Development of Stepping Figure 4. FT onset exhibited multiple trends. Data for an E18 (A) and E20 experiment (B) are shown. A1: FT bursts began in the first half of 4 TA cycles (3.960.5 Hz), accompanied by a 2nd burst in cycle 3 (q). A2: In the next RLM 30 s later, FT bursts began in the latter half of TA cycles 2–4 (3.060.8 Hz). A3: FT onset and cycle duration (top) moderately co-varied (slope = 0.59, R2 = 0.48). Relative onset (bottom) was broadly distributed at all frequencies. 55% of FT bursts began in the latter half of the TA cycle. B1: FT bursts began in the first half of cycles 2–6 (4.260.6 Hz). B2: FT onset weakly varied with cycle duration (slope = 0.33, R2 = 0.33) and 84% of FT bursts began in the first half of the TA cycle. doi:10.1371/journal.pone.0006111.g004 burst durations did not vary with TA cycle duration. Both slope and R2 were approximately 0.0 in 89% of all burst duration analyses (N = 76 regressions, E18 and E20 combined). Foot-free Following removal of egg shell anterior to the foot (foot-free), E20 embryos extended the leg beyond the egg and produced larger joint excursion ranges during some RLMs (9 embryos). Compare the joint angle amplitudes for 2 RLMs from a single experiment during control (Figure 5A) and foot-free conditions (Figure 5B), noting differences in scale. There was also an increase in cycle frequencies greater than 6 Hz. However, few differences were observed in EMG patterns between conditions. For example, in the experiment shown, SA burst onset latencies mostly fell between 0–0.1 s, weakly varying with cycle duration during both conditions (Figures 5A1, 5B1). Similar SA results were obtained in 6 of 6 foot-free experiments. LG burst onset varied closely with cycle duration during control and foot-free RLMs (Fig. 5A2, 5B2). LG onset closely varied with cycle duration and a late relative onset was the predominant pattern in 4 of 6 foot-free experiments. Participation rates for foot-free experiments (Figure 5C) were also similar to E20 control data (Figure 1D). FT onset was more likely to vary closely with TA cycle duration during foot-free experiments (5 of 7) than E20 control experiments (5 of 10). For example, FT burst onsets for the control data in the experiment shown in Figure 5 suggested there were 2 recruitment patterns (open and closed circles) across the entire range in cycle duration (Figure 5A3, left plot). The relative onset plot (Figure 5A3, right plot) revealed that the burst onsets sorted as early (open circles) and late (closed circles) in the TA cycle. The onset latency for early bursts was weakly associated with TA cycle duration; whereas, onset for late bursts was strongly associated. During foot- free RLM, early bursts were no longer observed at lowest RLM frequencies (Figure 5B3, right plot). Collectively, FT exhibited a distinct relative onset bias (.60% of sample) in 9 of 10 control experiments, and a preference for early onset (7 experiments). In contrast, only 4 of 7 foot-free experiments exhibited an FT relative onset bias, 2 early and 2 late (See Tables S2 and S3 for detailed summary comparisons). FT onset was also more likely to overlap TA activity during foot-free RLMs (33611% of cycles) compared PLoS ONE | www.plosone.org 5 July 2009 | Volume 4 | Issue 7 | e6111