Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.