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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Otx2 </it>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.</p> <p>Conclusion</p> <p>The GFP-tagged <it>Otx2 </it>mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of <it>Otx2 </it>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 <it>Otx2 </it>and the mutated gene.</p

Otx2 Gene Deletion in Adult Mouse Retina Induces Rapid RPE Dystrophy and Slow Photoreceptor Degeneration

International audienceBACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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

Otx2 promotes granule cell precursor proliferation and Shh-dependent medulloblastoma maintenance in vivo.

International audienceThe developmental gene OTX2 is expressed by cerebellar granule cell precursors (GCPs), a cell population which undergoes massive expansion during the early postnatal period in response to sonic hedgehog (Shh). GCPs are thought to be at the origin of most medulloblastomas, a devastating paediatric cancer that arises in the developing cerebellum. OTX2 is overexpressed in all types of medulloblastomas, except in Shh-dependent type 2 medulloblastomas, although it has GCPs as cell-of-origin. This has led to the current view that OTX2 is not involved in tumorigenesis of this subgroup. How OTX2 might contribute to normal or tumoral GCP development in vivo remains unresolved. Here, we have investigated, for the first time, the physiological function of this factor in regulating proliferation and tumorigenesis in the developing mouse cerebellum. We first characterized Otx2-expressing cells in the early postnatal cerebellum and showed that they represent a unique subpopulation of highly proliferative GCPs. We next performed in vivo loss-of-function analysis to dissect out the role of Otx2 in these cells and identified a novel, Shh-independent, function for this factor in controlling postnatal GCP proliferation and cerebellum morphogenesis. Finally, we addressed the function of Otx2 in the context of type 2 medulloblastomas by directing Shh-dependent tumour formation in Otx2+ cells of the developing cerebellum and assessing the effects of Otx2 ablation in this context. We unravel an unexpected, mandatory function for Otx2 in sustaining cell proliferation and long-term maintenance of these tumours in vivo, therefore bringing unpredicted insight into the mechanisms of type 2 medulloblastoma subsistence. Together, these data pinpoint, for the first time, a crucial Shh-independent role for Otx2 in the control of proliferation of normal and tumoral granule cell precursors in vivo and make it an attractive candidate for targeted therapy in Shh-dependent medulloblastomas

The structure of targeting vector (first line), wild type locus (second line), targeted allele after homologous recombination (third line) and allele after removal of the cassette by FLP recombinase (fourth line) is presented

<p><b>Copyright information:</b></p><p>Taken from "A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors"</p><p>http://www.biomedcentral.com/1471-213X/7/122</p><p>BMC Developmental Biology 2007;7():122-122.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2204009.</p><p></p> Gray boxes are coding regions, whites boxes are 5' and 3' UTR regions, yellow box is selection cassette, green box is cDNA. Red triangles are sites. Bent arrows symbolize the three main transcription starts sites known for gene [19, 32]. Dotted lines show BamH I fragments detected by southern blot analysis using probe represented by a thick line. PCR primers used are indicated by arrows. Product obtained with A and B primers is shown. Scale bar and sizes of fragments are indicated. PCR analysis of NeoES clones using primers A and B showing two non-homologous (N) and one homologous (H) recombinants. Southern blot analysis using BamH I digested genomic DNA and probe indicated in of wild type and homologous recombinant clones. Genotypes are indicated. PCR genotyping of mice produced from ES cells. Analyse was done using C, D and E primers. Sizes, schematic representations of amplified fragments (see part for legend) and deduced genotypes are indicated

GFP fluorescence and transmission observations of and control embryos at E6

<p><b>Copyright information:</b></p><p>Taken from "A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors"</p><p>http://www.biomedcentral.com/1471-213X/7/122</p><p>BMC Developmental Biology 2007;7():122-122.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2204009.</p><p></p>5 and E7.5. Anterior is leftward. Scale bars: 100 μm. Direct visualisation of GFP fluorescence (green) and immunofluorescent staining of F-actin (Phalloidin – red) and DNA (Hoechst – blue) on 10 μm thin uterus sections containing E6.5 embryo. Green staining in and around the extraembryonic part of the conceptus is not due to GFP fluorescence but to autofluorescence of decidual and blood cells that are present in the tissue. Images on the right correspond to magnification of white box. Arrow indicates the boundary between embryonic and extra-embryonic part of the conceptus. Dotted line delimits the frontier between anterior visceral endoderm (ave) and epiblast (ep). Anterior is leftward. Scale bars: 50 μm

A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors-3

<p><b>Copyright information:</b></p><p>Taken from "A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors"</p><p>http://www.biomedcentral.com/1471-213X/7/122</p><p>BMC Developmental Biology 2007;7():122-122.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2204009.</p><p></p>NA localisation detected by whole-mount hybridization on animals () at indicated stages. Anterior is leftward. The arrow indicates the position of the midbrain-hindbrain boundary. Lower right panels: direct detection of GFP fluorescence of transverse sections of E12.5 embryo at the level of nasal cavities (), eyecup () and inner ear (). di, diencephalon; mes, mesencephalon; oe, olfactory epithelium; ov, optic vesicle; tel, telencephalon. Scale bars: 500 μm in , 100 μm in