Ferdinando Ferdinando Scavizzi

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system’s functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of β-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

Acquisition of detailed structural and molecular information from intact biological samples, while preserving cellular three-dimensional structures, still represents a challenge for biological studies aiming to unravel system functions. Here we describe a novel X-ray-based methodology for analysis of gene expression pattern in intact murine brain ex vivo by microCT. The method relays on detection of bromine molecules in the products of enzymatic reaction generated by the β -galactosidase (lacZ) gene reporter. To demonstrate the feasibility of the method, the analysis of the expression pattern of tRNA endonuclease 54 (Tsen54)-lacZ reporter gene in the whole-mount murine brain in semi-quantitative manner is performed. Mutations in Tsen54 gene causes pontocerebellar hypoplasia (PCH), severe neurodegenerative disorder with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we have demonstrated that highest Tsen54 expression observed in anatomical brain substructures important for the normal motor and memory functions in mice. In the forebrain strong expression in perirhinal, retrosplenial and secondary motor areas was observed. In olfactory area Tsen54 is highly expressed in the nucleus of the lateral olfactory tract, anterior olfactory and bed nuclei, while in hypothalamus in lateral mammillary nucleus and preoptic area. In hindbrain Tsen54 is expressed in the reticular, cuneate and trigeminal nuclei of medulla, and in pontine gray of pons and in cerebellum, in the molecular and Purkinje cell layers. Delineating anatomical brain regions in which Tsen54 is strongly expressed will allow functionally address the role Tsen54 gene in normal physiology and in PCH disease. Significance Statement: Characterization of gene expression pattern in the brain of model organisms is critical for unravelling the gene function in normal physiology and disease. It is performed by optical imaging of the two-dimensional brain sections which then assembled in volume images. Here we applied microCT platform, which allows three-dimensional imaging of non transparent samples, for analysis of gene expression. This method based on detection by X-ray the bromine molecules presented in the products generated by enzymatic activity of b-galactosidase reporter gene. With this method we identify anatomical brain substructures in which Tsen54 gene, mutated in pontocerebellar hypoplasia disease, is expressed.