Abstract
BackgroundOtx genes, orthologues of the Drosophila orthodenticle gene (otd), play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b 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 Drosophila OTD, we performed an in vivo molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd.ResultsWe 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 vice versa. Moreover, the insertion of this box into Drosophila 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.ConclusionOur 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 Drosophila 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.
Highlights
Otx genes, orthologues of the Drosophila orthodenticle gene, play crucial roles in vertebrate brain development
A small 8–10 amino acid region confers specific activities on XOTX2 and XOTX5b in the Xenopus retina Previous lipofection experiments showed that two chimeric constructs, Xotx2/engR and Xotx5b/engR, in which most of the transactivation domain of either XOTX2 or XOTX5b was replaced with the repressor domain of the Drosophila Engrailed protein, had specific effects on either bipolar cells or photoreceptor cells, respectively, in this case leading to a decrease, rather than an increase, in their frequency [12]
This observation suggested that these XOTX/engR chimeric proteins might retain a region of XOTX2 or XOTX5b crucial for their specific activities, and focused our attention on the only part of their carboxyl termini that was included in the Xotx2/engR and Xotx5b/ engR constructs
Summary
Orthologues of the Drosophila orthodenticle gene (otd), play crucial roles in vertebrate brain development. Among the homeobox genes involved in retinal cell fate regulation are the Otx genes Otx and Crx/Otx5 These genes are related to the orthodenticle (otd) gene of Drosophila, required for normal anterior development of the fly [20,21]. Otx and Otx, were initially isolated in mouse [22] and shown to be essential for correct development of the rostral brain; the Otx2-/- phenotype is especially severe, leading to complete lack of anterior structures [23,24,25,26] This phenotype can be rescued by the Drosophila otd gene [27,28]. These data suggested an extensive functional conservation of the OTX/OTD class of proteins
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