice present a less intense staining.