Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of “sensors” in which green fluorescent protein translation is under control of the 3′ untranslated region (UTR), we found that the 3′ UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities

A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina

<p>Abstract</p> <p>Background</p> <p><it>Otx </it>genes, orthologues of the <it>Drosophila orthodenticle </it>gene (<it>otd</it>), play crucial roles in vertebrate brain development. In the <it>Xenopus </it>eye, <it>Xotx2 </it>and <it>Xotx5b </it>promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by <it>Drosophila </it>OTD, we performed an <it>in vivo </it>molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of <it>Xotx2</it>, <it>Xotx5b </it>and <it>otd</it>.</p> <p>Results</p> <p>We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and <it>vice versa</it>. Moreover, the insertion of this box into <it>Drosophila </it>OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box.</p> <p>Conclusion</p> <p>Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of <it>Drosophila </it>into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.</p

Dicer inactivation causes heterochronic retinogenesis in Xenopus laevis

Maturation of miRNAs by dicer is required in vertebrates for normal neural development. Here we report that dicer inactivation in Xenopus affects cell cycle progression, survival and timing of the generation of retinal cells, resulting in small retinas with lamination defects. In particular, dicer inactivation delays the exit from the cell cycle and the translation of key genes of late neurogenesis, highlighting a crucial role of miRNAs in retinal development

Xenopus Bsx links daily cell cycle rhythms and pineal photoreceptor fate

In the developing central nervous system, the cell cycle clock plays a crucial role in determining cell fate specification. A second clock, the circadian oscillator, generates daily rhythms of cell cycle progression. Although these two clocks interact, the mechanisms linking circadian cell cycle progression and cell fate determination are still poorly understood. A convenient system to address this issue is the pineal organ of lower vertebrates, which contains only two neuronal types, photoreceptors and projection neurons. In particular, photoreceptors constitute the core of the pineal circadian system, being able to transduce daily light inputs into the rhythmical production of melatonin. However, the genetic program leading to photoreceptor fate largely remains to be deciphered. Here, we report a previously undescribed function for the homeobox gene Bsx in controlling pineal proliferation and photoreceptor fate in Xenopus. We show that Xenopus Bsx (Xbsx) is expressed rhythmically in postmitotic photoreceptor precursors, reaching a peak during the night, with a cycle that is complementary to the daily rhythms of S-phase entry displayed by pineal cells. Xbsx knockdown results in increased night levels of pineal proliferation, whereas activation of a GR-Xbsx protein flattens the daily rhythms of S-phase entry to the lowest level. Furthermore, evidence is presented that Xbsx is necessary and sufficient to promote a photoreceptor fate. Altogether, these data indicate that Xbsx plays a dual role in contributing to shape the profile of the circadian cell cycle progression and in the specification of pineal photoreceptors, thus acting as a unique link between these two events

Xenopus Bsx links daily cell cycle rhythms and pineal photoreceptor fate

In the developing central nervous system, the cell cycle clock plays a crucial role in determining cell fate specification. A second clock, the circadian oscillator, generates daily rhythms of cell cycle progression. Although these two clocks interact, the mechanisms linking circadian cell cycle progression and cell fate determination are still poorly understood. A convenient system to address this issue is the pineal organ of lower vertebrates, which contains only two neuronal types, photoreceptors and projection neurons. In particular, photoreceptors constitute the core of the pineal circadian system, being able to transduce daily light inputs into the rhythmical production of melatonin. However, the genetic program leading to photoreceptor fate largely remains to be deciphered. Here, we report a previously undescribed function for the homeobox gene Bsx in controlling pineal proliferation and photoreceptor fate in Xenopus. We show that Xenopus Bsx (Xbsx) is expressed rhythmically in postmitotic photoreceptor precursors, reaching a peak during the night, with a cycle that is complementary to the daily rhythms of S-phase entry displayed by pineal cells. Xbsx knockdown results in increased night levels of pineal proliferation, whereas activation of a GR-Xbsx protein flattens the daily rhythms of S-phase entry to the lowest level. Furthermore, evidence is presented that Xbsx is necessary and sufficient to promote a photoreceptor fate. Altogether, these data indicate that Xbsx plays a dual role in contributing to shape the profile of the circadian cell cycle progression and in the specification of pineal photoreceptors, thus acting as a unique link between these two events

Evolution of highly repeated DNA within the genus Triturus (Amphibia Urodela)

Highly repeated DNA is a main feature of urodele amphibian genomes. In Triturus this class of DNA consists of several sequence families differently arranged at both the molecular and the chromosomal level, showing varying degrees of conservation across species. Present data on highly repeated DNA in Triturus are here summarized and discussed with regard to the evolution and possible functional role of these sequences