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 Myf5(Cre)-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 Myf5(Cre)-driven reporter when compared to the MyoD(Cre) driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5(Cre)-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 Myf5(Cre)-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.

Keywords: Extraocular muscles; Fibro/adipogenic progenitors; Myf5; MyoD; Pax3; Pericytes and vascular smooth muscle cells; Satellite cells; Sca-1; Wnt1; XLacZ4.

Fig. 1. Histological overview of periocular tissue preparations from MyoD^Cre, Myf5^Cre and Pax3^Cre reporter lines

Cross sections were obtained from periocular preparations isolated from (A) MyoD^Cre × R26^mTmG, (B) Myf5^Cre × R26^mTmG and (C) Pax3^Cre × R26^mTmG mice. (A–C) Images shown are from cross sections at the level of the optic nerve, immediately posterior to the eye. OpN, optic nerveN, peripheral nerveR, rectus muscleO, inferior oblique muscleRB, retractor bulbi muscle. Scale bar, 200µm. For a schematic overview of the mouse eye with attached rectus, oblique and retractor bulbi muscles refer to LifeMap (2013).

Fig. 2. Flow cytometric profiles of Cre-driven GFP⁺ cells isolated from muscle tissues of (A) MyoD^Cre and (B–D’) Myf5^Cre reporter lines

(A) and (B) Representative FACS overlay histograms analyzing Sca1 staining (X-axis) within GFP⁺ cells isolated from EOM, LIMB (hindlimb muscles: tibialis anterior, extensor digitorum longus and gastrocnemius) and DIA (diaphragm) of (A) MyoD^Cre × R26^mTmG and (B) Myf5^Cre × R26^mTmG mice. All populations were analyzed from G0-G1 cells after depletion of CD31⁺ and CD45⁺ cells. In each histogram the distribution of GFP⁺ cells (Y-axis) is depicted in a normalized fashion as %of Max based on the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells. (C–D’) Detailed flow cytometric characterization of Myf5^Cre-driven GFP⁺ cells isolated from EOM. (C) and (C’) Representative FACS plots showing (C) GFP staining among all G0-G1 cells and subsequent gating of GFP⁺ cells, and (C’) the distribution of the GFP⁺ population according to staining for CD31 and CD45 (Y-axis) versus Sca1 (X-axis). (D, D’) Representative FACS plots showing (D) the distribution of G0-G1 cells according to staining for CD31 and CD45 (Y-axis) versus Sca1 (X-axis) and subsequent gating of CD31⁻/CD45⁻/Sca1⁺ cells and (D’) the distribution of the CD31⁻/CD45⁻/Sca1⁺ cells according to GFP staining. In all plots, %values indicate the frequency of the highlighted population out of the total parent population analyzed as indicated at the top of each panel. Histograms and plots are all from one representative experiment of at least 3 independent experiments, which showed no significant differences.

Fig. 3. Sca1^±/GFP⁺ cells isolated from EOM preparations of the Myf5^Cre reporter line distinctively give rise to myogenic and non-myogenic progeny, respectively

Representative images of day 7 and day 14 cultures of (A–B’) GFP⁺/Sca1⁻ cells, (C–D’) GFP⁺/Sca1⁺ cells, and (E–F’) GFP⁻/Sca1⁺ cells, isolated by FACS as shown in Fig. 2C–D’. All populations were sorted from G0-G1 cells after depletion of CD31⁺ and CD45⁺ cells. Inserts in the lower left corner of (D) and (F) represent higher magnification views of the regions delineated by a white box in each corresponding panel, depicting adipogenic cells that develop spontaneously in the cultures of the non-myogenic cells with time. Scale bar, 100µm.

Fig. 4. Histological detection of Myf5^Cre-driven GFP⁺ cells in connective tissues of periocular preparations

(A–B”) Cross sections from Myf5^Cre × R26^mTmG mice analyzed for GFP and Tomato fluorescence. (C–D’) Cross sections from wildtype mice labeled by double-immunofluorescence with anti-Sca1 and anti-lamininSca1 marked cells within the periocular connective tissues and laminin identified the basal lamina of individual myofibers, delineating the rectus muscle. Unlike the straightforward Sca1 immunostaining of wildtype tissue, Sca1 immunolabeling of tissue sections from the Myf5^Cre × R26^mTmG reporter line required using the far-red or the blue channels, but these did not provide effective detection. First, as the red fluorescence bleeds into the far-red channel, and as both the Tomato and Sca1 signals are localized to the cytoplasmic membrane, determination of Sca1 staining in far-red is unreliable. Second the use of a number of secondary antibodies conjugated with different types of blue fluorophores yielded only extremely faint label with a low signal-to-noise ratio. Images shown in the figure are from cross sections at the level of the eyeball, either (A-A”) and (C-C’) posterior, or (B-B”) and (D-D’) anterior to the attachment of the EOM to the eye. R, rectus muscle. In all images DAPI staining is shown in blue. Scale bars in (A) and (B), 100µm, and in (A”), (B”), (C) and (D), 50µm.

Fig. 5. Histological detection of Myf5^Cre-driven GFP⁺ cells in the vasculature of periocular preparations

(A-C’) Cross sections from Myf5^Cre × R26^mTmG mice at the level of the optic nerve, immediately posterior to the eye. OpN, optic nerveN, peripheral nerveR, rectus muscleRB, retractor bulbi muscle; V, blood vessel. (D) and (D’) A Cross section from a wildtype mouse labeled by double-immunofluorescence with anti-Sca1 and anti-αSMA, (D) shows Sca1 staining aloneSca1 marked both the smooth muscle cells in the region closer to lumen, coinciding with α-smooth muscle actin (αSMA) staining (Skalli et al., 1986), and the perivascular cells in the more external adventitial region. In all images DAPI staining is shown in blue. Scale bars in (A) and (B), 100µm, in (A’), (B’), and (D), 50µm, in (C), 25µm, and in (A”), 10µm.

Fig. 6. Histological and cell culture analyses of the double reporter mouse Myf5^Cre × R26^mTmG × XLacZ4 establish a connection between the vascular-associated (Myf5^Cre-driven) GFP⁺ cells and the mural cell marker XLacZ4

(A-B”) Cross-section from periocular preparation at the level of the optic nerve, immediately posterior to the eye, depicting (A) X-gal+- cells at the wall of a blood vessel close to a rectus muscle and (B-B”) a higher magnification view of the same blood vessel, identifying DAPI-stained cells at the vessel wall that co-express nuclear β-gal and GFP (white arrowheads). R, rectus muscleV, blood vessel. (C-D’) Representative images of day 14 cultures of (C-C’) GFP⁺/Sca1⁺ cells and (D-D’) GFP/Sca1⁺ cells after staining with X-gal. The two populations, isolated from EOM preparations, were sorted by FACS from G0-G1 cells after depletion of CD31⁺ and CD45⁺ cells. Inserts in the lower left corner of (C) and (D) represent higher magnification views of the regions delineated by a white box in each corresponding panel, depicting adipogenic cells that develop spontaneously in these cultures with time. Scale bars in (A) and (C), 50µm, and in (B), 25µm.

Fig. 7. Histological detection of Pax3^Cre-driven GFP⁺ cells in connective tissues and vasculature of periocular preparations

(A–E) Cross sections from Pax3^Cre × R26^mTmG mice at the level of the eye, either (A-A’) posterior, or (B-B’) anterior to the attachment of the EOM to the sclera, or (C-E) at the level of the optic nerve, immediately posterior to the eye. OpN, optic nerveN, peripheral nerveR, rectus muscleV, blood vessel. Noticeably, the connective tissues which originate from the neural crest (as discussed in the “Results”section) and most nervous tissues appeared to be from Pax3 lineage origin (GFP⁺), with the exception of the optic nerve (seen in D and D’) which derived from the neural ectoderm that form the optic stalk (Evans and Gage, 2005; Pei and Rhodin, 1970; Schwarz et al., 2000). In all images, DAPI staining is shown in blue. Scale bars in (A), (D) and (E), 25µm, in (B) and (C), 50µm.

Fig. 8. Histological and cell culture analyses of periocular and EOM preparations from the double reporter mouse Pax3^Cre × R26^mTmG × XLacZ4

(A) and (A’) Cross-section from periocular preparation at the level of the optic nerve, immediately posterior to the eye, depicts a blood vessel close to a rectus muscle identifying GFP⁺ cells at the vessel wall that co-express nuclear β-gal. R, rectus muscleRB, retractor bulbi muscleV, blood vessel. DAPI staining is shown in blue in (A’). (B-C’) Representative images of day 14 cultures of (B-B’) GFP⁻/Sca1⁻ cells and (C-C’) GFP⁺/Sca1⁺ cells after staining with X-gal. Cells were isolated from EOM preparations by FACS and the specified populations were isolated from G0-G1 cells depleted of CD31⁺ and CD45⁺ cells. Insert in the lower left corner of (C) represents a higher magnification view of the region delineated by a white box in each corresponding panel, depicting an adipogenic cell expressing the XLacZ4 transgene. Scale bars in (A), 50µm, and in (B) and (C), 100µm.