Neurofilament content is correlated with branch length in developing collateral branches of Xenopus spinal cord neurons

During development, axons form interstitial collateral branches, which are initially dynamic but gradually stabilize as the projection sharpens. The initial outgrowth of collaterals is characterized by transitions in growth dynamics that occur at different lengths. Below 10 μm, collateral branches start out as unstable, thin filopodia. Above 30 μm, the branches stabilize. Although the relationship between branch length and the presence of microfilaments and microtubules has been well characterized, relatively less is known about the development of the neurofilament cytoskeleton in collateral branches. In the main axon, successive stages of outgrowth are accompanied by changes in the polypeptide composition of neurofilaments (NFs), which shifts from being rich in Type III neuronal intermediate filament proteins (nIFs) to progressively favoring Type IV subunits. To characterize the NF composition of developing collateral branches, antibodies to peripherin (a Type III nIF) and NF-M (a Type IV nIF) were used to stain newly differentiating embryonic Xenopus laevis spinal cord neurons in culture. In contrast to what happens in the main axon, staining for both subunits coincided in collaterals. Branches shorter than 10 μm seldom had NFs, whereas all branches longer than 30 μm did. In branches that had NFs staining either extended all the way to branch tip or terminated ∼10 μm from it. These lengths correspond remarkably well with lengths associated with branch stabilization. Given that NFs are the most stable of the cytoskeletal polymers, we speculate that they may contribute to this stabilization.