Molecular and Cell Biology of Nerve Development, Growth, and Regeneration in the Zebrafish Visual Pathway.
The research focus of this laboratory is to discover proteins which regulate and support the development and growth of neurons. As our initial model system, we used the visual pathway of goldfish which displays continuous growth and development throughout life. In addition, functional regeneration of the goldfish optic nerve occurs after injury. This is important clinically because functional regeneration does not occur in the mammalian central nervous system. More recently, we shifted our research focus to the zebrafish model system. Although zebrafish are physiologically similar to goldfish, proteins that are crucial to development can be studied in the transparent zebrafish embryos. Furthermore, the zebrafish is amenable to genetic manipulation. Thus, the zebrafish allows us to combine the techniques of molecular biology with those of cell biology to discover how specific proteins regulate and support neurogenesis.
Plasticin and gefiltin are two intermediate filament (IF) proteins that we discovered in goldfish and have subsequently characterized in zebrafish. The expression of these proteins is correlated with the development, growth and regeneration of the optic nerve. Furthermore, plasticin and gefiltin are structurally related to IF proteins that are expressed in the mammalian visual pathway during development. Since plasticin is expressed in newer retinal ganglion cells and is seen early in response to injury, we hypothesize that plasticin supports the initial growth phase of the optic nerve. On the other hand, gefiltin is expressed in older cells and during later phases of regeneration. Thus, we hypothesize that it is more essential to the formation of terminals of the retinal projections. Currently, we are using cultured cells to determine the impact of plasticin and gefiltin expression on the assembly of the IF network. In addition, we are using zebrafish embryos to determine the regulatory mechanisms by which a given cell type (such as retinal ganglion cells) can trigger the sequential expression of these structurally similar proteins from the same super gene family.
We also discovered two homeobox genes, Vsx-1 and Vsx-2, that were originally cloned from adult goldfish retina. These proteins are members of the paired-like:CVC subclass of homeobox genes. Paired-like:CVC proteins contain a 54-58 amino acid region, termed the CVC domain, which is adjacent to the C-terminus of the homeodomain. The expression of these transcription factors is linked to retinal development. In addition, a mutation in the mouse homologue of Vsx-2 results in ocular retardation.
Histological analysis in goldfish and zebrafish suggests roles for Vsx-1 and Vsx-2 in the differentiation of bipolar cells and in their stabilization within the laminated retina. Initially, Vsx-1 and Vsx-2 are expressed in a complementary fashion, but later their expression patterns become superimposed. This sequential change in expression pattern suggests that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during retinal development. This dynamic expression of Vsx-1 and Vsx-2 suggests that these transcription factors must have a rapid turnover to permit precise regulation during development. One mechanism by which this might occur is via the ubiquitin/ proteasome pathway. Current research is determining the roles of the CVC domain and putative phosphorylation sites in the ubiquitination of Vsx-1 and Vsx-2. A related project is investigating the role of a ubiquitin-like conjugating enzyme, Ubc9, in the transport of Vsx-1 and Vsx-2 to the nucleus. All of our experiments utilize the techniques of molecular and cell biology to determine how specific homeobox transcription factors regulate cell fate during development.