Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of <i>Xenopus laevis</i> embryonic spinal cord

Abstract

The molecular subunit composition of neurofilaments (NFs) progressively changes during axon development. In developing Xenopus laevis spinal cord, peripherin emerges at the earliest stages of neurite outgrowth. NF-M and XNIF (an alpha-internexin-like protein) appear later, as axons continue to elongate, and NF-L is expressed after axons contact muscle. Because NFs are the most abundant component of the vertebrate axonal cytoskeleton, we must understand why these changes occur before we can fully comprehend how the cytoskeleton regulates axon growth and morphology. Knowing where these proteins are localized within developing neurites and how their expression changes with cell contact is essential for this understanding. Thus, we examined by immunofluorescence the expression and localization of these NF subunits within dissociated cultures of newly differentiating spinal cord neurons. In young neurites, peripherin was most abundant in distal neuritic segments, especially near branch points and extending into the central domain of the growth cone. In contrast, XNIF and NF-M were usually either absent from very young neurites or exhibited a proximal to distal gradient of decreasing intensity. In older neurites, XNIF and NF-M expression increased, whereas that of peripherin declined. All three of these proteins became more evenly distributed along the neurites, with some branches staining more intensely than others. At 24 h, NF-L appeared, and in 48-h cultures, its expression, along with that of NF-M, was greater in neurites contacting muscle cells, arguing that the upregulation of these two subunits is dependent on contact with target cells. Moreover, this contact had no effect on XNIF or peripherin expression. Our findings are consistent with a model in which peripherin plays an important structural role in growth cones, XNIF and NF-M help consolidate the intermediate filament cytoskeleton beginning in the proximal neurite, and increased levels of NF-L and NF-M help further solidify the cytoskeleton of axons that successfully reach their targets.