Nisson Schechter

Vsx‐1 and Vsx‐2: Differential expression of two Paired‐like homeobox genes during zebrafish and goldfish retinogenesis

Vsx‐1 and Vsx‐2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired‐like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx‐1 and Vsx‐2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx‐2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down‐regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx‐1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up‐regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx‐2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx‐2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx‐2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx‐1 and Vsx‐2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina. J. Comp. Neurol. 388:495–505, 1997.

A type II keratin is expressed in glial cells of the goldfish visual pathway

The predominant intermediate filament proteins of the goldfish visual pathway consist of neuronal and non-neuronal isoelectric variants (58 kd). We have isolated a cDNA clone for the glial intermediate filament protein (ON3) from an optic nerve expression library. The predicted amino acid sequence of this clone reveals that it codes for a type II keratin representing the goldfish equivalent of mammalian keratin K8. K8 has been shown to be associated with embryogenesis and development. Unlike the mammalian visual system, the goldfish visual pathway displays a remarkable capacity for functional regeneration. The expression of K8, a protein not usually expressed in glial cells but shown to be associated with development, in the goldfish optic nerve may be involved with the processes of growth and regeneration in the goldfish visual pathway.

Experimental Febrile Convulsions: Long-Term Effects of Hyperthermia-Induced Convulsions in the Developing Rat

Summary: The susceptibility of infant rats to experimental febrile convulsions was investigated. Rats were subjected to a single hyperthermia convulsion at 5, 10, 15, or 20 days of age or to a series of convulsions from 5 to 20 days of age. Susceptibility to the experimental febrile convulsion decreased with age in all rats except those subjected to multiple convulsions. In this group, susceptibility tended to increase. This result is discussed in terms of its similarity to the kindling phenomenon and to the incidence of recurrent febrile convulsions in the human infant. Sex differences in susceptibility to the convulsion were examined, but none were found. Mature rats that had been subjected to experimental febrile convulsions as infants were found to be significantly more susceptible to a convulsive dose of pentylenetetrazol than controls. The results of this study indicate that even a single experimental febrile convulsion during infancy can exert a long‐lasting, if not permanent, enhancement in seizure susceptibility. The similarities between the present findings and human infantile febrile convulsions are discussed.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.

58,000 dalton intermediate filament proteins of neuronal and nonneuronal origin in the goldfish visual pathway

Abstract: A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two‐dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal‐enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one‐dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one‐dimensional gel electrophoresis.

VSX-1 AND VSX-2: TWO CHX10-LIKE HOMEOBOX GENES EXPRESSED IN OVERLAPPING DOMAINS IN THE ADULT GOLDFISH RETINA

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax‐6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired‐class homeobox gene, Vsx‐1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx‐2, in the adult goldfish retina. Like Vsx‐1, Vsx‐2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx‐1 expression in the mature INL. At the peripheral margin, Vsx‐2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx‐2, Vsx‐1, Chx10, and C. elegans ceh‐10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx‐2, Vsx‐1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates. J. Comp. Neurol. 387:439–448, 1997.

Survey of Intermediate Filament Proteins in Optic Nerve and Spinal Cord: Evidence for Differential Expression

Abstract: The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two‐dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145–150K range and in the 70–85K range. In addition, predominant neurofilament proteins in the 58–62K molecular‐weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low‐molecular‐weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.

Recovery of tectal nicotinic-cholinergic receptor sites during optic nerve regeneration in goldfish

The concentration of cholinergic nicotinic-like sites as measured by alphabungarotoxin (alphaBuTX) binding, decreased in the goldfish (Carassius auratus) optic tectum after optic nerve disconnection. Initially, the rate of loss of sites is greater than the rate of tissue or protein degradation in experiments where disconnection was achieved either by unilateral optic nerve crush or by enucleation of one eye. When the crushed optic nerve is allowed to regenerate and form behaviorally potent connections, the number and concentration of these sites appears restored. Pharmacological studies indicate that the alphaButTX binding site in the goldfish optic tectum has a drug binding profile similar to that seen at central or peripheral alphaBuTX sites in other species.

Molecular cloning of gefiltin (ON1): serial expression of two new neurofilament mRNAs during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins of this pathway do not match the intermediate filament protein composition of adult higher vertebrate neurons, which lack the capacity for growth and development. Using a goldfish retina lambda gt10 library we isolated cDNA clones representing the predominant goldfish optic nerve neurofilament protein, ON1. The mRNA for this protein is abundant in retinal ganglion cells, and its level increases slowly during optic nerve regeneration. The rate of ON1 mRNA accumulation after optic nerve crush was compared with that of plasticin, a previously described novel type III neurofilament from goldfish retinal ganglion cells. Plasticin mRNA is normally expressed at low steady state levels, but accumulates dramatically and rapidly, preceding gefiltin mRNA, in response to optic nerve crush. The predicted amino acid sequence for ON1 indicates that it is a novel intermediate filament protein. We have named it gefiltin, for goldfish eye intermediate filament protein. The serial expression of plasticin and gefiltin is discussed with respect to the diversity of neurofilament proteins during neurogenesis.

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.