Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin

Abstract

Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5Cre-driven GFP expression, we identified in addition to the expected GFP+ myogenic cells (presumably satellite cells), a second dominant GFP+ population distinguished as being Sca1+, non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5Cre-driven reporter when compared to the MyoDCre driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5Cre-driven GFP+ cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP+ cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5Cre-driven GFP+ cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.

Uncoupling protein 1 expression in adipocytes derived from skeletal muscle fibro/adipogenic progenitors is under genetic and hormonal control

“…However, recent studies showed that Myf5 can be more widely distributed than previously appreciated and also labels white adipocytes 28. Moreover, a subpopulation of Myf5 + cells from extraocular muscle expresses Sca1 and is adipogenic 50. In our study, we relied on a prospective isolation strategy to obtain MPs from skeletal muscle by FACS based on the absence of CD31, CD45, and Sca1 and the presence of alpha 7‐integrin, which results in highly pure myogenic cultures 35.…”

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

“…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)].…”

“…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). A typical EOM preparation for histology comprised the EOMs, the retractor bulbi muscle (an ocular accessory muscle involved in retracting the eye into the orbit forcing the mouse third eyelid across the cornea), the optic nerve and the associated periocular connective tissues, all attached to the eyeball (LifeMap, 2013), while for cell isolation only the EOMs were collected.…”

“…Harvested tissues were dissociated according to our two established methods that rely either on Pronase digestion (Danoviz and Yablonka-Reuveni, 2012; Yablonka-Reuveni, 2004) or collagenase/dispase digestion (Ieronimakis et al, 2010; Stuelsatz et al, 2014). When digested with Pronase, the isolated muscles were thoroughly cleaned while gently tearing the muscles apart into small pieces with forceps.…”

“…From this point, two different protocols, detailed in our previous publications, were used to isolate SCs by flow cytometry (Day et al., 2007; Stuelsatz et al, 2014). When working with Pronase-digested preparations from Nestin-GFP mice, the Hoechst-labeled cells were directly sorted based on GFP expression (Day et al, 2007).…”