3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.
The Leigh syndrome is a severe inborn neurodegenerative encephalopathy commonly associated with pyruvate metabolism defects. The transcription factor E4F1, a key regulator of the pyruvate dehydrogenase (PDH) complex (PDC), was previously found to be mutated in Leigh syndrome patients, but the molecular mechanisms leading to cell death in E4F1-deficient neurons remain unknown. Here, we show that E4F1 directly regulates Dlat and Elp3, two genes encoding key subunits of the PDC and of the Elongator complex, to coordinate AcetylCoenzyme A production and its utilization to acetylate tRNAs. Genetic inactivation of E4f1 in neurons during mouse embryonic development impaired tRNAs editing and induced an ATF4-mediated integrated stress response (ISR), leading to neuronal cell death and microcephaly. Furthermore, our analysis of PDH-deficient cells unraveled a crosstalk linking the PDC to ELP3 expression that is perturbed in Leigh syndrome patients. Altogether, our data support a model where pyruvate metabolism regulates the epitranscriptome to ensure protein translation fidelity.
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