The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and geneticcomplementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.cone ͉ Danio rerio ͉ T-box ͉ mosaic V ertebrates have evolved 2 major classes of retinal photoreceptors: rods, which mediate dim light vision, and cones, which detect light of greater intensity, have a faster temporal resolution, and mediate color vision. Largely from the analysis of mutations in mice and humans, a transcriptional network regulating photoreceptor cell development has been proposed. The presumptive photoreceptor progenitors sequentially express the homeobox transcription factors (TFs) Otx2 and Crx (1-3), and in their absence, photoreceptor precursors are not specified or fail to differentiate. Rod specification requires the additional expression of the Maf-family TF Nrl and its target Nr2e3 (4, 5). Nrl acts as a molecular switch; in its absence, precursors adopt the short-wavelength (S) opsin cone fate (5, 6), and mis-and over-expression of Nrl transforms most if not all cone precursors into functional rods (7,8). NR2E3 expression, which is disrupted in enhanced S-cone syndrome in humans and the rd7 mouse, is required for the repression of cone-specific genes in rod precursors (4, 9, 10). However, it remains to be determined if a reciprocal system exists in cone precursors for repressing rodspecific genes.The zebrafish retina, in addition to rods, possesses 4 cone subtypes, each with a distinct morphology and expressing a unique opsin (11-17). The spatial...
Chromatin immunoprecipitation assays were employed to assess the kinetics of transcription factor assembly and histone modifications that occur during gamma interferon (IFN-␥) induction of CIITA gene expression. CIITA is the master regulator of major histocompatibility complex class II transcription. Promoter IV (PIV), the major IFN-␥ responsive promoter for CIITA expression, requires both STAT1 and IFN regulatory factor 1 (IRF-1) for induction by IFN-␥. STAT1 binding to PIV was detected first and was accompanied by a modest acetylation of histones H3 and H4 that were associated with the region. Despite these changes, which occurred within 30 min of IFN-␥ treatment, CIITA mRNA was not detected until IRF-1 protein was synthesized and bound to its site, a process that required >120 min. In contrast to these events, fetal trophoblast-like cell lines, which are refractory to CIITA induction by IFN-␥, failed to assemble the above factors or modify their chromatin, suggesting that accessibility to the promoter is blocked. Bisulfite sequencing of PIV showed strong hypermethylation of PIV, providing a link between methylation, chromatin structure, and factor binding. Together, this analysis provides a kinetic view of the activation of the CIITA gene in response to IFN-␥ and shows that regulatory factor assembly, chromatin modification, and gene expression proceed in discrete steps.
The XOPS-mCFP transgene causes selective degeneration of rods without secondary loss of cones in animals up to 7 months of age. This raises important questions about the significance of rod-cone interactions in zebrafish and their potential as a model of human inherited retinal degenerations.
The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.
Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.
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