Positionally Selective Growth of Embryonic Spinal Cord Neurites on Muscle Membranes

Wang, H.; Chadaram, Srinivas; Norton, Ann; Lewis, Renate; Boyum, J.; Trumble, William R.; Sanes, Joshua R.; Laskowski, Michael B.

doi:10.1523/jneurosci.19-12-04984.1999

Cited by 9 publications

(25 citation statements)

References 39 publications

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“…We have shown previously that embryonic spinal cord neurites prefer to grow on membranes of muscles from a similar axial position (Wang et al, 1999). These data suggest but do not prove that the ability of axons to distinguish ephrins on muscle membranes may help explain their ability to innervate selectively muscles or portions of muscles in vivo.…”

Section: Introductionmentioning

confidence: 79%

“…The general morphology and location of the labeled neurons were similar to those for spinal motor neurons at this stage of development in vivo, as were many of the physiological properties. In addition, we have previously shown that virtually all of the outgrowing neurites in such culturesstainwithacetylcholinetransferase( Fig.1F) (Wang et al, 1999). We have further increased the likely percentage of motor neurons in our spinal cord explants by using only the ventral halves of the spinal cord slices.…”

Section: Technical Limitations Of Selective Outgrowthmentioning

confidence: 96%

“…Within 24 h of their birth, the pups were anesthetized by putting them on ice until all movement ceased, usually 10 min. Then the pups were decapitated and placed in dissecting medium for muscle removal (2.0 M NaCl, 1.0 M KCl, 1.0 M MgCl 2 , 0.5 M CaCl 2 , 0.3 M HEPES, 0.011 mg/ml glucose, 0.3 mg/ml penicillin, and 0.1 mg/ml streptomycin in deionized water, pH 7.2-7.3) (Wang et al, 1999). The serratus anterior, gluteus maximus and diaphragm muscles were removed.…”

Section: P0 Muscle Membrane Preparationmentioning

confidence: 99%

“…Pictures of the cocultures were also taken at this point. The cocultures were stained by the neurofilament immunocytochemistry method using either the fluorescein-labeled secondary anti-rabbit IgG or the biotinylated HRP anti-rabbit IgG against the primary anti-neurofilament antibody, which is the IgG fraction of rabbit antiserum (Donoghue et al, 1996;Wang et al, 1999). The stained cocultures were observed under a Bio-Rad MRC 1000 argon/krypton laser confocal microscope using fluorescein and rhodamine optics as well as under transmitted light.…”

Section: Staining Of Neurites and Scoring Of Growth Preferencesmentioning

confidence: 99%

“…Thus, rostral portions of a motor pool form synaptic contact with fibers in rostral portions of a muscle, whereas caudal motor neurons synapse caudally (Brown and Booth, 1983a,b;Bennett and Lavidis, 1984;Wigston and Sanes, 1985;Laskowski and Sanes, 1987;Laskowski and High, 1989). This ability of spinal neurites to distinguish axial origin of muscle regions is inherent to muscle because membranes of transgenic muscle cell lines induce similar growth preferences (Wang et al 1999).…”

Section: Introductionmentioning

confidence: 99%

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Topographic Specificity within Membranes of a Single Muscle DetectedIn Vitro

Chadaram¹,

Laskowski²,

Madison³

2007

J. Neurosci.

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Spinal motor pools project to target muscles forming distinct rostrocaudal topographic maps during development and regeneration. To define the mechanisms underlying these neuromuscular maps we studied the preferential outgrowth of embryonic spinal cord neurites on muscle membranes from different axial positions and explored the role of ephrin A ligands. We found all five ephrin As (EphAs) expressed in serratus anterior, gluteus maximus and diaphragm muscles. In the diaphragm, four of the five ephrin As are expressed as a caudal to rostral gradient. When ephrin A function is disrupted in muscle membranes by deletion of glycosyl-phosphatidylinositol anchored ephrin A ligands with phosphatidylinositol-specific phospholipase C enzyme treatment or by blocking of ephrin A ligands with EphA fusion proteins, or by genetic manipulation leading to ephrin A2/A5 mutant mice, the spinal cord neurites loose their preference for the membranes of corresponding axial position; suggesting a significant role for ephrins in topographic choices made by growing motor neurons. To closely approximate topographic choices presented to embryonic neurites in vivo, neurites within the phrenic motor pool were challenged to make outgrowth choices on membranes of their normal target, the diaphragm muscle. We observed that neurites from rostral cervical segments (C 1 and C 2 ) prefer to grow on rostral diaphragm membranes; caudal cervical neurites (C 6 -C 8 ) choose caudal diaphragm membranes; a transition of positional preference occurs at C 4 and this ability is lost in ephrin A2/A5 mutant mice. These results demonstrate for the first time topographical outgrowth of axons from within a motor pool onto a single target muscle in vitro.

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Section: Introductionmentioning

confidence: 79%

Section: Technical Limitations Of Selective Outgrowthmentioning

confidence: 96%

Section: P0 Muscle Membrane Preparationmentioning

confidence: 99%

Section: Staining Of Neurites and Scoring Of Growth Preferencesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Topographic Specificity within Membranes of a Single Muscle DetectedIn Vitro

Chadaram¹,

Laskowski²,

Madison³

2007

J. Neurosci.

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Denervation and age modify neuromuscular positional selectivity

Chadaram¹,

Laskowski²

2003

J. Neurobiol.

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The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.

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

et al. 2001

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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.

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Positionally Selective Growth of Embryonic Spinal Cord Neurites on Muscle Membranes

Cited by 9 publications

References 39 publications

Topographic Specificity within Membranes of a Single Muscle DetectedIn Vitro

Topographic Specificity within Membranes of a Single Muscle DetectedIn Vitro

Denervation and age modify neuromuscular positional selectivity

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

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