Plasticin, a type III neuronal intermediate filament protein, assembles as an obligate heteropolymer: implications for axonal flexibility.

The assembly characteristics of the neuronal intermediate filament protein plasticin were studied in SW13 cells in the presence and absence of a cytoplasmic filament network. Full-length plasticin cannot polymerize into homopolymers in filament-less SW13c1.2Vim(-) cells but efficiently coassembles with vimentin in SW13c1.1Vim(-) cells. By cotransfecting plasticin and vimentin in SW13c1.1Vim(-) cells, we show that plasticin assembly requires vimentin in noncatalytic amounts. Differing effects on assembly were seen with point mutations of plasticin monomers that were analogous to the keratin mutations that cause epidermolysis bullosa simplex (EBS). In particular, plasticin monomers with point mutations analogous to those in EBS do not uniformly inhibit neurofilament (NF) network formation. A point mutation in the helix termination sequence resulted in complete filament aggregation when coexpressed with vimentin but showed limited coassembly with low- and medium-molecular-weight NF proteins (NF-L and NF-M, respectively). In transfected SW13c1.1Vim(+) cells, a point mutation in the first heptad of the alpha-helical coil region formed equal amounts of filaments, aggregates, and a mixture of filaments and aggregates. Furthermore, coexpression of this point mutation with NF-L and NF-M was associated with a shift toward increased numbers of aggregates. These results suggest that there are important structural differences in assembly properties between homologous fish and mammalian intermediate filament proteins. These structural differences may contribute to the distinctive growth characteristics of the teleost visual pathway.

Gefiltin in zebrafish embryos: sequential gene expression of two neurofilament proteins in retinal ganglion cells.

Neurogenesis is correlated with the progressive synthesis of diverse neuronal intermediate filaments (IF) proteins. This apparent developmental regulation of IF protein gene expression suggests that specific neurofilament proteins impart unique structural attributes that support the staged growth of the neuron. In the teleost visual pathway, the sequential expression of two IF genes, plasticin and gefiltin, is linked to the age of retinal ganglion cells (RGCs) and to the regeneration of optic axons after nerve injury. Given this pattern of plasticin and gefiltin expression, we hypothesized that the two proteins would be sequentially expressed in zebrafish retina during development. We analyzed the pattern of gefiltin expression during zebrafish development and compared it to our previous determination of plasticin expression (Canger et al. 1998). Gefiltin is expressed after plasticin, during the later stages of retinal development when axons grow past the optic chiasm and innervate their targets. Thus, during RGC development, expression of plasticin and gefiltin resembles that with optic nerve regeneration. Outside of the visual pathway, gefiltin is predominantly expressed in the central nervous system whereas plasticin is primarily expressed in the peripheral nervous system. These results suggest that the expression of these genes is regulated in a neuron-specific manner. In addition, since plasticin and gefiltin are co-expressed during RGC development, these findings suggest a more complex mechanism of transcriptional regulation which orchestrates the sequential expression of these genes.

Cloning of zebrafish vsx1: expression of a paired-like homeobox gene during CNS development.

vsx1 is a homeobox gene encoding a paired-type homeodomain and a CVC domain that was originally cloned from an adult goldfish retinal library. We previously reported the spatiotemporal expression pattern of vsx1 in the adult and developing retina of zebrafish and goldfish, and we suggested that vsx1 plays a role in determining the cell fate and maintenance of retinal interneurons. Other related genes encoding a CVC domain, such as vsx2 (alx) and chx10, are expressed both within and outside the retina during development. In this study, we report the cloning of zebrafish vsx1 and its developmental expression in both retinal and nonretinal regions of the CNS in zebrafish embryos. vsx1 expression was detected in a subset of hindbrain and spinal cord neurons before it was expressed in the retina. At about the same time that retinal expression began, the level of vsx1 was decreased in the spinal cord. The expression of vsx1 was progressively restricted, and eventually it was detected only in the inner nuclear layer (INL) of the developing retina. The combined expression patterns of teleost vsx1 and vsx2 (alx) during early zebrafish development encompasses the expression pattern observed for murine Chx10, and indicates a partitioning of function for CVC genes in lower vertebrates.

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development.

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons.

Cloning of zebrafish neurofilament cDNAs for plasticin and gefiltin: increased mRNA expression in ganglion cells after optic nerve injury.

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.