Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis 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.

A Role for Intermediate Filaments in Determining and Maintaining the Shape of Nerve Cells

“…In contrast, peripherin-positive cells extended neurites that were at least four to five times longer (Table 2). These observations are also supported by the findings that there are dramatic increases in peripherin expression during the initiation and outgrowth of axons that take place in early developing nervous systems of mammals, amphibians, and fish (Troy et al, 1990b;Gervasi et al, 2000;Undamatla and Szaro, 2001). Similarly, peripherin expression increases significantly during NGF-induced neurite outgrowth in PC12 cells (Parysek and Goldman, 1987; Aletta et al, 1988;Troy et al, 1990a).…”

“…These two structural forms, thought to be prescursors to long IF, are found in all regions of growing neurites, including the central and peripheral domains of growth cones (Helfand et al, 2003). Peripherin expression has also been correlated with the initiation and outgrowth of axons that take place within the developing nervous systems of vertebrate animals (Gervasi et al, 2000;Troy et al, 1990b;Undamatla and Szaro, 2001 onal regeneration, while at the same time NF protein expression decreases (Oblinger et al, 1989b). The resulting regenerating peripherin-rich axons are of notably smaller diameter relative to mature axons.…”

A crucial role for hnRNP K in axon development inXenopus laevis

“…Each culture was prepared from a single embryo and grown at 22.5°C on 35 mm polystyrene dishes (Δ plastic, Nunclon Nalge International), as described (Tabti and Poo, 1991;Undamatla and Szaro, 2001). …”

Post-transcriptional regulation mediated by specific neurofilament introns in vivo

“…The test construct expresses a red fluorescent protein (DsRed2) and contains the cis-regulatory regions of interest, whereas the reference plasmid expresses a green fluorescent protein (glGFP) to serve both as a control for selecting successfully injected embryos and as a normalization factor in subsequent biochemical analyses. In the current study, injected embryos were raised to late tailbud stages (stages 43-44), by which time they are completely transparent, and Xenopus nefm exhibits a mature expression pattern (Szaro et al, 1989;Szaro and Gainer, 1988;Undamatla and Szaro, 2001).…”

“…light (NEFL), medium (NEFM), and heavy (NEFH) neurofilament proteins] responds to cues encountered by growing axons (Schwartz et al, 1990;Undamatla and Szaro, 2001;Zhao and Szaro, 1995). For example, during developmental and regenerative axon outgrowth, increased expression that is stimulated by axons making appropriate contacts is principally due to dynamic changes in nucleocytoplasmic export, stability, and translation of neurofilament RNAs, as opposed to changes in gene transcription (Ananthakrishnan et al, 2008;Ananthakrishnan and Szaro, 2009;Schwartz et al, 1994).…”