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A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors.

IGFL, UMR CNRS 5242-INRA 1237-ENS, IFR128 Lyon-Gerland, 46 allée d'Italie, 69364 Lyon Cedex 07, France.
BMC Developmental Biology (Impact Factor: 2.75). 02/2007; 7:122. DOI: 10.1186/1471-213X-7-122
Source: PubMed

ABSTRACT Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing.
We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type.
The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.

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    ABSTRACT: The Otx2 gene encodes a paired-type homeobox transcription factor that is essential for the induction and the patterning of the anterior structures in the mouse embryo. Otx2 knockout embryos fail to form a head. Whereas previous studies have shown that Otx2 is required in the anterior visceral endoderm and the anterior neuroectoderm for head formation, its role in the anterior mesendoderm (AME) has not been assessed specifically. Here, we show that tissue-specific ablation of Otx2 in the AME phenocopies the truncation of the embryonic head of the Otx2 null mutant. Expression of Dkk1 and Lhx1, two genes that are also essential for head formation, is disrupted in the AME of the conditional Otx2-deficient embryos. Consistent with the fact that Dkk1 is a direct target of OTX2, we showed that OTX2 can interact with the H1 regulatory region of Dkk1 to activate its expression. Cross-species comparative analysis, RT-qPCR, ChIP-qPCR and luciferase assays have revealed two conserved regions in the Lhx1 locus to which OTX2 can bind to activate Lhx1 expression. Abnormal development of the embryonic head in Otx2;Lhx1 and Otx2;Dkk1 compound mutant embryos highlights the functional intersection of Otx2, Dkk1 and Lhx1 in the AME for head formation.
    Development 09/2014; 141(20). DOI:10.1242/dev.114900 · 6.27 Impact Factor
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    DESCRIPTION: ABSTRACT DLX HOMEOBOX TRANSCRIPTIONAL REGULATION OF CRX AND OTX2 GENE EXPRESSION DURING VERTEBRATE RETINAL DEVELOPMENT Objectives: We are interested in identifying and characterizing DLX transcriptional targets during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 double knockout (mutant) mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Methods: CRX and OTX2 expression in mutants and wild-type littermates was detected at RNA and protein levels. Chromatin immunoprecipitation (ChIP) of embryonic retina was utilized to identify DLX protein-genomic DNA complexes in situ. Quantification of expression was assessed by qRT-PCR and cell counting. In vitro assays such as electrophoretic mobility shift assays (EMSA) and luciferase reporter assays were used to detect the direct binding and activity, respectively, of DLX2 on the Crx and Otx2 promoters in vitro. Results: Qualitative and quantitative assessment of the temporal and spatial expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression, thereby restricting CRX expression to the outer nuclear layer. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. ChIP assays demonstrated that DLX proteins are bound to specific regions of the Crx and Otx2 promoters in situ, supporting a direct role for Dlx genes in repressing Crx and Otx2 expression during retinal development. Conclusion: The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data supports our hypothesis that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.

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May 17, 2014