Live cell tracking assay of migratory muscle progenitors (n = 5) isolated from forelimb tissue of E12.5 Lbx1^{EGFP/+}|Pitx2^{+/+} (WT), Lbx1^{EGFP/+}|Pitx2^{LacZ/+} (HET), or Lbx1^{EGFP/+}|Pitx2^{LacZ/LacZ} (MUT) embryos. Migration pathway of migratory muscle progenitors over a 2 hour period for WT (**A**), HET (**B**), MUT (**C**). **(D)** Mean total distance travelled of WT (53±4 micrometer), HET (74±10 micrometer), and MUT (23±1 micrometer). (**E**) Mean velocity of movement of WT (0.5±0.1 micrometer/min), HET (0.6±0.1 micrometer/min) and MUT (0.2±0.02 micrometer/min). (**F**) Mean time spent moving vs. paused for WT moving (92±16.4 min) and paused (32±16.8 min), HET moving (101±7.4 min) and paused (24±7.4 min) and MUT moving (59±14 min) and paused (66±14 min). Using Dunnett's ANOVA test setting WT as control, the MUT MMPs were found to be significantly different in distance travelled, velocity, and time moving vs. paused. Following Dunnett's ANOVA an unpaired T-test between WT and MT determined significance values for distance traveled (p = 0.0001), velocity (p = 0.0001), time moving (p = 0.0089) and time paused (p = 0.0082). (**G**) Quantitation of persistent migratory directionality. Relative ratios of direct distance from start point to end point (D) divided by the total pathway distance traveled (T), ratios expressed as relative to WT (value set = 1.0). The MMPs from HET had a ratio of 63% and MUT MMPs had a ratio of 127%. (**H**) The mean square displacement of total pathway distance traveled (T^{2}) measured every 20 min. The x-intercept HET (diamonds, black dotted line) and WT (light grey squares, solid light grey line) cells were as close to the origin as the intercept for MUT (dark grey triangles, solid dark grey line), indicating that cells from all genotypes exhibit similar migration behaviors.

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