Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions.

Abstract

Abstract. After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890. This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in norreal muscle.Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli/~-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogeous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site.In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells >200 #m from synaptic sites were lacZ positive. These result indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.

Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions

“…The exact disposition of 'perisynaptic fibroblasts' is unclear from previous reports, since they were depicted diagrammatically to be near to, but not overlying, neuromuscular junctions (Murray and Robbins, 1982;Gatchalian et al, 1989;Weis et al, 1991). No discernible immunofluorescence of NMJ-capping cells was detected using Thy-1 antibody, although axons, which also express Thy-1 antigen were immunolabelled (Reynolds and Woolf, 1992;Feng et al, 2000;Van der Putten et al, 2000).…”

“…However, similar non-neural cells were identified in scanning electron micrographs by Desaki and Uehara (Desaki and Uehara, 1981), who described them as 'flattened stellate cells of unknown nature'. Other investigators recognised the association of fibroblast-like cells to NMJs (Murray and Robbins, 1982;Connor and McMahan, 1987;Connor, 1997;Caroni and Schneider, 1994) in their description of 'perisynaptic fibroblasts' (Covault and Sanes, 1985;Sanes et al, 1986;Connor and McMahan, 1987;Connor, 1997;Gatchalian et al, 1989;Weis et al, 1991). Perhaps perisynaptic fibroblasts and NMJ-capping cells are the same as the endothelial cells of Robertson (Robertson, 1956), but this remains unproven.…”

“…Previous data suggest that reactive perisynaptic fibroblasts express several adhesion molecules including tenascin-C, N-CAM, fibronectin and heparan sulphate proteoglycan and that perisynaptic fibroblasts might be one source of tenascin-C (Covault and Sanes, 1985;Sanes et al, 1986;Gatchalian et al, 1989;Weis et al, 1991). These molecules have also been implicated in nerve sprouting and regeneration (Cifuentes-Diaz et al, 2002).…”

“…This arose from a serendipitous initial finding: whole-mount immunofluorescent staining of skeletal muscle with a novel polyclonal antibody (2166) produced vivid cytoskeletal labelling of a sub-set of cells capping the NMJ but lying outside the synaptic basal lamina. These NMJ-capping cells may correspond to 'endoneurial cells' first identified in the neighbourhood of NMJs in pioneering ultrastructural studies of the NMJ by Robertson (Robertson, 1956), or to cells of similar appearance referred to by Weis et al (Weis et al, 1991) as 'perisynaptic fibroblasts'. However, the data here suggest a much tighter association of NMJ-capping cells than indicated by these previous studies.…”

Changing intracellular compartmentalization of β‐galactosidase in the ROSA26 reporter mouse during embryonic development: A light‐ and electron‐microscopic study

“…X-gal, which yields a brighter blue reaction product than Bluo-gal, is used for light-microscopic observations. Bluo-gal is applied for transmission electron microscopy because it produces a heavy electron-dense precipitate and is less diffusable than X-gal (Weis et al, 1991;Sekerkova et al, 1997). The outcome of the X-gal method varies according to various fixation factors (e.g., fixative type, fixation time, and tissue block size), washing protocols (frequency and duration), and incubation procedures (e.g., incubation time and temperature, and pH of the reaction buffer and X-gal) (Ave et al, 1997;Couffinhal et al, 1997;Weiss et al, 1997;Cheng et al, 1999;Weiss et al, 1999;SanchezRamnos et al, 2000;Gioglio et al, 2002).…”