Ablation of the fetal mouse spinal cord: The effect on soleus muscle cytoarchitecture

Abstract

A technique is described whereby it is possible to surgically ablate the lumbosacral spinal cord of a developing mouse fetus without interfering with fetal viability. The lumbosacral spinal cords of 14-day in utero, 129ReJ mice were ablated with a Cooper Nd-YAG laser, and the fetuses, enclosed in their membranes and attached to the uterus by their placentae, were allowed to develop in the abdominal cavity of the dam. The cytoarchitecture and the temporal pattern of organogenesis of aneural soleus muscles were studied in spaced, serial, transverse, ultrathin sections of muscles of 16-and 18-day gestation and newborn (20-day gestation) mice. At the time of surgery, the soleus muscle was a discrete mass consisting of primary myotubes and a pleomorphic population of mononucleated cells. Axon bundles and blood vessels were found at the muscle's periphery, but had not penetrated throughout the muscle mass. The organogenesis of the aneural muscle was remarkably similar to that of the innervated soleus muscle (Ontell et al., Am J Anat 181: [267][268][269][270][271][272][273][274][275][276][277][278]1988). In the aneural muscle, as in the innervated muscle, significant numbers of secondary myotubes formed all along the lengths of primary myotubes. Moreover, the time course of myotube formation, the dynamics of cluster formation and cluster dispersal, and the ultrastructural appearance of the myotubes mimicked that observed in innervated muscle. The frequency of necrotic myotubes was no greater in the aneural muscle than in the innervated soleus muscle. Myotube maturation was similar in aneural and innervated soleus muscles until 18 days gestation. However, at birth, aneural myotubes appeared to be slightly less mature than innervated myotubes. Thus, the major morphogenic phenomena that characterize the development of the soleus muscle appear to be independent of innervation.

Modulation of contractile protein gene expression in fetal murine crural muscles: Emergence of muscle diversity

“…To determine whether differences exist in the program for contractile protein gene expression between primary and secondary myotubes, it will be necessary to evaluate fetal mouse muscles either with in situ hybridization using probes that provide increased resolution (as compared with 35S-labeled riboprobes) without sacrificing the sensitivity of the system, or with appropriate antibodies. In order to determine the effects of innervation on contractile protein modulation during the fetal period, we will carry out similar in situ hybridization studies with muscles derived from fetal mice whose lumbosacral spinal cord has been laser ablated during the embryonic period (Hughes et al, 1992).…”

Morphometric analysis of the developing, murine aneural soleus muscle

“…De novo myotube formation is reflected in the -60% increase in small-diameter (< 5 km) myotubes at the aneural muscle's widest girth over this interval. The extent of the de novo myotube formation is suggested, but not fully reflected, in the doubling of the number of myotubes found at the muscles' widest girths, because secondary myotubes are short and are distributed all along the lengths of the primary myotubes (Hughes et al, 1992). The observation that 97% of the myotubes that are < 5 km are found as cluster constituents confirms our observation (Hughes et al, 1992) that secondary myotubes form in aneural muscle (as in innervated muscle) using primary myotubes as scaffolds.…”

“…Comparison of the results of this study with a previous study of the morphogenesis of inner-16 days gestation diameter (pm) 20 days gestation (birth) diameter (pm) vated muscle (Ontell et al, 1988b) permits a quantitative assessment of the effect of the absence of innervation on normal muscle development. A descriptive study of the cytoarchitecture of the developing, aneural murine soleus muscle has been reported (Hughes et al, 1992).…”

“…These problems are avoided using a recently described denervation technique that permits laser ablation of lumbosacral spinal cords of mouse fetuses without interfering with fetal viability and development (Hughes et al, 1992). In a previous study (Hughes et al, 1992), the three-dimensional cytoarchitecture of the murine soleus muscle, rendered aneural by this technique, has been described. In the present study, a morphometric analysis of the aneural soleus muscle has been undertaken.…”

“…The use of P-bungarotoxin is inherently problematic as it is directly myotoxic (Creazzo and Sohal, 1981), and injected fetuses are not viable at birth (Harris, 1981;Condon et al, 1990). These problems are avoided using a recently described denervation technique that permits laser ablation of lumbosacral spinal cords of mouse fetuses without interfering with fetal viability and development (Hughes et al, 1992). In a previous study (Hughes et al, 1992), the three-dimensional cytoarchitecture of the murine soleus muscle, rendered aneural by this technique, has been described.…”