Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

Spatio-temporal regulation of Rx and mitotic patterns shape the eye-cup of the photoreceptor cells in Ciona

The ascidian larva has a pigmented ocellus comprised of a cup-shaped array of approximately 30 photoreceptor cells, a pigment cell, and three lens cells. Morphological, physiological and molecular evidence has suggested evolutionary kinship between the ascidian larval photoreceptors and vertebrate retinal and/or pineal photoreceptors. Rx, an essential factor for vertebrate photoreceptor development, has also been suggested to be involved in the development of the ascidian photoreceptor cells, but a recent revision of the photoreceptor cell lineage raised a crucial discrepancy between the reported expression patterns of Rx and the cell lineage. Here, we report spatio-temporal expression patterns of Rx at single-cell resolution along with mitotic patterns up to the final division of the photoreceptor-lineage cells in Ciona. The expression of Rx commences in non-photoreceptor a-lineage cells on the right side of the anterior sensory vesicle at the early tailbud stage. At the mid tailbud stage, Rx begins to be expressed in the A-lineage photoreceptor cell progenitors located on the right side of the posterior sensory vesicle. Thus, Rx is specifically but not exclusively expressed in the photoreceptor-lineage cells in the ascidian embryo. Two cis-regulatory modules are shown to be important for the photoreceptor-lineage expression of Rx. The cell division patterns of the photoreceptor-lineage cells rationally explain the generation of the cup-shaped structure of the pigmented ocellus. The present findings demonstrate the complete cell lineage of the ocellus photoreceptor cells and provide a framework elucidating the molecular and cellular mechanisms of photoreceptor development in Ciona.     

Conserved roles of ems/Emx and otd/Otx genes in olfactory and visual system development in Drosophila and mouse

The regional specialization of brain function has been well documented in the mouse and fruitfly. The expression of regulatory factors in specific regions of the brain during development suggests that they function to establish or maintain this specialization. Here, we focus on two such factors—the Drosophila cephalic gap genes empty spiracles (ems) and orthodenticle (otd), and their vertebrate homologues Emx1/2 and Otx1/2—and review novel insight into their multiple crucial roles in the formation of complex sensory systems. While the early requirement of these genes in specification of the neuroectoderm has been discussed previously, here we consider more recent studies that elucidate the later functions of these genes in sensory system formation in vertebrates and invertebrates. These new studies show that the ems and Emx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective olfactory systems. Moreover, they demonstrate that the otd and Otx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective visual systems. Based on these recent experimental findings, we discuss the possibility that the olfactory and visual systems of flies and mice share a common evolutionary origin, in that the conserved visual and olfactory circuit elements derive from conserved domains of otd/Otx and ems/Emx action in the urbilaterian ancestor.     

Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor-specific gene expression

RX, a homeodomain-containing protein essential for proper eye development (Mathers, P. H. Grinberg, A., Mahon, K. A., and Jamrich, M. (1997) Nature 387, 603-607), binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells (Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gillbert, D. J. Jenkins, N. A., and Zack, D. J. (1997) Neuron 19, 1017-1030; Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531-541). Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages.     

A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera

Corals exhibit circadian behaviors, but little is known about the molecular mechanisms underlying the regulation of these behaviors. We surveyed the recently decoded genome of the coral, Acropora digitifera, for photoreceptor and circadian genes, using molecular phylogenetic analyses. Our search for photoreceptor genes yielded seven opsin and three cryptochrome genes. Two genes from each family likely underwent tandem duplication in the coral lineage. We also found the following A. digitifera orthologs to Drosophila and mammalian circadian clock genes: four clock, one bmal/cycle, three pdp1-like, one creb/atf, one sgg/zw3, two ck2alpha, one dco (csnk1d/cnsk1e), one slim/BTRC, and one grinl. No vrille, rev-ervα/nr1d1, bhlh2, vpac2, adcyap1, or adcyaplr1 orthologs were found. Intriguingly, in spite of an extensive survey, we also failed to find homologs of period and timeless, although we did find one timeout gene. In addition, the coral genes were compared to orthologous genes in the sea anemone, Nematostella vectensis. Thus, the coral and sea anemone genomes share a similar repertoire of circadian clock genes, although A. digitifera contains more clock genes and fewer photoreceptor genes than N. vectensis. This suggests that the circadian clock system was established in a common ancestor of corals and sea anemones, and was diversified by tandem gene duplications and the loss of paralogous genes in each lineage. It will be interesting to determine how the coral circadian clock functions without period.     

Evolutionarily conserved and divergent regulatory sequences in the fish rod opsin promoter

Fish have multiple types and subtypes of opsin genes that are expressed in a highly regulated manner in retinal photoreceptor cells. In the rod opsin proximal promoter region (RPPR) of zebrafish (Danio rerio), the BAT 1 regulatory region contains highly conserved OTX (GATTA) and OTX-like (TATTA) sequences that can be recognized by the mammalian cone–rod homeobox (CRX) protein. However, binding of zebrafish crx to the OTX sequence has remained elusive. In contrast to the BAT 1 region, the Ret 1 region, located approximately 20 bp upstream of the BAT 1 region in mammals, is not conserved in zebrafish. In the Ret 1 region, even the core OTX-like sequence (AATTA sequence in mammals) is destructed. We show in this study that a region between Ret 1 and BAT 1 (denoted IRB, Inter-Ret 1-BAT 1) is highly conserved among fish species. Using electrophoretic mobility shift assay (EMSA), we show that zebrafish crx binds to the conserved OTX sequence and that the fish-specific IRB region specifically binds elements present in both retinal and brain nuclear extracts of zebrafish. These results imply that the regulatory mechanisms of opsin gene expression consist not only of evolutionarily conserved but also of divergent machinery among different animal taxa.     

Divergent functions of orthodenticle, empty spiracles and buttonhead in early head patterning of the beetle Tribolium castaneum (Coleoptera)

The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.