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: 2.67). 02/2007; 7(1):122. DOI: 10.1186/1471-213X-7-122
Source: PubMed


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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Available from: Gilles Chatelain, Oct 04, 2015
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    • "At E12.5, photoreceptor and bipolar cell fate is determined by the expression of Otx2 in retinal progenitor cells [7], which controls the subsequent induction of the related Crx transcription factor. Expression of both Otx2 and Crx is then maintained throughout life in photoreceptor and bipolar cells [8]–[10]. "
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    ABSTRACT: During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.
    PLoS ONE 02/2014; 9(2):e89110. DOI:10.1371/journal.pone.0089110 · 3.23 Impact Factor
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    • "In order to unambiguously confirm the identity of the differentiated cells as post-mitotic rod photoreceptors, staining was performed for other markers of the retinal lineage at 6 weeks of differentiation (supplementary material Fig. S2). Markers of mature RPE cells (RPE65+), Müller glia (CRALBP+), mature bipolar cells or immature rod/bipolar progenitors (Otx2+Rho-) (Nishida et al., 2003; Fossat et al., 2007), and horizontal cells (calbindin+) were present in prominent levels in 1% FBS cultures (supplementary material Table S1), consistent with previous reports of the differentiation potential of RSCs (Tropepe et al., 2000; Coles et al., 2004). The expression of these markers was reduced significantly in taurine/RA cultures at 40 days. "
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    ABSTRACT: Self-renewing, multipotential retinal stem cells (RSCs) reside in the pigmented ciliary epithelium of the peripheral retina in adult mammals. RSCs can give rise to rhodopsin positive-cells, which can integrate into early postnatal retina, and represent a potentially useful option for cellular therapy. The ability to purify a stem cell population and direct the differentiation toward a particular cell lineage is a challenge facing the application of stem cells in regenerative medicine. Here we use cell sorting to prospectively enrich mouse RSCs based on size, granularity and low expression of P-cadherin and demonstrate that only rare cells with defined properties proliferate to form colonies. We show that clonally-derived mouse and human RSC progeny are multipotent and can differentiate into mature rhodopsin-positive cells with high efficiency using combinations of exogenous culture additives known to influence neural retinal development, including taurine and retinoic acid. This directed RSC differentiation follows the temporal sequence of photoreceptor differentiation in vivo, and the cells exhibit morphology, protein and gene expression consistent with primary cultures of rods in vitro. These results demonstrate that the RSC, an adult stem cell, can be enriched and directed to produce photoreceptors as a first step toward a targeted cell replacement strategy to treat retinal degenerative disease.
    Biology Open 03/2012; 1(3):237-46. DOI:10.1242/bio.2012027 · 2.42 Impact Factor
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    • "These studies have proven very informative for the role of Otx2 in development of the retina. Still, little is known about its function in the adult, although it is strongly expressed in the adult retina [10], [12], [13]. "
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    ABSTRACT: Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells. Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease. Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.
    PLoS ONE 07/2010; 5(7):e11673. DOI:10.1371/journal.pone.0011673 · 3.23 Impact Factor
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