OTD/OTX2 functional equivalence depends on 5' and 3' UTR-mediated control of Otx2 mRNA for nucleo-cytoplasmic export and epiblast-restricted translation.

How gene activity is translated into phenotype and how it can modify morphogenetic pathways is of central importance when studying the evolution of regulatory control mechanisms. Previous studies in mouse have suggested that, despite the homeodomain-restricted homology, Drosophila orthodenticle (otd) and murine Otx1 genes share functional equivalence and that translation of Otx2 mRNA in epiblast and neuroectoderm might require a cell type-specific post-transcriptional control depending on its 5' and 3' untranslated sequences (UTRs). In order to study whether OTD is functionally equivalent to OTX2 and whether synthesis of OTD in epiblast is molecularly dependent on the post-transcriptional control of Otx2 mRNA, we generated a first mouse model (otd(2)) in which an Otx2 region including 213 bp of the 5' UTR, exons, introns and the 3' UTR was replaced by an otd cDNA and a second mutant (otd(2FL)) replacing only exons and introns of Otx2 with the otd coding sequence fused to intact 5' and 3' UTRs of Otx2. otd(2) and otd(2FL) mRNAs were properly transcribed under the Otx2 transcriptional control, but mRNA translation in epiblast and neuroectoderm occurred only in otd(2FL) mutants. Phenotypic analysis revealed that visceral endoderm (VE)-restricted translation of otd(2) mRNA was sufficient to rescue Otx2 requirement for early anterior patterning and proper gastrulation but it failed to maintain forebrain and midbrain identity. Importantly, epiblast and neuroectoderm translation of otd(2FL) mRNA rescued maintenance of anterior patterning as it did in a third mouse model replacing, as in otd(2FL), exons and introns of Otx2 with an Otx2 cDNA (Otx2(2c)). The molecular analysis has revealed that Otx2 5' and 3' UTR sequences, deleted in the otd(2) mRNA, are required for nucleo-cytoplasmic export and epiblast-restricted translation. Indeed, these molecular impairments were completely rescued in otd(2FL) and Otx2(2c) mutants. These data provide novel in vivo evidence supporting the concept that during evolution pre-existing gene functions have been recruited into new developmental pathways by modifying their regulatory control.

Forebrain and midbrain development requires epiblast-restricted Otx2 translational control mediated by its 3' UTR.

Otx genes play an important role in brain development. Previous mouse models suggested that the untranslated regions (UTRs) of Otx2 mRNA may contain regulatory element(s) required for its post-transcriptional control in epiblast and neuroectoderm. In order to study this, we have perturbed the 3' UTR of Otx2 by inserting a small fragment of DNA from the lambda phage. Otx2(lambda) mutants exhibited proper gastrulation and normal patterning of the early anterior neural plate, but from 8.5 days post coitum they developed severe forebrain and midbrain abnormalities. OTX2 protein levels in Otx2(lambda) mutants were heavily reduced in the epiblast, axial mesendoderm and anterior neuroectoderm but not in the visceral endoderm. At the molecular level, we found out that the ability of the Otx2(lambda) mRNA to form efficient polyribosome complexes was impaired. Sequence analysis of the Otx2-3' UTR revealed a 140 bp long element that is present only in vertebrate Otx2 genes and conserved in identity by over 80%. Our data provide experimental evidence that murine brain development requires accurate translational control of Otx2 mRNA in epiblast and neuronal progenitor cells. This leads us to hypothesise that this control might have important evolutionary implications.

OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression.

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Binding properties of the human homeodomain protein OTX2 to a DNA target sequence.

OTX2, a homeodomain protein essential in mouse for the development of structures anterior to rhombomere 3, binds with high affinity to a DNA element (called OTS) present in the human tenascin-C promoter. Here we investigate the binding properties of the full length recombinant human OTX2 and of several deletion mutants to the OTS element. We demonstrate that, upon binding of the protein to its DNA target site, a second molecule of OTX2 is recruited to the complex and that a nearby second binding site is not necessary for this interaction. OTX2 sequences located within a region carboxyl-terminal to the homeodomain are necessary in addition to the homeodomain for binding to DNA. Furthermore, OTX2 dimerization requires the same protein domains necessary for DNA binding.

Mapping of a potent transcriptional repression region of the human homeodomain protein EVX1.

The human homeodomain protein EVX1 is a transcriptional repressor in transfected mammalian cells and this function depends on a region carboxyl-terminal to the homeodomain. In this study, we transiently expressed several deletions of the EVX1 C-terminal region in mammalian cells and investigated their effect on the transcription of a reporter gene directed by different promoters. We show that the repressor activity maps to a region of 51 amino acids with a high abundance of alanine and proline residues. This region is able to transfer the repressor function to either the entire HOXC6 or CREB transcription factors, or to the GAL4 DNA binding domain.

Transcriptional repression by the human homeobox protein EVX1 in transfected mammalian cells.

The human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. In this study, the effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters we tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. We speculate that the repressor function of EVX1 contributes to its proposed role in embryogenesis.