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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    • "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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    • "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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    • "Based on the degree of head truncation, embryos of various genotypes were assigned to one of the five phenotype categories: [I] normal head size (0%) and morphology comparable to the wild-type embryo, [II] ≤25 % reduction of the forebrain, [III] 26-75% reduction of the forebrain, [IV] >75% reduction but with recognisable remnant of the forebrain and [V] phenocopy of the complete head truncation (100%) of Dkk1-null embryo (Fig. 4B). In situ hybridisations with Six3, Fgf8, Emx2, Hesx1, Tcf4, En2, Cer1, Foxa2, Lhx1 and Shh riboprobes were performed on between two and eight specimens of each specified genotype as described by Simeone et al. (Simeone et al., 1992), Fossat et al. (Fossat et al., 2007), Lewis et al. (Lewis et al., 2008) and Lavado et al. (Lavado et al., 2008). "
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