Arrested differentiation and epithelial cell degeneration in zebrafish lens mutants.

In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities.

Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors.

To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kilobase (kbp) zebrafish rod opsin genomic clone, which consists of 18 kbp of 5'-flanking region, the entire coding region, and 0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, Southern blotting, and DNA sequencing revealed the rod opsin gene lacks introns. The transcription start site was localized 94 nucleotides upstream of the translation initiation site. Sequence alignment with orthologous promoters revealed conserved cis-elements including glass, NRE, OTX/Bat-1, Ret-1/PCE-1, Ret-4, and TATA box. A 1.2-kbp promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1- to 2-cell stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by polymerase chain reaction amplification of fin genomic DNA, exhibited EGFP expression in the retina, confirming the germline transmission of the transgene. Frozen eye sections demonstrated the EGFP expression was rod-specific and exhibited a similar developmental expression profile as the rod opsin protein. This stable transgenic line provides a novel tool for identification of genes regulating development and maintenance of rod photoreceptors.

Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.

Light-induced photoreceptor cell degeneration has been studied in several species, but not extensively in the teleost fish. Furthermore, the continual production of rods and cones throughout the teleost's life may result in regeneration of lost rods and cones. We exposed adult albino zebrafish to 7 days of constant darkness, followed by 7 days of constant 8000 lux light, followed by 28 days of recovery in a 14-h light:10-h dark cycle. We characterized the resulting photoreceptor layer cell death and subsequent regeneration using immunohistochemistry and light microscopy. Within the first 24 h of constant light, the zebrafish retina exhibited widespread rod and cone cell apoptosis. High levels of cell proliferation within the inner nuclear layer (INL) were observed within the first 3 days of constant light, as assessed by immunodetection of proliferating cell nuclear antigen and BrdU labeling. The proliferating cells within the INL were closely associated with the radial processes of Müller glia, similar to the pluripotent retinal stem cells observed during embryonic development. Using antibodies generated against the individual zebrafish opsins, we determined that rods and the green, blue, and ultraviolet cone cells were replaced within the 28 day recovery period. While both rods and cones were replaced, the well-ordered cone cell mosaic was not reestablished.

Cloning and tissue localization of a novel zebrafish RdgB homolog that lacks a phospholipid transfer domain.

The retinal degeneration B (RdgB) protein family is characterized by an amino-terminal phosphatidylinositol transfer protein (PITP) domain, several hydrophobic domains, and a highly conserved carboxyl terminus. We identified a zebrafish RdgB homolog (pl-RdgB) that lacks the amino-terminal PITP domain, while retaining over 45% amino acid identity with the two mouse RdgB proteins (M-RdgB1 and M-RdgB2). Unlike the widespread retinal expression observed for other vertebrate RdgB homologs, pl-RdgB is restricted in the retina to the cone cell inner segments. The pl-RdgB protein is also expressed in the brain, although its distribution is different than the other RdgB homologs. Analogous to M-RdgB2, pl-RdgB protein is extracted from a retinal homogenate by guanidine and not by Triton X-100. Thus, pl-RdgB and likely all the identified RdgB homologs are not integral membrane proteins, but may associate with the membrane through protein-protein interactions. While expression of either murine RdgB homolog restored the defective light response and prevented retinal degeneration in rdgB mutant flies, expressing zebrafish pl-RdgB in Drosophila rdgB2 null mutants slowed retinal degeneration without restoring the electrophysiological light response. Thus, pl-RdgB may define a previously unrecognized protein family, which includes the other RdgB homologs, that act through a protein complex to maintain photoreceptor viability.

A neuronal-specific mammalian homolog of the Drosophila retinal degeneration B gene with expression restricted to the retina and dentate gyrus.

Mutations in the Drosophila retinal degeneration B (rdgB) gene cause a rapid loss of the electrophysiological light response and subsequent light-enhanced photoreceptor degeneration. The rdgB gene encodes a protein with an N-terminal phosphatidylinositol transfer protein domain, a large C-terminal segment, and several hydrophobic regions thought to multiply span the subrhabdomeric cisternal membrane. A mammalian rdgB homolog (m-rdgB1) was previously identified and shown to exhibit widespread tissue distribution and functionally rescue the Drosophila rdgB mutant phenotypes. We describe a second mammalian rdgB homolog (m-rdgB2) that possesses 46% amino acid identity to Drosophila RdgB and 56% identity to M-RdgB1. M-RdgB2 possesses a neuronal-specific expression pattern, with high levels in the retina and the dentate gyrus mossy fibers and dendritic field. Using M-RdgB2-specific antibodies and subcellular fractionation, we demonstrate that M-RdgB2 is not an integral membrane protein but is stably associated with a particulate fraction through protein-protein interactions. Although transgenic expression of M-RdgB2 in rdgB2 null mutant flies suppressed the retinal degeneration, it failed to fully restore the electrophysiological light response. Because transgenic expression of M-RdgB2 does not restore the wild-type phenotype to rdgB2 mutant flies to the same extent as M-RdgB1, functional differences likely exist between the two M-RdgB homologs.

Cloning and characterization of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins.

Zebrafish (Danio rerio) represents an excellent genetic model for vertebrate visual system studies. Because the opsin proteins are ideal markers of specific photoreceptor cell types, we cloned six different zebrafish opsin cDNAs. Based on pairwise alignments and phylogenetic comparisons between the predicted zebrafish opsin amino acid sequences and other vertebrate opsins, the cDNAs encode rhodopsin, two different green opsins (zfgr1 and zfgr2), a red, a blue, and an ultraviolet opsin. Phylogenetic analysis indicates the zfgr1 protein occupies a well-resolved dendrogram branch separate from the other green opsins examined, while zebrafish ultraviolet opsin is closely related to the human blue- and chicken violet-sensitive proteins. Polyclonal antisera were generated against individual bacterial fusion proteins containing either the red, blue, or ultraviolet amino termini or the rod or green opsin carboxyl termini. Immunolocalization on adult zebrafish frozen sections demonstrates the green and red opsins are each expressed in different members of the double cone cell pair, the blue opsin is detected in long single cones, and the ultraviolet opsin protein is expressed in the short single cones. In 120-h postfertilization wholemounts, green, red, blue, and ultraviolet opsin-positive cells are detected in an orderly arrangement throughout the entire retina. The antibodies' photoreceptor-type specificity indicates they will be useful for characterizing both wild-type and mutant zebrafish retinas.

Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein.

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.

Isolation and characterization of the Drosophila retinal degeneration B (rdgB) gene.

Retinal degeneration-B (rdgB) mutants of Drosophila melanogaster undergo rapid light-induced retinal degeneration. We conducted a molecular characterization of the rdgB gene to examine the nature of the gene product. Through the isolation and analysis of X-ray-induced rdgB alleles, the cytogenetic position of the gene was determined to be the 12C1 salivary region. Genomic DNA corresponding to this region was isolated by a chromosomal walk. The chromosomal aberrations associated with the three X-ray-induced rdgB alleles were shown to be within a 5-kb genomic region. A single transcription unit was affected by the alleles, identifying it as the rdgB gene. RNA-RNA Northern hybridization indicated the rdgB gene transcribed five mRNAs ranging in size from 3.9 to 9.5 kb. These mRNAs were expressed in adult heads, but not detected in bodies. Analysis of RNA isolated from wild-type and eyes absent heads indicated that rdgB mRNA expression was not restricted to the retina. DNA sequence analysis of the transcription unit revealed an open reading frame capable of encoding a 116-kD transmembrane protein. The deduced protein shows no overall homology to previously described proteins, but has sequences in common with proposed functional domains of Ca(2+)-ATPase.

Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin.

From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner.