Change in Nox4 expression is accompanied by changes in myogenic marker expression in differentiating C2C12 myoblasts.

Myoblast differentiation is mediated by a cascade of changes in gene expression including transcription factors such as myogenin. Subsequent to myoblast differentiation, there is an increase in expression of the transmembrane protein NADPH oxidase (Nox). Nox is one of the primary factors for the generation of reactive oxygen species (ROS) in myogenic (C2C12) cells. Recently, ROS have been shown to be important regulators of several intracellular signaling pathways, and the full extent of their regulatory roles is yet to be discovered. In the present study, qRT PCR analysis demonstrated that Nox4 isoform is primarily expressed in differentiating C2C12 cells and contributes to the generation of ROS in C2C12 myoblast during differentiation. Over-expression and silencing of Nox4 expression during myoblast differentiation was accompanied by a reduction in intracellular ROS concentrations and an alteration in the expression patterns of Myf5, Pax7, MyoD1, and myogenin. This modulation was found to be associated with ERK1/2 phosphorylation. In both over-expression and reduced expression of Nox4, we found significant reductions in ERK1/2 phosphorylation. This indicates that cellular differentiation may be affected by Nox4-mediated endogenous ROS generation. These data suggest a new opportunity to study the temporal expression of Nox4 in the generation of ROS accompanying changes in myogenic differentiation.

Positionally selective growth of embryonic spinal cord neurites on muscle membranes.

Motor neurons from distinct positions along the rostrocaudal axis generally innervate muscles or muscle fibers from corresponding axial levels. These topographic maps of connectivity are partially restored after denervation or transplantation under conditions in which factors of timing and proximity are eliminated. It is therefore likely that motor neurons and some intramuscular structures bear cues that bias synapse formation in favor of positionally matched partners. To localize these cues, we studied outgrowth of neurites from embryonic spinal cord explants on carpets of membranes isolated from perinatal rat muscles. Neurites from rostral (cervical) and caudal (lumbar) spinal cord slices exhibit distinct growth preferences. In many instances, rostrally derived neurites grew selectively on membranes from forelimb muscles or from a single thoracic muscle (the serratus anterior) when given a choice between these membranes and membranes from hindlimb muscles or laminin. Caudally derived neurites almost never exhibited such rostral preferences, but instead preferred membranes from hindlimb muscles or a single hindlimb muscle (the gluteus) to rostral muscles or laminin. Likewise, spinal neurites exhibited distinct position-related preferences for outgrowth on membranes of clonal myogenic cell lines derived from specific rostral and caudal muscles. Taken together these results suggest that the membranes of motor axons and myotubes bear complementary labels that vary with rostrocaudal position and regulate neuromuscular connectivity.

A morphological technique for exploring neuromuscular topography expressed in the mouse gluteus maximus muscle.

Motor neuron pools innervate muscle fibers forming an ordered topographic map. In the gluteus maximus (GM) muscle, as well as additional muscles, we and others have demonstrated electrophysiologically that there exists a rostrocaudal distribution of axon terminals on the muscle surface. The role of muscle fiber type in determining this topography is unknown. A morphological approach was designed to investigate this question directly. We combined three different methods in the same muscle preparation: (1) the uptake of activity-dependent dyes into selected axon terminals to define the spinal segmental origin of a peripheral nerve terminal; (2) the fluorescent labeling of nicotinic acetylcholine receptors to determine motor endplate size; (3) the immunocytochemical staining of skeletal muscle to determine fiber subtype. We applied these methods to the mouse GM muscle to determine the relationship between muscle fiber type and the topographic map of the inferior gluteal nerve (IGN). Results from this unique combination of techniques in the same preparation showed that axon terminals from more rostral spinal nerve segments of origin are larger on rostral muscle fibers expressing myosin heavy chain (MyHC) IIB epitope than caudal type IIB fibers. Because type IIB fibers dominate the GM, this suggests that for these rostral axons terminal size is independent of fiber type. How this axon terminal size is related to the topographic map is the next question to be answered.

Ephrin-A5 overexpression degrades topographic specificity in the mouse gluteus maximus muscle.

Motor neurons project onto specific muscles with a distinct positional bias. We have previously shown using electrophysiological techniques that overexpression of ephrin-A5 degrades this topographic map. Here, we show that positional differences in axon terminal areas, an entirely different parameter of neuromuscular topography, are also eliminated with ephrin-A5 overexpression. Therefore, we now have both morphological and electrophysiological approaches to explore the mechanisms of neuromuscular topography.

Morphometric analysis of neuromuscular topography in the serratus anterior muscle.

Groups of neurons form ordered topographic maps on their targets, and defining the mechanisms that develop such maps, and re-connect them after disruption, has biological as well as clinical importance. The neuromuscular system is an accessible and well-studied model for defining the principles that guide map formation, both during its development and its reformation after motor nerve damage. We present evidence for the expression of this map at the level of nerve terminal morphology and muscle fiber type in the serratus anterior muscle. Morphometric analyses indicate, first, a rostrocaudal difference in nerve terminal size depending on the ventral root of origin of the axons. Second, motor endplates are larger on type IIB than type IIA muscle fibers. Third, whereas IIB muscle fibers are distributed rather evenly along the rostrocaudal axis of the muscle, the more rostral type IIB fibers are preferentially innervated by anteriorly derived (C6) motor neurons, and more caudal IIB fibers are preferentially innervated by posteriorly derived (C7) motor neurons. This inference is supported by analysis of the size of nerve terminals formed in each muscle sector by rostral and caudal roots, and by evidence that the larger terminals are on IIB fibers. These results demonstrate a subcellular expression of neuromuscular topography in the serratus anterior muscle (SA) muscle in the form of differences in nerve terminal size. These results provide deeper insights into the organization of a neuromuscular system. They also offer a rationale for a topographic map, that is, to allow spinal motor centers to activate selectively different compartments within a muscle.

Branching patterns of the rat phrenic nerve during development and reinnervation.

Previous studies have shown that the phrenic motor nucleus in the rat projects onto the diaphragm muscle, forming an orderly topographic map. Moreover, this topography is partially restored upon reinnervation. This orderly map is expressed prior to birth, suggesting that early contacts between nerve and muscle are topographically appropriate. The phrenic divides during embryonic development into rostral and caudal branches, and motor axons preferentially enter the appropriate branch. In an effort to understand the mechanisms that underlie the choices growing phrenic neurons make in selecting their appropriate muscle targets, we examined the patterns of branching displayed by the phrenic nerve during development and reinnervation. In all muscles studied the phrenic nerve splits into three primary branches, rostral, caudal, and crural. At a coarse level the pattern of branching of the phrenic is remarkably consistent from animal to animal and at all ages of development. At a finer level of resolution, however, there is an asymmetry between right and left hemidiaphragms. Moreover, the precise emergence of any particular branch is unpredictable, resulting in an overall incongruence in branching architecture from animal to animal. The hemidiaphragm muscle grows unevenly, particularly on the right side, resulting in greater muscle fiber elongation medially. Upon reinnervation, the same coarse pattern of branching is reestablished, but the higher order pattern is much simpler and muscle growth is slower than in controls. These results suggest that very early in development primary branches of the phrenic funnel axons into three well-defined zones in the muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Regeneration by skeletomotor axons in neonatal rats is topographically selective at an early stage of reinnervation.

The seven sectors of the rat's serratus anterior (SA) muscle are innervated topographically by motor neurons of spinal cord segments C6 and C7 whose axons travel in the long thoracic (LT) nerve. That pattern is roughly mapped early in development and gains its final precision postnatally. The segmentotopic pattern is reestablished better in neonates than adults after cutting the LT nerve. We examined the process of reinnervation to see whether segmental selectivity is reestablished at the outset or whether it arises by rearrangement of the regenerated axons. Recordings were made from muscle fibers 1 to 70 days following a cryogenic lesion of the LT nerve done within 48 h of birth, as well as from sham-operated and unoperated control rats. Reinnervation of all sectors of SA occurred within a week after freezing the nerve. Reinnervation by C6 and C7 motor neurons was topographically selective though not quite to the degree found in controls. The precision observed during the first week of reinnervation did not improve over the next 9 weeks. Thus, selectivity exists from the start rather than being a more random reinnervation subsequently sharpened by elimination of inappropriate connections. The number of muscle fibers innervated by both C6 plus C7 motor neurons was greater after reinnervation than in controls. There was a significant decrease in the percentage of these dually innervated fibers over the initial few weeks of reinnervation but there was no difference among the reinnervated sectors of SA. Reinnervation of SA under optimal conditions resembles normal development in that there is a degree of topographic selectivity of (re)innervation that is present even at the earliest time periods studied. Unlike normal development the topographic selectivity after neonatal reinnervation does not improve over time, and fibers receiving a dual segmental innervation are not preferentially located in sectors where there is the most overlap in segmental projection.

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites.

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.