NEUROFILAMENTS HELP MAINTAIN NORMAL MORPHOLOGIES AND SUPPORT ELONGATION OF NEURITES IN XENOPUS-LAEVIS CULTURED EMBRYONIC SPINAL-CORD NEURONS

Neurofilament number and subunit composition, which are highly regulat ed during development, have been proposed to help regulate axonal diam eter and stability. From experiments on dissociated cell cultures of X enopus laevis embryonic spinal cord, we have obtained direct evidence that neurofilaments help maintain the structural integrity of newly de veloping axons. An anti-neurofilament monoclonal antibody specific for Xenopus NF-M and the cell lineage tracer, lysinated FITC-dextran, wer e coinjected into a single blastomere of 2-cell stage embryos. Within neurons descended from the injected blastomere, this antibody specific ally confined neurofilaments to the cell body for the first two days o f culture, as assayed by immunocytochemical staining with antiserum ag ainst the low molecular weight neurofilament protein XNIF. Although wh ole IgGs and Fab fragments both affected neurofilament distribution, t he whole IgGs were more effective. For the first 9 hr of culture, neur ites containing anti-NF-M developed normally. By 21 hr, they were shor ter than those of sibling control neurons within the same dish, and ma ny became morphologically abnormal. Defects included large variations in diameter, poorly defined separations between the growth cone and ne urite, and more collateral branching. Despite these abnormal features, neurons containing anti-NF-M had normal distributions of alpha-tubuli n immunoreactivity and phalloidin-stained F-actin. These latter observ ations argued that defects resulted from the absence of neurofilaments rather than from interference of the movement of other structural mat erials essential for axonal growth. These results support the hypothes is that neurons use neurofilaments to help maintain the characteristic shapes of axons against the increasing structural demands placed upon the elongating process.