Mouse chick chimera: a new model to study the in ovo developmental potentialities of mammalian somites.

Chimeras were prepared by transplanting somites from 9-day post-coïtum mouse embryos or somitic dermomyotomes from 10-day post-coïtum mouse embryos into 2-day-old chick embryos at different axial levels. Mouse somitic cells then differentiated in ovo in dermis, cartilage and skeletal muscle as they normally do in the course of development and were able to migrate into chick host limb. To trace the behavior of somitic myogenic stem cells more closely, somites arising from mice bearing a transgene of the desmin gene linked to a reporter gene coding for Escherichia coli beta-galactosidase (lacZ) were grafted in ovo. Interestingly, the transgene was rapidly expressed in myotomal muscles derived from implants. In the limb muscle mass, positive cells were found several days after implantation. Activation of desmin nls lacZ also occurred in in vitro cultures of somite-derived cells. Our experimental method facilitates investigation of the mechanisms of mammalian development, allowing the normal fate of implanted mouse cells to be studied and providing suitable conditions for identification of descendants of genetically modified cells.

Interactions between spinal cord stimulation and activity blockade in the regulation of synaptogenesis and motoneuron survival in the chick embryo.

The present study investigated the effects of spinal cord stimulation, neuromuscular blockade, or a combination of the two on neuromuscular development both during and after the period of naturally occurring motoneuron death in the chick embryo. Electrical stimulation of the spinal cord was without effect on motoneuron survival, synaptogenesis, or muscle properties. By contrast, activity blockade rescued motoneurons from cell death and altered synaptogenesis. A combination of spinal cord stimulation and activity blockade resulted in a marked increase in motoneuron death, and also altered synaptogenesis similar to that seen with activity blockade alone. Perturbation of normal nerve-muscle interactions by activity blockade may increase the vulnerability of developing motoneurons to excessive excitatory afferent input (spinal cord stimulation) resulting in excitotoxic-induced cell death.

Influence of spinal cord stimulation on the innervation pattern of muscle fibers in vivo.

In chick embryo, chronic stimulation of the brachial spinal cord at a fast rhythm from days 7 to 18 of development induced an increase in AChE activity sites and ACh receptor (AChR) clusters in slow anterior latissimus dorsi (ALD) muscle. Most AChR clusters and AChE spots were contacted by nerve endings. A previous study showed that such spinal cord stimulation causes changes in ALD muscle properties, especially the appearance of a high proportion of fast type II fibers (Fournier Le Ray et al., 1989). Analysis of the synaptic pattern in different fiber types of experimental ALD muscle indicated a decrease in the distance between successive AChE spots in slow type III fibers compared to controls, whereas the intersynaptic distance in fast type II fibers was very similar to that in the rare fast fibers developing in control ALD. Fast fibers of experimental muscles exhibited less AChR than did slow fibers. The increased number of neuromuscular junctions in ALD muscle after spinal cord stimulation appeared to be preferentially located in slow fibers. Electron microscopy showed no change in the number of axons in ALD nerve after spinal cord stimulation. The activity imposed on brachial motoneurons apparently caused terminal sprouting of ALD nerve in target muscle, thus accounting for the increase in neuromuscular contacts in ALD muscle fibers. Differences in the distribution of nerve contacts indicate that the type of muscle fiber innervated may play a critical role in the synaptic pattern during chick embryogenesis.

Change in motor neurone activity modifies the differentiation of a slow muscle in chick embryo.

Slow-tonic anterior latissimus dorsi (ALD) muscle properties were studied following chronic spinal cord stimulation in chick embryo. Stimulation at a fast rhythm was applied from day 7, 8 or 10 of development until the end of embryonic life. When stimulation was applied from day 7 up to day 18 of development, ALD muscle exhibited at day 18 a large decrease in half time to peak of tetanic contraction, a large proportion of fast type II fibres and an increase in fast myosin light chain content as compared to control muscle. When stimulation started at day 8 of development, changes in properties of ALD muscle were reduced when compared to the previous experimental series. Indeed, no fast type II fibres were observed within the muscle, even when stimulation was prolongated until the 20th day of embryonic development. In addition, chronic stimulation at a fast rhythm initiated at day 10 of development did not modify ALD muscle differentiation. The present results indicate that a fast pattern of motor neurone activity can induce some slow-to-fast transformations of ALD muscle fibres. However, after the first week of embryonic life, ALD myotubes appeared refractory to these transformations. The possible mechanisms responsible for the transformation of slow myotubes and for their further loss of plasticity are discussed.

Effects of low and high frequency patterns of stimulation on contractile properties, enzyme activities and myosin light chain accumulation in slow and fast denervated muscles of the chicken.

The effects of denervation and direct stimulation in fast and slow latissimus dorsii muscles were investigated in chicken. In slow ALD muscle, denervation resulted in an incompleteness of the relaxation, a decrease in MDH and CPK activities and an increase in fast myosin light chains (MLC) accumulation. Direct stimulation at either fast or slow rhythm prevented the effects of denervation on relaxation and CPK activity but was ineffective on MDH activity and fast MLC accumulation. Moreover, direct stimulation of denervated ALD caused rhythm-dependent change in tetanic contraction. In fast PLD muscle, the main changes in muscle properties following denervation were a slowing down of the time course of the twitch and an incompleteness of the relaxation, a decrease in LDH and CPK activities and in LC3F accumulation. Stimulation at a high frequency partly prevented the effects of denervation and resulted in a large accumulation of LC3F, while a low frequency stimulation did not restore the twitch time to peak, increased MDH activity and induced synthesis of slow MLC. This study emphasizes the role of muscle activity and its pattern in some properties of slow and fast chicken muscles following denervation.

Effect of spinal cord stimulation on the metabolism of developing latissimus dorsii muscles in chick embryo.

Influence of chronic spinal cord stimulation upon some characteristic enzyme activities of energy metabolism was investigated in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii muscles of the chick embryo. During embryonic life, oxidative metabolism (as evaluated by the activity of malate dehydrogenase (MDH] represents the main energetic pathway in both slow and fast muscles. At the end of embryonic life, an increase in anaerobic (as evaluated by the activity of lactate dehydrogenase (LDH] and creatine phosphokinase (CPK) activities occurs in PLD muscle. Chronic spinal cord stimulation at a low frequency was performed from the 10th day to the 16th day of embryonic development. In ALD, the enzyme activities were unaffected, while in PLD a concomitant decrease in LDH and CPK activities was observed.