We modified a murine BAC containing the Pax7 locus by replacing the Pax7 coding sequence from exon 1 with a sequence encoding ZsGreen, and we generated transgenic mice by pronuclear injection. To confirm whether green fluorescence recapitulated expression of Pax7, we analyzed the ZsGreen expression pattern during embryonic development. Clearly detectible green signal was observed in the somites, frontonasal processes, and the neural tube, as early as 9.5 days of gestation (Fig. 1A). The intensity of the signal increased at embryonic day 10.5 and following, and the signal remained localized to the defined Pax7-expressing areas . To further validate specificity, we evaluated expression in various adult tissues. Skeletal muscle, heart, lung, spleen, liver, intestine, bladder, uterus, and kidney were enzymatically digested to single-cell suspensions and analyzed by flow cytometry. Fluorescent cells were detected only in skeletal muscle (data not shown), and they were homogeneous in size and granularity (Fig. 1B). At 1 month of age, the frequency of positive cells within the mononuclear cell fraction from total hind limb muscles digests averaged 0.8%. Pax7-ZsGreen+ cells from the diaphragm displayed the same small size and low granularity as those from the hind limb and were present at half the frequency (Fig. 1B). To further confirm that ZsGreen expression mirrored that of Pax7, we sorted 10,000 positive and negative cells from 3-month-old male mice (n = 3) and investigated Pax7 expression by reverse transcription (RT)-PCR. We found expression of Pax7 only in the ZsGreen+ fraction (Fig. 1C). Analyses of other myogenic transcripts, satellite cell markers, and genes characteristic of quiescent cells showed that green cells expressed significantly more Myf5, MyoD, Syn4, and Lbx1 compared with the negative cells (Fig. 1C). The expression levels of CD34, Syn1, Msx1, Foxk1, Foxo1, and Foxo3 in the positive cells were comparable to those in the negative cells. Many of these markers are expected to be expressed by nonmuscle cells (e.g., CD34 by endothelial cells, Foxo factors by various quiescent cells). Finally, significant overexpression of Sca1 and CXCR4 was detected in negative cells. These results correlate with the fluorescence-activated cell sorting analyses, which showed that a high percentage of Pax7neg mononuclear cells were labeled with CD34, CXCR4, and Sca1 (Figs. 1C, 3). In addition, basal (very low) expression of Pax3 was detected in both negative and positive fractions. (Fig. 1C) [24, 29, 30]. We evaluated muscle sections but were not able to detect ZsGreen by immunohistochemistry, a result that we attribute in part to the quality of the available ZsGreen antibodies.
Figure Figure 1.. ZsGreen fluorescence recapitulates Pax7 expression. (A): ZsGreen expression in embryos from E9.5 to E12.5. Note that green florescence is exclusively localized in the somite, frontonasal processes, and the neural tube. (B): Fluorescence-activated cell sorting profile of muscle digests from Pax7-ZsGreen mice. The upper left panel shows the FSC/SSC profile of total cells. The upper right panel shows fluorescence of live cells (gated using propidium iodide, not shown) in the FSC/SSC gate indicated at the left. Green fluorescence is shown on the x-axis, and red fluorescence is shown on the y-axis. This uncompensated two-dimensional representation allows maximum separation of autofluorescent cells, which track along the diagonal, from the weak true green fluorescent population, which is shifted to the right of the diagonal. The lower panel shows the FSC/SSC profile of total live cells in the ZsGreen gate indicated. Note that green cells were homogeneous in size (FSC) and granularity (SSC). (C): Real-time reverse transcription-polymerase chain reaction on ZsGreen+ or -neg cells directly sorted from hind limb muscle of 3-month-old male mice. Results are presented as fold difference comparing ZsGreen-positive with -neg cells; data represent mean ± SE (n = 3). Abbreviations: E, embryonic day; FSC, forward scatter; neg, negative; SSC, side scatter.
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We further tested the ZsGreen-positive and -negative sorted fractions by culturing sorted cells in vitro under conditions that allow expansion of myogenic progenitors (medium containing serum, chick embryonic extract, and bFGF). ZsGreen+ sorted cells plated on a gelatin-coated surface expanded as small round cells, which grew into three-dimensional colonies and sporadically fused to form multinucleated myotubes (Fig. 2A). On the other hand, cells from the negative fraction exhibited a fibroblastic morphology and grew strongly attached to the plastic (Fig. 2A). When medium was switched to promote differentiation, ZsGreen+ sorted cells fused and formed large numbers of typical multinucleated myotubes and a residual population of undifferentiated cells, whereas negative-sorted cells did not form myotubes (Fig. 2A). The myogenic character of the ZsGreen+ sorted fraction and lack thereof in the ZsGreenneg fraction was supported by RT-PCR analyses of these cultures for specific myogenic genes (Fig. 2B). To further evaluate the myogenic potential of the Pax7-ZsGreen population, we established single-cell clones by sorting individual ZsGreen+ cells directly from muscle digests into gelatin-coated 96-well dishes. All clones that arose (192 of 588 wells) had the morphology described above. On the other hand, under the same culture conditions, ZsGreenneg cells were not able to expand from single cells (Fig. 2D). To evaluate the frequency of myogenic progenitors in the ZsGreen-negative fraction, we therefore plated increasing numbers of cells in each well and found that at 500 cells per well, approximately one-third of wells had growth after 8 days. We then analyzed individual cultures derived from single ZsGreen+ cells or 500 ZsGreenneg cells by immunostaining for Pax7, MyoD, or MyHC (Fig. 2C, 2D). All of the clones expanded from ZsGreen+ cells contained a population of MyoD- and MyHC-positive cells, and 79% had some Pax7-positive cells (Fig. 2D). On the other hand, the majority of the cultures (90%) obtained from the negative cells were nonmyogenic, based on the absence of immunoreactivity for Pax7, MyoD, or MyHC (Fig. 2D). This means that although the negative fraction does have some myogenic progenitors, the positive fraction is approximately 5,000-fold enriched.
Figure Figure 2.. Direct isolation of satellite cells from Pax7-ZsGreen mice. (A): Morphology of ZsGreen+ and ZsGreenneg sorted cells from limb and diaphragm of 3-month-old male and female mice, 3 (top row) and 9 (middle row) days after sorting and expansion in proliferation medium. Bottom row: Morphology of the expanded cells after 2 days in differentiation medium. Note that only ZsGreen+ cells differentiated into myotubes. (B): Real-time reverse transcription-polymerase chain reaction for myogenic genes in Pax7-Zsgreen+ (+) or Pax7-Zsgreen-neg sorted cells expanded in vitro for 7 days in proliferation medium. (C): Immunofluorescence for Pax7, MyoD, and MyHC (in red) in clones obtained from single ZsGreen+ cells. DAPI (blue) was used for counterstaining of the nuclei. Clones were expanded on gelatin-coated surfaces in myogenic proliferation medium for 8 days. (D): Quantification of the plating efficiency of ZsGreen+ and ZsGreenneg sorted cells (top panel). Note that the ZsGreenneg cells could not expand from single cells. All of the colonies obtained from the ZsGreen+ cells were myogenic based on the presence of Pax7-, MyoD- or MyHC-expressing cells within the clone (middle panel). The bottom panel represents the percentage of Pax7-, MyoD-, and MyHC-pos cells within each clone derived from ZsGreen+ cells. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MyHC, myosin heavy chain; neg, negative; pos, positive.
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