Early effects of embryonic movement: 'a shot out of the dark'

Abstract

It has long been appreciated that studying the embryonic chick in ovo provides a variety of advantages, including the potential to control the embryo's environment and its movement independently of maternal influences. This allowed early workers to identify movement as a pivotal factor in the development of the locomotor apparatus. With an increasing focus on the earliest detectable movements, we have exploited this system by developing novel models and schemes to examine the influence of defined periods of movement during musculoskeletal development. Utilizing drugs with known neuromuscular actions to provoke hyperactivity (4-aminopyridine, AP) and either rigid (decamethonium bromide, DMB) or flaccid (pancuronium bromide, PB) paralysis, we have examined the role of movement in joint, osteochondral and muscle development. Our initial studies focusing on the joint showed that AP-induced hyperactivity had little, if any, effect on the timing or scope of joint cavity elaboration, suggesting that endogenous activity levels provide sufficient stimulus, and additional mobilization is without effect. By contrast, imposition of either rigid or flaccid paralysis prior to cavity formation completely blocked this process and, with time, produced fusion of cartilaginous elements and formation of continuous single cartilaginous rods across locations where joints would ordinarily form. The effect of these distinct forms of paralysis differed, however, when treatment was initiated after formation of an overt cavity; rigid, but not flaccid, paralysis partly conserved precavitated joints. This observation suggests that 'static' loading derived from 'spastic' rigidity can act to preserve joint cavities. Another facet of these studies was the observation that DMB-induced rigid paralysis produces a uniform and specific pattern of limb deformity whereas PB generated a diverse range of fixed positional deformities. Both also reduced limb growth, with different developmental periods preferentially modifying specific osteochondral components. Changes in cartilage and bone growth induced by 3-day periods of flaccid immobilization, imposed at distinct developmental phases, provides support for a diminution in cartilage elaboration at an early phase and for a relatively delayed influence of movement on osteogenesis, invoking critical periods during which the developing skeleton becomes receptive to the impact of movement. Immobilization also exerts differential impact along the proximo-distal axis of the limb. Finally, our preliminary results support the possibility that embryonic hyperactivity influences the potential for postnatal muscle growth.

Fig. 1

Diagram to demonstrate the scope to influence distinct phases of cavitation in particular joints. This provides details on the days of gestation, the relative stages of chick development as well as the time at which cavitation commences in the knee (stifle), tibiotarsal (TT) and metatarsophalangeal (MTP) joints. Also shown are the ‘treatment windows’ (stages 36–39 and 39–41) that allow distinct phases of cavitation (before, during or after) of these joints to be selectively targeted. In most studies, treatment with drugs used to evoke immobilization has started relatively early during development and only the relatively long-term effects of treatment have been examined.

Fig. 2

Diagrammatic representation of distinct targeting strategies used to treat embryonic chicks to establish the effects of flaccid immobilization on hind-limb development. (a) Dosing strategy used to establish the short- and long-term effects of flaccid, relaxed paralysis (induced by daily treatment with pancuronium bromide) on skeletal development. Treatment commenced at stage 36 (day 10) and effects examined in chicks at stages 37, 40 and 44 (days 11, 14 and 18, respectively). (b) Dosing strategy used to establish the stage at which the effects of pancuronium bromide-induced flaccid paralysis exert their impact on skeletal development. Treatment commenced at stages 26, 43, 37 and 40 (days 5, 8, 11 and 14, respectively) and the effects on development examined at stage 44 (day 18). Also shown for reference is the stage at which tibial osteogenesis commences. Together, these ‘treatment windows’ (a and b) will allow the phase at which flaccid immobilization exerts influence on particular facets of skeletal development to be determined.

Fig. 3

AP induces rapid and sustained embryonic hyperactivity. Changes in the duration of limb movement (s min⁻¹) at various times after treatment of stage 36 chick embryos with a single in ovo dose of 4-aminopyridine (AP). Control (Tyrode's solution, black circles) and AP-induced hyperactivity (green circles). Data are expressed as mean ± SEM (n = 3 for each treatment).