Precocious Locomotor Behavior Begins in the Egg: Development of Leg Muscle Patterns for Stepping in the Chick Young U. Ryu, Nina S. Bradley* Division of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles, California, United States of America Abstract Background: The chicken is capable of adaptive locomotor behavior within hours after hatching, yet little is known of the processes leading to this precocious skill. During the final week of incubation, chick embryos produce distinct repetitive limb movements that until recently had not been investigated. In this study we examined the leg muscle patterns at 3 time points as development of these spontaneous movements unfolds to determine if they exhibit attributes of locomotion reported in hatchlings. We also sought to determine whether the deeply flexed posture and movement constraint imposed by the shell wall modulate the muscle patterns. Methodology/Principal Findings: Synchronized electromyograms for leg muscles, force and video were recorded continuously from embryos while in their naturally flexed posture at embryonic day (E) 15, E18 and E20. We tested for effects of leg posture and constraint by removing shell wall anterior to the foot. Results indicated that by E18, burst onset time distinguished leg muscle synergists from antagonists across a 10-fold range in burst frequencies (1–10 Hz), and knee extensors from ankle extensors in patterns comparable to locomotion at hatching. However, burst durations did not scale with step cycle duration in any of the muscles recorded. Despite substantially larger leg movements after shell removal, the knee extensor was the only muscle to vary its activity, and extensor muscles often failed to participate. To further clarify if the repetitive movements are likely locomotor-related, we examined bilateral coordination of ankle muscles during repetitive movements at E20. In all cases ankle muscles exhibited a bias for left/right alternation. Conclusions/Significance: Collectively, the findings lead us to conclude that the repetitive leg movements in late stage embryos are locomotor-related and a fundamental link in the establishment of precocious locomotor skill. The potential importance of differences between embryonic and posthatching locomotion is discussed. Citation: Ryu YU, Bradley NS (2009) Precocious Locomotor Behavior Begins in the Egg: Development of Leg Muscle Patterns for Stepping in the Chick. PLoS ONE 4(7): e6111. doi:10.1371/journal.pone.0006111 Editor: Michael Hendricks, Harvard University, United States of America Received May 12, 2009; Accepted June 8, 2009; Published July 3, 2009 Copyright: 2009 Ryu, Bradley. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NIH National Institute of Child Health and Human Development grant R01 HD-053367 awarded to NS Bradley. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nbradley@usc.edu Introduction Chicks emerge from the egg after 21 days of incubation equipped to walk, swim and airstep [1–3]. Chicks begin moving embryonic day (E) 3. The movement (e.g., embryonic motility) is spontaneously generated throughout embryogenesis until hatching [4–6], which appears to be the only sensory-triggered behavior during embryonic development [5,7,8]. Embryonic motility is episodically generated by a recurrently connected excitatory network within the spinal cord that is transiently silenced by activity-dependent depression [9,10]. Motility is initially driven by acetylcholine, then glutamate by E8-E10 [11]. The temporal features of the activity play an instructive role in motor neuron pathfinding [12,13], and possibly the flexor-extensor and interlimb alternations for stepping [14]. However the relationship between the early network for motility and the locomotor network is uncertain [15,16]. Leg movements during motility at E9 are characterized by alternating flexion and extension muscle synergies [17,18] and joint excursions [19,20]. Yet, electromyographic (EMG) and kinematic patterns appear to break down between E12 and E15 [17,19,21]. Recent studies indicate that a distinctly different pattern of repetitive leg movements (RLMs) emerges between E15 and E18 [22,23]. RLMs are also composed of alternating flexion and extension, but the frequency range (1–10 Hz) far exceeds that for E9 motility (0.2–2 Hz). Interestingly, the RLM frequency range is similar to the combined ranges for three locomotor forms in hatchlings: walking, swimming and airstepping [2]. Together these findings raise the possibility that RLMs are an essential link between early motility and locomotion. Leg muscles also express membership in either the flexor synergy for leg protraction (swing) or the extensor synergy for leg retraction (stance) during walking, swimming and airstepping [2]. Variations in muscle burst patterns across these behaviors appear to be due to differences in limb loading. For example, extensor burst duration exhibits a close association with step cycle duration during stance (walking), but a weak association during buoyancy (swimming) and limb suspension (airstepping). Differences in knee PLoS ONE | www.plosone.org 1 July 2009 | Volume 4 | Issue 7 | e6111