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. 2012 Apr 27;7(4):e35822. doi: 10.1371/journal.pone.0035822

Figure 1. Regulation of Shape and Size of Limb Muscle Anlagen by Pitx2.

Figure 1

(A–H) Whole mount X-gal staining of Pitx2LacZ/+ and Pitx2LacZ/LacZ knock-in mice from E10.5–E14.5. Pitx2 was expressed throughout muscle anlagen but not in epidermis, mesenchyme or bone anlagen. Most distal limb muscle anlagen were mildly deformed. Muscle groups are outlined. (I) Percentage of EGFP+ cells collected from Pax3Cre|ROSAEGFP|Pitx2LacZ/+ (HET) and Pax3Cre|ROSAEGFP|Pitx2LacZ/LacZ (MUT) embryos at E12.5 forelimb tissue dissociated into single cell suspension. The percentage of EGFP+ cells had a mean ± standard error of the mean (SEM) of 17±0.6% for HET (n = 8) and 11±1% for MUT (n = 7). This 26% reduction in EGFP+ cells in the MUT forelimb was considered to be significant using unpaired t-test with a p-value = 0.0001. (J) Example histograms of propidium iodide (PI) staining of HET and MUT EGFP+ cells isolated from forelimb tissue at E12.5. (K) Results of cell cycle analysis using PI staining showing distribution (mean ± SEM) of the EGFP+ cell population between HET (n = 5) and MUT (n = 4) during G1 (69±0.007% HET, 74±0.009% MUT); S (18±0.005% HET, 17±0.006% MUT); and G2 (13±0.003% HET, 9±0.015%MUT) phases. The increase in MUT cells during G1 phase was determined to be significant using unpaired t-test with a p-value  = 0.0102.