A Contemporary Atlas of the Mouse Diaphragm

Abstract

The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this “contemporary atlas” can be further explored in the context of diaphragm pathology and genetic disorders.

Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency

“…1A), previously reported by us as a characteristic of SCs in LIMB and DIA (Day et al., 2007; Day et al, 2010; Fig. S1 in Stuelsatz et al, 2012). Pax7/Nestin-GFP co-expression was additionally established in freshly isolated SC preparations from EOM, LIMB and DIA (Fig.…”

“…S2). Also, in common with SCs in all muscle groups examined to date (Beauchamp et al, 2000; Day et al, 2010; Ono et al, 2010; Stuelsatz et al, 2012), myogenic progenitors in adult EOM are β-gal positive when analyzed using the Myf5 nLacZ knockin reporter mouse (Fig. S3).…”

“…Most studies were performed with adult mice (4–6 month old) but as indicated in the Results, some studies were done with younger (3-week old) or older (12–24 month old) mice, and unless otherwise noted, only males were used. The following strains (all described in our previous studies) were used for tissue and cell isolation: (i) Knockin heterozygous Cre males MyoD Cre [Myod1 tm2.1(icre)Glh , (Kanisicak et al, 2009)], and Pax3 Cre [(Pax3 tm1(cre)Joe /J, (Engleka et al, 2005)] were crossed with knockin reporter females R26 mTmG [Gt(ROSA)26Sor tm4(ACTB-tdTomato,–EGFP)Luo /J, (Muzumdar et al, 2007)] to generate F1 animals (Stuelsatz et al, 2012; Stuelsatz et al, 2014). (ii) Transgenic, heterozygous Nestin-GFP (Mignone et al, 2004) and MLC3F-nLacZ [MLC3F=muscle-specific myosin light chain 3F; AKA 3F-nlacZ-2E and Tg(Myl1-lacZ) 1Ibdml/J, (Beauchamp et al, 2000; Kelly et al, 1995)].…”

“…Hindlimb muscles (LIMB; consisting of tibialis anterior [TA], extensor digitorum longus [EDL] and gastrocnemius) and whole diaphragm (DIA; includes the costal and crural muscles and the central tendon) were harvested according to our routine procedures for cell isolation or tissue sectioning (Danoviz and Yablonka-Reuveni, 2012; Day et al, 2007; Stuelsatz et al, 2012). For the EOM we processed two types of preparations, one for tissue sections and one for cell isolation as detailed in our recent publication (Stuelsatz et al, 2014).…”

“…β-galactosidase (β-gal) activity in cells expressing lacZ reporter was detected by X-gal staining in cultured cells or whole TA muscles, as previously described (Day et al, 2010; Stuelsatz et al, 2012). Cell cultures were fixed in 2% PFA for 10 min at room temperature as described above for cell culture immunostaining.…”