Expression of the inactivating deiodinase, Deiodinase 3, in the pre-metamorphic tadpole retina

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A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain

RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor α1 (TRα1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain

Thyroid Hormone Signaling and Adult Neurogenesis in Mammals

The vital roles of thyroid hormone in multiple aspects of perinatal brain development have been known for over a century. In the last decades, the molecular mechanisms underlying effects of thyroid hormone on proliferation, differentiation, migration, synaptogenesis and myelination in the developing nervous system have been gradually dissected. However, recent data reveal that thyroid signalling influences neuronal development throughout life, from early embryogenesis to the neurogenesis in the adult brain. This review deals with the latter phase and analyses current knowledge on the role of T3, the active form of thyroid hormone, and its receptors in regulating neural stem cell function in the hippocampus and the subventricular zone, the two principal sites harbouring neurogenesis in the adult mammalian brain. In particular, we discuss the critical roles of T3 and TRα1 in commitment to a neuronal phenotype, a process that entails the repression of a number of genes, notably that encoding the pluripotency factor, Sox2. Furthermore, the question of the relevance of thyroid hormone control of adult neurogenesis is considered in the context of brain aging, cognitive decline and neurodegenerative disease

A summary of p<i>dio3</i>-GFP expression and T₃—responsiveness.

<p>A summary of p<i>dio3</i>-GFP expression and T₃—responsiveness.</p

Comparison of p<i>dio3</i>-GFP and <i>TH/bZIP</i>-GFP expression pattern in NF41-42 reporter tadpoles.

<p>Fig 2A–2F: p<i>dio3</i>-GFP transgenic reporter tadpoles, Fig 2G–2L: <i>THbZIP</i>-GFP (T₃ sensor) reporter tadpoles. Fig 2A–2I: lateral plane sections. Fig 2J–2L: median plane section. Fig 2A, 2G and 2J: DAPI/GFP co-staining, Fig 2B and 2H: GABA labeling in horizontal neurons (magenta arrow heads) and amacrine cells (white arrow heads). Fig 2C and 2I: GFP/GABA co-labeling in horizontal neurons (magenta arrow heads) and amacrine cells (white arrow heads). Fig 2D and 2K: Parvalbumin PARV labeling in amacrine cells (white arrow head) and ganglion cells (asterisks). Fig 2E and 2L: GFP/PARV co-labeling in amacrine cells (white arrow head) and ganglion cells (asterisks). Fig 2F: merge of all channels shown in Fig 2A–2E. Yellow arrow heads indicate photoreceptors and blue arrow heads indicate the bipolar neurons. Scale bars: 20 microns.</p

Multiple double-immuno-labeling with GFP (green) of <i>dio3</i> transgenic reporter line and Opsin-Blue or Rhodopsin or ChX10 (red) in NF 48 tadpoles.

<p>Opsin S cones co-expressing GFP and Opsin-S (Fig 3A–3C; magenta arrow: Opsin S cones; Fig 3B, 3C cone expressing GFP and apical cone expressing Opsin S). Rod cells co-expressing GFP and Rhodopsin (Fig 3E–3G; white arrow: rod Fig 3F and 3G body rod expressing GFP and apical rod expressing Rhodopsin). Bipolar neurons co-expressing GFP and ChX10 (white head arrow: Fig 3D, yellow cells); ChX10 channel from Fig 3D in Fig 3H. Scale bars: 20 microns (Fig 3A, 3B, 3D, 3E, 3F and 3H). Scale bars: 10 microns (Fig 3C and 3G).</p

Retinal cell type neurogenesis in pre-metamorphic <i>Xenopus</i> from stage NF33 to stage NF48.

<p>The schema is based on the following references: Chang WS. and Harris WA. 1998, for photoreceptors determination from stages NF33 to NF41; Bilitou A. and Ohnuma S. 2010, for stem cells and retinoblasts from stages NF33 to NF41; Parker RO. et al. 2010, for violet cone opsin from stages NF35 to NF55; Dullin JP. et al. 2007, for horizontal and amacrine cells for stage NF40-41; Bessodes N. et al. 2017, for amacrine cells for NF41; <a target="_blank">Álvarez-Hernán G</a>. et al. 2013, for ganglion cells for stage NF35-36; <a target="_blank">D'Autilia S</a>. et al. 2006, for bipolar neurons for stage NF35-36 (in toto ISH). Results from the current study are used for the different retinal cell-types for stages NF41-48. The key to the different cell types legends is given within the schema.</p

Expression of <i>dio3</i> contributes to modulate T₃ transcriptional response in the developing retina.

<p>Fig 4A. Real-time q-PCR analysis of <i>eGFP</i>, <i>dio3</i>, <i>klf9</i>, <i>thibz</i> and <i>thrb</i> for their T₃ transcriptional response in NF48 eye from reporter transgenic line p<i>dio3</i>-GFP. Gene expression was normalized against <i>odc</i>. mRNA levels from vehicle controls (CTL) were used as reference values. Results pooled from two to three independent experiments are represented as scatter dot plots mean with SD. 14≥n≥6 per group. Non-parametric ANOVA, Kruskall Wallis with uncorrected Dunn’s test (PRISM7) was used to assess statistical significance. *, p<0.05, **, p<0.01; ***, p<0.001. Fig 4B. Working model for the local control of T₃ local availability. Fig 4C. Heatmap of mean expression for each group illustrating the correlation between endogenous <i>dio3</i> expression and <i>eGFP</i> expression in p<i>dio3</i>-GFP tadpole retina.</p