brain sciences

Article Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression

Olga Ermakova 1,2,*,†, Tiziana Orsini 1,2,† , Paolo Fruscoloni 1,2 , Francesco Chiani 1,2, Alessia Gambadoro 1,2, Sabrina Putti 1,2, Maurizio Cirilli 2, Alessio Mezzi 3 , Saulius Kaciulis 3 , Miriam Pasquini 1,2, Marcello Raspa 1,2, Ferdinando Scavizzi 1,2 and Glauco P. Tocchini-Valentini 1

1 Adriano Buzzati-Traverso Campus, European Mouse Mutant Archive (EMMA), INFRAFRONTIER, Monterotondo Mouse Clinic (MMC), Italian National Research Council (CNR), Via Ramarini, 32, Monterotondo, 00015 Rome, Italy; [email protected] (T.O.); [email protected] (P.F.); [email protected] (F.C.); [email protected] (A.G.); [email protected] (S.P.); [email protected] (M.P.); [email protected] (M.R.); [email protected] (F.S.); [email protected] (G.P.T.-V.) 2 Adriano Buzzati-Traverso Campus, Institute of Biochemistry and Cell Biology (IBBC), Italian National Research Council (CNR), Via Ramarini, 32, Monterotondo Scalo, 00015 Rome, Italy; [email protected] 3 Institute for the Study of Nanostructured Materials, ISMN–CNR, Monterotondo Staz., 00015 Rome, Italy;


[email protected] (A.M.); [email protected] (S.K.)
 * Correspondence: [email protected] † These authors contributed equally to this work. Citation: Ermakova, O.; Orsini, T.; Fruscoloni, P.; Chiani, F.; Gambadoro, A.; Putti, S.; Cirilli, M.; Mezzi, A.; Abstract: Acquisition of detailed anatomical and molecular knowledge from intact biological samples Kaciulis, S.; Pasquini, M.; et al. while preserving their native three-dimensional structure is still a challenging issue for imaging Three-Dimensional X-ray Imaging of studies aiming to unravel a system’s functions. Three-dimensional micro-CT X-ray imaging with a β-Galactosidase Reporter Activity by high spatial resolution in minimally perturbed naive non-transparent samples has recently gained Micro-CT: Implication for increased popularity and broad application in biomedical research. Here, we describe a novel X- Quantitative Analysis of Gene ray-based methodology for analysis of β-galactosidase (lacZ) reporter-driven gene expression in an Expression. Brain Sci. 2021, 11, 746. intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in https://doi.org/10.3390/ the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed brainsci11060746 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 Academic Editor: Allan Bieber activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To

Received: 5 April 2021 demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ Accepted: 27 May 2021 reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 Published: 4 June 2021 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 Publisher’s Note: MDPI stays neutral that the highest Tsen54 expression is observed in anatomical brain substructures important for the with regard to jurisdictional claims in normal motor and memory functions in mice. published maps and institutional affil- iations. Keywords: mouse brain; gene expression; lacZ reporter; X-ray imaging; pontocerebellar hypoplesia; Tsen54; mouse phenotyping

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Molecular phenotyping of gene expression patterns without perturbing the three- distributed under the terms and dimensional (3D) cellular organization in tissue samples is a critical step for unraveling conditions of the Creative Commons gene functions in normal physiology and disease [1]. Usually, histological analysis of gene Attribution (CC BY) license (https:// expression on consecutive 2D sections using light or electron microscopy is the method creativecommons.org/licenses/by/ of choice to accomplish such task. The 2D information is then digitally assembled into 4.0/).

Brain Sci. 2021, 11, 746. https://doi.org/10.3390/brainsci11060746 https://www.mdpi.com/journal/brainsci Brain Sci. 2021, 11, 746 2 of 22

a 3D volume. This methodology suffers from substantial loss of volume information in terms of shape and morphology [2,3]. The application of sophisticated reconstruction methods is then required to correct a distorted 3D volume image obtained from a 2D stack. This computationally demanding step hampers an extensive implementation of the methodology both in research and clinical laboratories [4–7]. Innovative methodologies, such as two-photon microscopy, optical coherence tomog- raphy and confocal microscopy have been recently implemented for imaging of optically transparent tissues obtained by tissue clearing. Tissue clearing techniques make tissues transparent, thus reducing light scattering and absorption. This enables a deeper image acquisition, empowering the volumetric imaging of large-scale biological tissue samples, organs and even entire organisms [8–17]. The combination of tissue clearing with molec- ular labeling methods adds a cell specificity to whole organ and whole body imaging at cellular or subcellular resolution [18]. Despite the fact that tissue clearing methods are constantly evolving, they still suffer from several limitations. The small depth of light penetration through the samples requires the acquisition of images through multiple planes and, consequently, the necessity for the development and application of special algorithms and software in order to achieve correct 3D reconstructions. Sample treatment with harsh reagents might also lead to morphological changes within the anatomical structures [19,20]. Bioluminescence (BLI) is another optical imaging method applied for studies of tissues in normal development and during their pathological transformation [21,22]. It is based on monitoring of luciferase reporter gene expression in genetically modified cells or organisms. The luciferase reporter gene is expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. The variety of engineered forms of luciferase genes from fireflies, beetles and marine organisms and many available substrates provides possibilities for simultaneous labeling of several genes [23]. Thus, it allows visualizing and quantifying molecular and cellular processes such as gene expression, protein–protein interactions, cell proliferation, migration and differentiation in different organs, including the brain, and in small animals in vivo [24]. The current limitation of BLI, however, is the low resolution, making it difficult to discriminate between transduced tissues. This limitation is currently being addressed, and promising studies to improve the resolution in 3D bioluminescence tomography have been recently presented [25]. Hard X-rays, which are characterized by high energy of >10 keV, have the potential to penetrate and image non-transparent thick specimens with high spatial resolution. As a result, X-ray analysis is an attractive and fast-developing method for non-invasive imaging of non-transparent tissues, organs and even whole organisms both in clinics and in research laboratories. At first, X-ray microtomography (micro-CT) imaging was used to analyze mineralized tissues which are characterized by high endogenous X-ray absorption or tissue radiodensity [26,27]. Imaging of soft organs and tissues by hard X-ray remains a challenging task due to the low endogenous radiodensity of soft tissues [28]. Treatment with contrasting stains containing high-atomic number elements and able to diffuse into tissues is necessary to increase the X-ray absorption properties of non-mineralized samples. At present, the most widely used contrasting agent is an iodine-based solution, I2KI or Lugol’s solution. The iodide and triiodide anions interact with (poly-)cationic molecules within the cells and increase the radiodensity of the tissue due to the high atomic number of iodine (Z = 65). Lugol’s solution became one of the most favored stains for X-ray imaging because of its ease of use, low cost and reversibility of the staining. This method was successfully applied for X-ray imaging of most organs and entire embryos of different model organisms [29–31]. Recently, eosin stain was implemented as an effective X-ray contrasting agent for ex vivo imaging of the murine kidney and brain. As with Lugol’s solution, negatively charged eosin molecules non-covalently interact with cationic amino acid side chains of proteins/peptides. The four bromine (Z = 35) atoms contained in the eosin molecule ensure the detectable increase in the radiodensity of stained tissues [32,33]. Several laboratories have demonstrated that fixed and dehydrated soft tissue samples embedded in paraffin, as routinely prepared for light microscopy–based histology, can be non- Brain Sci. 2021, 11, 746 3 of 22

destructively imaged without the need for any contrasting stains either by conventional X-ray attenuation–based micro-CT or by X-ray phase-contrast synchrotron (XPCT) [34–38]. XPCT imaging allows increasing the resolution to the subcellular level with and without contrasting agents [39]. The main anatomical brain structures such as the cortex, hippocampus, thalamus, brain ventricular system and cerebellar layers (molecular, granular and Purkinje cell layers) as well as blood vessels can be visualized by X-ray and segmented based on their endogenous contrasting properties [39,40]. The advances in sample preparation methodologies proceed in parallel with tech- nological developments in hard X-ray imaging modalities, which allow for imaging of biological samples with subcellular, approaching electron microscopy, resolution [39–46]. The next challenge is to add cell specificity potential to X-ray imaging procedures. This can be achieved by implementation of cell-specific labels with different X-ray-detectable radiodensities [47]. There are only a few successful examples of tissue-specific labeling with radiopaque probes published thus far. Two methods are based on metal detection of the peroxidase enzyme, conjugated to antibodies (metal immunodetection). Peroxi- dase reduces silver or nickel ions to an elemental state, resulting in precipitation of metal particles at labeled sites. The silver-based approach was used to visualize regions ex- pressing acetylated-tubulin in the chick developing nervous system and type-II collagen in developing limbs by X-ray imaging [48]. The second is based on nickel-enhanced 3,3-diaminobenzidine (Ni-DAB) detection followed by staining with osmium tetraoxide and uranyl acetate to provide intrinsic contrast and enhance the DAB stain. Such protocol was applied for analysis of Ret expression in wild type and Ret mutant developing (E9,75) embryos [49]. The third report describes a genetic cell-specific targeting of the peroxidase reporter gene to the cellular compartments: peroxidase catalyzes the H2O2-dependent polymerization of 3,30-diaminobenzidine (DAB) into a localized precipitate detectable by X-ray after treatment with OsO4. This gene reporter labeling scheme was used to enable X-ray imaging analysis of neuronal cell types and cellular substructures both in drosophila legs and in the murine brain [39]. A reporter gene assay, based on enzymatic features of a bacterial β-galactosidase (β- gal) enzyme encoded by the bacterial lacZ gene, was first described in the early 1980s [50]. Since then, lacZ is a very common gene reporter used for studies of cell-specific gene expression in bacteria, yeasts, mammalian cell lines and many model organisms. The lacZ reporter assay gained broad popularity in life science mainly due to the high stability of β-gal throughout histological fixation and tissue processing steps. Furthermore, enzymatic products of the β-gal reaction could be detected by a variety of substrates with specific chemical properties, allowing for a remarkable flexibility in the choice of detection methods. Both chromogenic and fluorogenic substrates to detect β-gal activity have been developed over the years and implemented for light-based imaging of different model organisms in vitro and in vivo [51,52]. Furthermore, lacZ reporter gene activity can be detected by magnetic resonance imaging (MRI). For this purpose, a gadolinium-based probe, EgadMe, was synthesized. β-gal cleaves this probe and releases the paramagnetic ion Gd3+, which, via direct interaction with water protons, increases the MR signal. This contrast agent was successfully used to follow lacZ expression in living Xenopus laevis embryos [53]. Among available β-gal substrates, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) is routinely used for reporter expression analysis. β-gal converts the X-gal substrate into 5,50-dibromo-4,40-dichloro-indigo, a blue stable and insoluble chromogenic product. This compound labels the cells expressing the promoter-driven lacZ reporter gene within tis- sues and organs, providing an effective anatomical and cell type-specific labeling of gene expression [54]. Pontocerebellar hypoplasia (PCH) is a heterogeneous group of autosomal recessive neurodegenerative disorders characterized by a wide diagnostic spectrum including delay in cognitive and motor development, seizures and death in early childhood. PCH is morphologically characterized by hypoplasia of the cerebellum and ventral pons, and progressive microcephaly with neocortical atrophy [55]. Brain Sci. 2021, 11, 746 4 of 22

Genetic analysis of PCH patients identified several pathological mutations in a small numbers of genes. The majority of PCH cases have been linked to mutations in genes important for tRNA metabolism. In particular, 60% of all genetically defined cases of PCH are caused by mutations in the genes encoding for protein subunits of the TSEN complex [56–60]. The TSEN complex is composed of four subunits: TSEN54, TSEN34, TSEN15 and TSEN2. The main function of the complex is to remove introns from intron- containing pre-tRNA genes, a critical step for the production of functional tRNA [61]. Recessive mutations in all four subunits of the TSEN complex have been identified in PCH patients. Among these, mutations in the TSEN54 subunit account for 90% of all PCH patients with a mutation in the TSEN complex [57–59,62,63]. In the human developing brain, the TSEN54 gene is highly expressed in the telencephalon, which gives rise to the cerebral cortex and basal ganglia within the cerebral hemispheres and metencephalon, which eventually forms the pons and cerebellum. TSEN54 expression in the telencephalon and metencephalon was detected already at 8 weeks of gestation [62]. Later in development, at 23 weeks of gestation, a strong and specific expression of TSEN54 in human cerebellar neurons has been reported [62]. While TSEN54 mRNA has been extracted and detected in the adult human cortex and cerebellum (https://www.genecards.org/cgi- bin/carddisp.pl?gene=Tsen54 (accessed on 31 May 2021), and www.human.brain-map.org (accessed on 31 May 2021)), histological characterization of the TSEN54 expression pattern in the adult brain was not performed. In model organisms, expression studies demonstrated that the Tsen54 gene was ubiquitously expressed in the developing zebrafish (Danio rerio)[64]. In the murine brain, in situ hybridization data from the Allen Brain Atlas database suggest a very low, difficult to interpret signal (https://mouse.brain-map.org/experiment/show/69352788 (accessed on 31 May 2021). Here, we developed a novel X-ray imaging-based analysis of gene expression of the lacZ reporter gene. We demonstrate that the product of the β-gal/X-gal reaction is a radiodense label for X-ray imaging of lacZ activity in situ. Our results show that the X-ray- detectable probes are the bromine atoms of the β-gal/X-gal reaction product. They produce an elevated local radiodensity within the tissues visualized as an increase in opaqueness. We applied this novel approach and evaluated its feasibility for the analysis of Tsen54-lacZ reporter gene activity in the murine brain. Using laboratory micro-CT, we analyzed murine brains expressing the Tsen54-lacZ reporter gene, previously stained with X-gal, dehydrated and embedded in paraffin. We observed that the combination of β-gal/X-gal staining and paraffin embedding of murine brains allows for both visualization and mapping of lacZ reporter expression to the anatomical brain substructures and regions. Furthermore, we demonstrate that density-based X-ray imaging allows performing a relative quantitative comparison of lacZ presence in different brain subregions within the same brain sample. Using the imaging platform presented here, we mapped and built a 3D view of the highest Tsen54-lacZ expression. These findings will allow focusing further studies on specific brain regions where the Tsen54 gene is expressed with the aim to understand its function in normal development and in PCH disorder.

2. Results 2.1. Three-Dimensional Imaging of Tsen54-lacZ Reporter Gene Expression in Whole-Mount Mouse Brain by X-ray First, we wanted to demonstrate the feasibility of X-ray micro-CT imaging for volumetric studies of gene expression. We analyzed the whole-mount mouse brains from the heterozy- gous Tsen54-lacZ (IKMC-Tsen54Tm1b/+ line) animals. This reporter allele is also a knockout of the Tsen54 gene (Supplementary Figure S1). We previously demonstrated that Tsen54 is an essential gene. Homozygous Tsen54-lacZ animals die immediately at the post-implantation stage [30]. Heterozygous Tsen54-lacZ animals were analyzed by the International Mouse Phenotyping Consortium (IMPC). IMPC performs high-throughput phenotyping of mutant animals, assess- ing many physiological parameters (behavioral, metabolic, immunological, anatomical, etc.) with the goal to reveal as many phenotypes relevant to human disease as possible. The IMPC phenotyping data demonstrated that in the heterozygous Tsen54-lacZ animals, no patholog- Brain Sci. 2021, 11, x FOR PEER REVIEW 5 of 22

2. Results 2.1. Three-Dimensional Imaging of Tsen54-lacZ Reporter Gene Expression in Whole-Mount Mouse Brain by X-ray First, we wanted to demonstrate the feasibility of X-ray micro-CT imaging for volu- metric studies of gene expression. We analyzed the whole-mount mouse brains from the heterozygous Tsen54-lacZ (IKMC-Tsen54Tm1b/+ line) animals. This reporter allele is also a knockout of the Tsen54 gene (Supplementary Figure S1). We previously demonstrated that Tsen54 is an essential gene. Homozygous Tsen54-lacZ animals die immediately at the post-implantation stage [30]. Heterozygous Tsen54-lacZ animals were analyzed by the In- Brain Sci. 2021, 11, 746 ternational Mouse Phenotyping Consortium (IMPC). IMPC performs5 of high 22 -throughput phenotyping of mutant animals, assessing many physiological parameters (behavioral, metabolic, immunological, anatomical, etc.) with the goal to reveal as many phenotypes relevant to human disease as possible. The IMPC phenotyping data demonstrated that in ical phenotypesthe were heterozygous detected. The Tsen54 comprehensive-lacZ animals IMPC, no phenotypingpathological phenotypes data can be found were detected. The on the IMPC webcomprehensive page: (https://www.mousephenotype.org/data/genes/MGI:1923515 IMPC phenotyping data can be found on the IMPC web page: (accessed on 31( Mayhttps://www.mousephenotype.org/data/genes/MGI:1923515 2021)). (accessed on 31 May 2021)). We stained wholeWe stained brains withwhole X-gal brain tos visualizewith X-gal the to brainvisualize regions the brain in which regionslacZ inis which lacZ is expressed. Theexpressed. reaction wasThe reaction performed was in performed the presence in the of presence potassium of potassium ferri- and ferri ferro-- and ferro-cya- cyanides, whichnide ares, necessary which are as necessary electron acceptorsas electron (X-gal/FeCN acceptors (X- staininggal/FeCN protocol) staining inprotocol) the in the re- reaction [54]. Theaction X-gal/FeCN [54]. The staining’sX-gal/FeCN blue staining’s precipitated blue productprecipitated is strong product and is visible strong in and visible in brains from thebrainsTsen54-lacZ from themice,Tsen54 but-lacZ not inmice, wild but type not controls in wild (Figuretype controls1A,B). (Figure 1A,B).

Figure 1. ThreeFigure-dimensional 1. Three-dimensional imaging of Tsen54 imaging-lacZ of expressionTsen54-lacZ in expressionintact adult inmouse intact brain adult by mousemicro-CT brain. (A) by Dorsal and ventral viewsmicro-CT. of Tsen54 (A-)lacZ Dorsal mouse and brain ventral stained views with of Tsen54-lacZ X-gal/FeCNmouse and visualized brain stained by stereomicroscope; with X-gal/FeCN (B and) dorsal and ventral views of littermate control whole mouse brain stained with X-gal/FeCN and visualized by stereomicroscope. (C) visualized by stereomicroscope; (B) dorsal and ventral views of littermate control whole mouse Representative maximum intensity projection (MIP) micro-CT images scanned with the resolution 5 m/voxel of whole brain stained with X-gal/FeCN and visualized by stereomicroscope. (C) Representative maximum intensity projection (MIP) micro-CT images scanned with the resolution 5 µm/voxel of whole brain from Tsen54-lacZ (N = 3) and wild type (WT) (N = 3) animals. (D) Sagittal view of MIP images of the brains represented in panel (C). Additional regions of increased density (indicated by white arrows) in Tsen54-LacZ brains correspond to precipitated product of X-gal substrate converted by β-gal. White arrows indicate the regions of the elevated density in Tsen54-lacZ brains imaged by micro-CT and corresponding regions in the brains imaged by microscopy. The arrows filled with black indicate the endogenous densities detected both in the wild type and experimental brains.

We examined whether the deposited products of the β-gal reaction provide sufficient X-ray radiopacity. Hence, we performed micro-CT scans of the whole-mount X-gal/FeCN- stained, dehydrated and paraffin-embedded brains from Tsen54-lacZ and wild type animals. Micro-CT imaging detected the specific radiopaque regions of increased density in the Tsen54-lacZ brains, which were not observed in wild type controls (Figure1C,D). Strikingly, our maximum intensity projection (MIP) of micro-CT analysis showed an overall good agreement with light stereomicroscopy images of the mouse brains (Figure1A), both in Brain Sci. 2021, 11, 746 6 of 22

frontal and sagittal 2D (Figure1C,D) and in 3D views (Movies S1 and S2). This inspection revealed the differences in the distribution of X-ray-detected opaqueness when wild type and Tsen54-lacZ brains were compared. We hypothesize that additional regions of X-ray opaqueness in Tsen54-lacZ brains are due to X-ray detection of the lacZ/X-gal reaction product deposited in situ.

2.2. Comparative Analysis of Virtual 2D Micro-CT and Histological Sections in the X-gal/FeCN Tained Brains The micro-CT image is a 3D matrix of density measurements, expressed in grayscale/ brightness values calculated for every voxel. The volume image is digitally reconstructed from multiple 2D sections. Therefore, the brain volume can be computationally sliced, and corresponding 2D sections derived from different scans can be compared (Figure2). To determine the differences in radiopaqueness between Tsen54-lacZ and wild type brains, we compared virtual 2D cerebrum sagittal sections from Tsen54-lacZ and wild type scans. Additional areas of high opaqueness in Tsen54-lacZ images absent in the wild type images were observed (Figure2A). Then, we performed comparative quantitative analysis of Tsen54-lacZ images (Figure2B). We subtracted the grayscale values calculated from wild type sections from the Tsen54-lacZ images using ImageJ. To compute this analysis, every image was segmented based on endogenous gray value differences in the following substructures: cortex, central brain region, hippocampus and amygdala. For each region, the mean and standard deviation of grayscale values were calculated separately. The sum of the mean and one standard deviation of wild type calculated grayscale values was set as the background correction threshold and subtracted from the mean grayscale values of every corresponding region of the Tsen54-lacZ image. The gray values obtained by this subtraction are a measure of the brightness differences between Tsen54-lacZ and analogues wild type 2D images (Figure2B). To demonstrate that the X-ray opaqueness is indeed produced by the β-gal reaction products deposited in situ, we performed a histological analysis of the Tsen54-lacZ brain sample. For this purpose, X-gal/FeCN-stained and paraffin-embedded brains, previously analyzed by micro-CT, were sectioned and reanalyzed by light microscopy (Figure2C). We aligned corresponding 2D virtual micro-CT with histological sections. Our analysis demonstrated there is an agreement between areas having high X-ray opaqueness and those showing the presence of β-gal/X-gal/FeCN blue chromogenic precipitate (Figure2B,C). Importantly, the histological analysis of wild type littermates’ brains did not show any chromogenic product of lacZ activity (data not shown) in agreement with previously published data [56]. We have to mention here that histological analysis provides cellular resolution, while the micro-CT analysis implemented here detects only the regions in which lacZ expression can be detected. This limitation can be overcome by using a synchrotron-based X-ray imaging methodology combined with lacZ reporter expression analysis. Next, we analyzed the 2D micro-CT sections from the hindbrain region (Figure3A) . The hindbrain was manually segmented in two subregions: (1) cerebellar lobes and (2) brainstem. We found that separate analyses of these two segments were necessary because of the endogenous substantial divergence of the medium gray values between the two hindbrain substructures. The background correction scheme described above was ap- plied: the sum of the mean and one standard deviation of the wild type’s grayscale values of each of the two subregions was subtracted from the mean value of the corresponding subregion of the Tsen54-lacZ image. The background-corrected grayscale values are the X-ray-detected opaqueness above the background (Figure3B). Comparison of this resulting image to the best corresponding histological section demonstrates that this high density results from in situ deposition of the β-gal/X-gal reaction product (Figure3C). Brain Sci. 2021, 11, 746 7 of 22 Brain Sci. 2021, 11, x FOR PEER REVIEW 7 of 22

Figure 2.2. ComparisonComparison of of virtual virtual X-ray X-ray sections sections to histologicalto histological sections sections of Tsen54-lacZ of Tsen54cerebrum-lacZ cerebrum reveals reveals X-ray-detected X-ray-detected regions ofregions reporter of reporter gene expression. gene expression. (A) Two-dimensional (A) Two-dimensional micro-CT-derived micro-CT sections-derived from sections cerebrum from ofcerebrum wild type of ( aw–cild) and typeTsen54- (a–c) lacZand Tsen54(d–f) animals.-lacZ (d– (fB) )animals. Segmentation (B) Segmentation analysis of theanalysis virtual of micro-CT-derivedthe virtual micro-CT sections-derived from sectionsTsen54-lacZ from Tsen54brains- (lacZd0–f 0brains). The lines(d′–f′ delineate). The lines the delineate segmentation the segmentation regions: yellow—cortex; regions: yellow red—hippocampal—cortex; red— plate;hippocampal blue—amygdala; plate; blue green—central—amygdala; brain. green The— Lookupcentral brain. Table The (LUT) Lookup function Table was (LUT) set from function a 0 to was 155 grayscaleset from a value 0 to 155 interval. grayscale (C) Correspondingvalue interval. ( histologicalC) Corresponding sections histo- from thelogicalTsen54-lacZ sectionsbrain from whole-mountthe Tsen54-lacZ stained brain withwhole X-gal-mount and stained counterstained with X- withgal and eosin counterstained solution (d00– fwith00). Micro-CT eosin solution scans were(d′′–  performedf′′). Micro-CT with sc theans resolutionwere performed 20 µm/voxel. with the resolution 20 m/voxel.

To demonstrate further verify that that the the X increase-ray opaqueness in contrast is indeed produced produced by X-gal/FeCN by the β-gal staining reaction is productsdetected bydeposited X-ray, we in focused situ, we on performed a smaller regiona histological within molecularanalysis of and the granular Tsen54-lacZ cerebellar brain sample.layers (Figure For this3D). purpose, To do this, X-gal/FeCN we applied-stained alternative and paraffin experimental-embedded analysis. brains, The previously regions analyzedof molecular by micro and granular-CT, were layers sectioned of the and simple reanalyzed cerebellar by lobe light were microscopy compared (Figure using 2C the). WeBruker aligned CTAN corresponding program (Figure 2D virtual3E). The micro gray-CT pixel with values histological were firstsections. corrected Our a fornalysis the demonstratedbackground caused there byis paraffinan agreement in which between the samples areas were having embedded. high X-ray The opaqueness average paraffin and thosebackground showing gray the value presence was calculated of -gal/X- fromgal/FeCN a 300 blueµm-long chromogenic stretch of precipitate pixels taken (Figure in an 2B,C).area occupied Importantly, by paraffin the histological only. In orderanalysis to obtainof wild background-corrected type littermates’ brains gray did values not show for anymolecular chromogenic and granular product layers of lacZ of the activity cerebellar (data lobe, not shown) we selected in agreement three parallel with 300 previouslyµm-long publishedand 1 voxel-wide data [56]. stretches We have separated to mention by 2 here voxels that (10 histologicalµm) crossing analysis the boundary provides between cellular resolution,molecular and while granular the micro layers-CT (Supplementary analysis implemented Figure S1). here The detects paraffin only background the regions pixel in whichaverage lacZ value expression was subtracted can be from detected. every This pixel limitation in each stretch, can be the overcome mean and by standard using a errorsyn- chrotronwere calculated-based X for-ray three imaging related methodology adjacent pixels combined in each with line lacZ and reporter final values expression were plotted anal- ysis.(Figure 3E). This analysis demonstrated an increase in gray values in the molecular layer, withNext, the maximum we analyzed reached the at2D the micro boundary-CT sections between from molecular the hindbrain and granular region (Figure layers in 3A). the TheTsen54-lacZ hindbraincerebellum. was manually No differences segmented in in brightness two subregions: between (1) wild cerebellar type and lobesTsen54-lacZ and (2) brainstem.cerebellar lobesWe found were that observed separate in theanalyses granular of these layer two (Figure segments3E). The were micro-CT necessary grayscale because ofvalues the endogenous comparison issubstantial in agreement divergence with the histologicalof the medium analysis gray showing values thatbetween deposition the two of hindbrainthe chromogenic substructures. product ofThe the backgroundβ-gal/X-gal correction reaction occurs scheme within described the internal above molecular was ap- plied:layer andthe sum between of the molecular mean and and one granularstandard layersdeviation (Figure of the3F). wild In conclusion,type’s grayscale our results values Brain Sci. 2021, 11, x FOR PEER REVIEW 8 of 22

Brain Sci. 2021, 11, 746 8 of 22

of each of the two subregions was subtracted from the mean value of the corresponding subregion of the Tsen54-lacZ image. The background-corrected grayscale values are the X- raydemonstrate-detected opaqueness that the product above of the the background biochemical (Figureβ-gal/X-gal 3B). Comparison reaction performed of this resulting in the imagepresence to ofthe FeCN best corresponding acceptors produces histological a radiodense, section contrasting demonstrate agents that in situthis detectablehigh density by resultsX-ray imaging. from in situ deposition of the -gal/X-gal reaction product (Figure 3C).

Figure 3. ComparativeComparative analysis of virtual X X-ray-ray and histological sections in hindbrain region. ( A) Representative Representative 2D virtual sections from hindbrain region of wild type (a) andand Tsen54-lacZTsen54-lacZ ((bb)) brains.brains. Segmentation lines are indicated: yellow yellow—— cerebellar lobes,lobes, and red—brainstem.red—brainstem. ResolutionResolution ofof thethe micro-CTmicro-CT scanscan isis 2020µ m/voxel.m/voxel. ( B)) Segmentation Segmentation analysis of the 2D micro-CTmicro-CT hindbrain section. The LUT function was set from a 0 to 155 grayscalegrayscale value interval. ( C) Histological section of hindbrain from the Tsen54-lacZ brain. The regions with the highest expression in brainstem are indicated: CU—cuneate hindbrain from the Tsen54-lacZ brain. The regions with the highest expression in brainstem are indicated: CU—cuneate nucleus; Sp5—spinal nucleus of trigeminal; LRN—lateral reticular nucleus. (D) Two-dimensional micro-CT-acquired im- nucleus; Sp5—spinal nucleus of trigeminal; LRN—lateral reticular nucleus. (D) Two-dimensional micro-CT-acquired age of the simple cerebellar lobule from the Tsen54-lacZ and wild type animals at 5 m/voxel resolution. The punctate lines µ imageindicate of the the regions simple selected cerebellar for lobule density from quantification. the Tsen54-lacZ Arrowsand wildindicate type the animals cerebellar at 5 lovem/voxel layers. resolution. (E) Average Thes of punctate bright- linesness values, indicate corrected the regions for selectedbackground, for density from Tsen54 quantification.-lacZ (N = Arrows3) and wild indicate type the(N = cerebellar 3) cerebellar love lobules, layers. (wereE) Averages plotted as of brightnessa function values,of distance. corrected (F) Histological for background, analysis from ofTsen54-lacZ the simple(N cerebellar = 3) and wildlobule type of (Ncerebellum = 3) cerebellar from lobules,the whole were-mount plotted X- asgal/FeCN a function stained of distance. brains. ML (F)— Histologicalmolecular layer analysis; PCL of— thePurkinje simple cell cerebellar layer; and lobule GL— ofgranular cerebellum layer from. the whole-mount X-gal/FeCN stained brains. ML—molecular layer; PCL—Purkinje cell layer; and GL—granular layer. To further verify that the increase in contrast produced by X-gal/FeCN staining is detected2.3. Relative by X Quantitative-ray, we focused Analysis on ofa smaller the Tsen54-lacZ region within Gene Activity molecular in Murine and granular Brain cerebel- lar layersX-ray-based (Figure imaging3D). To hasdo this a unique, we applied feature amongalternative imaging experimental techniques. analysis. X-ray imagingThe re- gionallowss of for molecular a direct volumetricand granular density layers quantification of the simple ofcerebellar an aggregated lobe were contrasting compared agent using in thesitu. Bruker In our CTAN case, this program means (Figure that the 3E). experimental The gray densitiespixel values are were directly first proportional corrected for to the backgroundlacZ reporter caused activity. by The paraffin reconstructed in which X-ray the brightnesssamples were value embedded. at each voxel The of average the β-gal/X- par- affingal-contrasted background sample gray isvalue the sum: was calculated (1) of the brightness from a 300 derived m-long from stretch deposited of pixels products taken ofin anthe areaβ-gal occupiedenzymatic by reaction paraffin and only. (2) In endogenous order to obtain radiopaqueness background of-corrected the dehydrated gray values tissue. forIn thismolecular instance, and the granular simple layers subtraction of the cerebellar of the grayscale lobe, we value selected of non-enhanced three parallel contrast300 m- longtissue and from 1 voxel contrast-enhanced-wide stretches tissue separated would by return 2 vox theels (10 absolute m) crossing quantity the of theboundary deposited be- tweencontrast molecular agent at each and voxel. granular layers (Supplementary Figure S1). The paraffin back- groundIn thispixel study, average we value used was a Bruker subtracted Skyscan from 1172 every X-ray pixel micro-CT in each stretch, system. the To mean perform and standarddensity comparison, error were calculated we used calibratedfor three related datasets. adjacent Every pixels dataset in waseach calibratedline and final using val- a uesphantom were plotted made of (Figure distilled 3E water,). This withanalysis a known demonstrated density of an Hounsfield increase in units gray (HU) values = 0.in Thethe molecularwater sample layer was, with scanned the maximum under the samereached conditions at the boundary used for brain between samples, molecular the mean and granulargrayscale layers value in of the Tsen54 water sample-lacZ cerebellum. was calculated No differences and the brightness in brightness values between of each wild scan typewere and normalized Tsen54-lacZ as describedcerebellar inlobe thes were Bruker observed manual in ( theBruker-micro-CT granular layer Method (Figure Note: 3E). The HU microcalibration,-CT gray 2005scale) both values for Tsen54-lacZ comparisonand is wild in agreement type brains. with In this the study,histological we adopted analysis a showingrelative comparison that deposition of brightness of the chromogenic values within product the same of Tsen54-lacZthe β-gal/Xbrain-gal reaction to evaluate occurs the gene expression activity among different brain regions of the same dataset. Brain Sci. 2021, 11, 746 9 of 22

Using the calibrated datasets, we were able to perform relative quantitative compari- son of the Tsen54-lacZ reporter activity. Furthermore, we conducted comparative analyses of densities’ distribution between wild type and experimental datasets. To accomplish the above tasks, the brains were first divided into two regions of interest (ROIs)—cerebrum (ROIc) and hindbrain (ROIh). Using CTAN Bruker software, means and standard devia- tions of gray value distributions for each ROI were calculated separately. The grayscale values of the paraffin background were excluded by applying the OTSU method (auto- matic image thresholding method) using Bruker software (CT-Analyser V1.13 UserManual). Then, the sum of the mean and one standard deviation calculated for the Tsen54-lacZ brain was defined as the threshold. The grayscale values above the threshold were subdivided into three grades: from 1SD to 2SD (low brightness values); 2SD to 3SD (intermediate); and from 3SD to maximum (highest). The two ROIs were treated independently and then assembled in a single volume image using Bruker CTVOL software (Figure4A). The same values were also used for segmentation analysis of wild type brains (Figure4B). Using this approach, we were able to perform a relative quantification of Tsen54-lacZ gene expression and classify the brain regions with high, medium and low activity of the reporter gene (Movie S3). These results demonstrate that the lacZ reporter enables identification of brain Brain Sci. 2021, 11, x FOR PEER REVIEWregions in which the Tsen54 gene is expressed within X-ray images and allows10 for of the22

relative comparison of the gene expression level in the specific brain regions.

FigureFigure 4.4. RelativeRelative quantification quantification of ofTsen54-lacZ Tsen54-lacZgene gene expression expression by micro-CT by micro analysis.-CT analysis (A,B) The. (A,B average) The average grayscale gray valuesscale werevalues calculated were calculated using three using datasets three datasets from the fromTsen54-lacZ the Tsen54brains-lacZ using brains CTAN using (Bruker) CTAN (Bruker) software. software. The sum The of thesum average of the meanaverage and mean one standard and one deviationstandard wasdeviation set up was as a backgroundset up as a background of non-contrasted of non tissues-contrasted (gray), tissues while values(gray), outsidewhile values of this outside of this range were color-coded: 1SD(Standard Deviation) to 2SD—blue (lowest brightness); 2SD to 3SD—magenta range were color-coded: 1SD(Standard Deviation) to 2SD—blue (lowest brightness); 2SD to 3SD—magenta (intermediate); (intermediate); and 3SD and above (maximum)—yellow. These calculated values were applied to the datasets obtained and 3SD and above (maximum)—yellow. These calculated values were applied to the datasets obtained from the Tsen54-lacZ from the Tsen54-lacZ (A) and wild type (B) brains. Resolution of the micro-CT original images is 20 m/voxel. (A) and wild type (B) brains. Resolution of the micro-CT original images is 20 µm/voxel. 2.4. Bromine Atoms in the -gal/X-gal/FeCN Reaction Product are the Radiodense Substrates for X-ray Imaging The methodology presented here couples the chemical detection of -gal enzymatic activity, that converts the X-gal substrate in chromogenic 5,5′-dibromo-4,4′-dichloro-in- digo in the presence of ferri- and ferro-cyanides, with an in situ molecular signal detected as an increase in the X-ray attenuation coefficient within the brain. Thereby, the attenua- tion coefficients can be digitally converted into gray or brightness values. We aimed to determine what molecular substrate is responsible for the radiodensity detected by X-ray imaging in our samples. The biochemistry of the -ga/X-gal/FeCN reac- tion has been extensively studied since the early 1950s [65,66]. In brief, under proper con- ditions, the -gal enzyme breaks the -D-glycosidic linkage of X-gal, releasing a soluble, colorless indolyl monomer. Subsequently, two liberated indolyl moieties can form a di- mer, if a proper acceptor, to which they can transfer an electron, is present in solution (Figure 5A). The ferric and ferrous ions are included in most X-gal reaction buffers exactly to provide the electron acceptor function to the solvent [67]. The above chemical steps produce a very stable, insoluble, bluish compound, which is an optically detectable -gal reporter at the site of reaction. We wondered which compound, among the -gal/X-gal reaction products, is able to adsorb the X-rays and acts as an X-ray reporter molecule in our experiments. The first hypothesis was that the presence of the electron transfer within the ferric/ferrous mix (FeCN) added to the X-gal reaction can lead to insoluble iron-con- Brain Sci. 2021, 11, 746 10 of 22

2.4. Bromine Atoms in the β-gal/X-gal/FeCN Reaction Product Are the Radiodense Substrates for X-ray Imaging The methodology presented here couples the chemical detection of β-gal enzymatic activity, that converts the X-gal substrate in chromogenic 5,50-dibromo-4,40-dichloro-indigo in the presence of ferri- and ferro-cyanides, with an in situ molecular signal detected as an increase in the X-ray attenuation coefficient within the brain. Thereby, the attenuation coefficients can be digitally converted into gray or brightness values. We aimed to determine what molecular substrate is responsible for the radiodensity detected by X-ray imaging in our samples. The biochemistry of the β-ga/X-gal/FeCN reaction has been extensively studied since the early 1950s [65,66]. In brief, under proper conditions, the β-gal enzyme breaks the β-D-glycosidic linkage of X-gal, releasing a soluble, colorless indolyl monomer. Subsequently, two liberated indolyl moieties can form a dimer, if a proper acceptor, to which they can transfer an electron, is present in solution (Figure5A). The ferric and ferrous ions are included in most X-gal reaction buffers exactly to provide the electron acceptor function to the solvent [67]. The above chemical steps produce a very stable, insoluble, bluish compound, which is an optically detectable β-gal reporter at the site of reaction. We wondered which compound, among the β-gal/X-gal reaction products, Brain Sci. 2021, 11, x FOR PEER REVIEW 11 of 22 is able to adsorb the X-rays and acts as an X-ray reporter molecule in our experiments. The first hypothesis was that the presence of the electron transfer within the ferric/ferrous mix (FeCN) added to the X-gal reaction can lead to insoluble iron-containing products, taining products, such as FeIII, [FeIIIFeII(CN)6]3, similar to the reaction which occurs dur- such as FeIII, [FeIIIFeII(CN)6]3, similar to the reaction which occurs during Prussian blue ing Prussian blue synthesis [68]. We wondered if the hypothetical production of FeIII dur- synthesis [68]. We wondered if the hypothetical production of FeIII during the reaction ing the reaction could be responsible for the increase in X-ray absorption at the sites of the β could-gal be reaction. responsible for the increase in X-ray absorption at the sites of the -gal reaction.

FigureFigure 5. XPS 5. XPS analysis analysis of of the the β-galactosidase-galactosidase enzymatic enzymatic reaction reaction products. products. (A) Schematic (A) Schematic representation representation of the -gal of reac- the β-gal tion in vitro. (B–D) X-ray photoemission spectroscopy (XPS) analysis of the -gal reaction products: black lines—-gal reaction in vitro.(B–D) X-ray photoemission spectroscopy (XPS) analysis of the β-gal reaction products: black lines—β-gal reaction performed with X-gal/FeCN substrate; red lines—X-gal/FeCN substrate only; (B) complete survey spectra of the reaction-gal performed reaction products; with X-gal/FeCN (C,D) regions substrate; of main photoemission red lines—X-gal/FeCN spectra Fe 2p substrate (C) and Br only; 3d ( (DB) peaks; complete black survey lines— spectrareaction of the β-galof reaction -gal with products; X-gal/FeCN (C,D substrate;) regions red of mainlines— photoemissionX-gal/FeCN substrate spectra only. Fe 2p KLL (C-) auger and Br transition; 3d (D) peaks; -gal- black-galactosidase. lines—reaction of β-gal with X-gal/FeCN substrate; red lines—X-gal/FeCN substrate only. KLL- auger transition; β-gal-β-galactosidase. To determine the molecules that could generate the high-contrast signal, we exam- ined the atomic composition of the -gal/X-gal/FeCN reaction products using X-ray pho- toemission spectroscopy (XPS). The atomic components of the reactions with and without pure -gal in the presence of the X-gal/FeCN substrate were analyzed in vitro (Figure 5B– D). The XPS measurements excluded the presence of FeIII in the products of the -gal reaction because Fe 2p3/2 peak at BE = 708.7 eV (Figure 5C) and there is absence of satellite peaks at a higher BE (lying at ~6 eV or ~8 eV from the main component), which are char- acteristic for FeII [69]. Indeed, no differences in the atomic composition between reactions performed in the presence of -gal and control (substrate only) were detected. To confirm this finding, we replaced FeCN solution with tetranitroblue tetrazolium chloride as an alternative electron acceptor substrate in the X-gal staining reaction. Tsen54- lacZ and wild type control brains were stained with X-gal/tetrazolium, dehydrated, em- bedded in paraffin and imaged by micro-CT (Figure 6). The datasets from Tsen54-lacZ/X- gal/tetrazolium-stained brains were calibrated using water as a phantom and segmented, using the CTAN program. The methodology for segmentation is described above. The grayscale values below the sum of the mean and 1SD were considered as a background, and values above were defined in three ranges: low from 1SD to 2SD; intermediate from Brain Sci. 2021, 11, 746 11 of 22

To determine the molecules that could generate the high-contrast signal, we examined the atomic composition of the β-gal/X-gal/FeCN reaction products using X-ray photoemis- sion spectroscopy (XPS). The atomic components of the reactions with and without pure β-gal in the presence of the X-gal/FeCN substrate were analyzed in vitro (Figure5B–D). The XPS measurements excluded the presence of FeIII in the products of the β-gal reaction because Fe 2p3/2 peak at BE = 708.7 eV (Figure5C) and there is absence of satellite peaks at a higher BE (lying at ~6 eV or ~8 eV from the main component), which are characteristic for FeII [69]. Indeed, no differences in the atomic composition between reactions performed in the presence of β-gal and control (substrate only) were detected. To confirm this finding, we replaced FeCN solution with tetranitroblue tetrazolium chloride as an alternative electron acceptor substrate in the X-gal staining reaction. Tsen54- lacZ and wild type control brains were stained with X-gal/tetrazolium, dehydrated, em- bedded in paraffin and imaged by micro-CT (Figure6). The datasets from Tsen54-lacZ/X- gal/tetrazolium-stained brains were calibrated using water as a phantom and segmented, using the CTAN program. The methodology for segmentation is described above. The grayscale values below the sum of the mean and 1SD were considered as a background, and values above were defined in three ranges: low from 1SD to 2SD; intermediate from 2SD to 3SD; and high above 3SD. We applied those calculations both for the analysis of virtual 2D micro-CT sections (Figure6) and to the entire brain datasets (Movie S4). The comparison between the X-gal/tetrazolium and X-gal/FeCN methods demonstrated similar patterns of brightness distributions in Tsen54-lacZ brains. This finding holds true both for the regions in which Tsen54-lacZ gene expression was detected and for the rela- tive quantitative estimation of intensities derived from reporter gene activity in specific brain areas (Movies S3 and S4). Importantly, the reproducible expression pattern of the Tsen54-lacZ reporter imaged by micro-CT was obtained irrespective of which staining methods and segmentation protocols were applied ( Figure3; Figure6, Movies S3 and S4). We conclude that the bromine atoms contained in the chromogenic product of the β- gal/X-gal reaction are in situ radiodense probes for X-ray imaging of lacZ reporter activity. Indeed, the mass attenuation coefficient of the Br atom at 40 kV (energy used in our micro-CT scan setting) is 6.832 µ/ρ, while the gray matter of the brain tissues is 25-fold lower and is 0.270 µ/ρ (https://www.nist.gov/pml/x-ray-mass-attenuation-coefficients (accessed on 31 May 2021)). This explains the elevated density detected by X-ray at the site of the 5,50-bromo-4,40-chloro-3-dichloro-indigo deposition in situ in the brain regions. To conclude, we demonstrate here that the β-gal/X-gal reaction product containing bromine atoms provides an in situ high-contrast enhancement measurable by X-ray imaging.

2.5. Three-Dimensional Map of the Brain Regions Characterized by Specific Tsen54-lacZ Reporter Gene Expression Based on the expression data presented in this manuscript, we performed a mapping of the brain regions with established Tsen54-lacZ expression. The 3D view of Tsen54-lacZ reporter expression was manually built by analyzing both 3D and 2D micro-CT imaging modalities and histological sections. The regions with Tsen54-lacZ expression were determined using the 2D and 3D Allen Brain Virtual Reference Atlas tool (http://atlas.brain-map.org/ (accessed on 31 May 2021)) (Movie S5). The anatomical structures characterized by the highest and intermediate levels of Tsen54 expression are summarized in Table1. Brain Sci. 2021, 11, x FOR PEER REVIEW 12 of 22

2SD to 3SD; and high above 3SD. We applied those calculations both for the analysis of virtual 2D micro-CT sections (Figure 6) and to the entire brain datasets (Movie S4). The comparison between the X-gal/tetrazolium and X-gal/FeCN methods demonstrated simi- lar patterns of brightness distributions in Tsen54-lacZ brains. This finding holds true both for the regions in which Tsen54-lacZ gene expression was detected and for the relative quantitative estimation of intensities derived from reporter gene activity in specific brain areas (Movies S3 and S4). Importantly, the reproducible expression pattern of the Tsen54- lacZ reporter imaged by micro-CT was obtained irrespective of which staining methods and segmentation protocols were applied ( Figure 3; Figure 6, Movies S3 and S4). We con- clude that the bromine atoms contained in the chromogenic product of the -gal/X-gal reaction are in situ radiodense probes for X-ray imaging of lacZ reporter activity. Indeed, the mass attenuation coefficient of the Br atom at 40 kV (energy used in our micro-CT scan setting) is 6.832 μ/ρ, while the gray matter of the brain tissues is 25-fold lower and is 0.270 μ/ρ (https://www.nist.gov/pml/x-ray-mass-attenuation-coefficients (accessed on 31/05/2021)). This explains the elevated density detected by X-ray at the site of the 5,5′- Brain Sci. 2021, 11, 746 bromo-4,4′-chloro-3-dichloro-indigo deposition in situ in the brain regions. To conclude,12 of 22 we demonstrate here that the -gal/X-gal reaction product containing bromine atoms pro- vides an in situ high-contrast enhancement measurable by X-ray imaging.

Brain Sci.Brain 2021 Sci., 11Brain2021, x ,FOR Sci.11, x 2021PEER FOR, 11 PEERREVIEW, x FOR REVIEW PEER REVIEW 13 of 1322 of 22 13 of 22 Brain Sci. 2021, 11 , x FOR PEER REVIEW 13 of 22

2.5. Three2.5. Three-Dimensional2.5.-Dimensional Three- MapDimensional ofMap the of Brain theMap Brain Regions of the Regions Brain Characteri RegionsCharacterized Characteri byz edSpecific by Specificz edTsen54 by SpecificTsen54-lacZ - Re-lacZTsen54 Re--lacZ Re- FigureFigure 6. VirtualVirtual2.5. Three micro-CTmicro-Dimensional-CT sectionssections Map fromfrom of thethe theTsen54-lacZ Tsen54 Brain -RegionslacZmurine murine Characteri cerebrum cerebrumzed stained stainedby Specific with with X-gal Tsen54 X-gal in the-inlacZ the presence Re-presence of tetrazoliumof tetrazo- lium as anporter electronporter Gene acceptor. GeneExpressionporter Expression ( AGene) Virtual Expression micro-CT sections of murine cerebrum from Tsen54-lacZ (a–c) and wild type (d– as an electronporter acceptor. Gene Expression (A) Virtual micro-CT sections of murine cerebrum from Tsen54-lacZ (a–c) and wild type (d–f) brains. f) brains. Whole-mount brains were stained with X-gal in the presence of tetratzolium, and volume images were acquired Whole-mount brainsBasedBased wereon the stainedonBased expression the expression withon the X-gal data expression inpresenteddata the presented presence data in presentedthis of in tetratzolium,manuscript, this manuscript, in this andmanuscript,we performed volume we performed images we a performedmapping were a mapping acquired a mapping with with the resolutionBased of 20 on  them/voxel. expression (B) Segmentation data presented analysis in of this the manuscript, micro-CT-imaged we performed brains was aperformed mapping using CTAN of theof brain the brain regionsof the regions brain with regions withestablished established with Tsen54established Tsen54-lacZ - expressionlacZTsen54 expression-lacZ. Theexpression. 3DThe view 3D. Theview of Tsen54 3D of view Tsen54-lacZ of - lacZTsen54 -lacZ (Bruker)the resolution softwareof the of brain 20onµ 2Dm/voxel. regions sections with (B from) Segmentation established Tsen54-lacZ analysisTsen54 (a′–c′) -andlacZ of the wild expression micro-CT-imaged type brains. The (d 3D′–f brains′ ).view Sum was ofof Tsen54mean performed and-lacZ one using standard CTAN reporterreporter expressionreporter expression was expression manuallywas manually was built manually 0 builtby0 analyzing by built analyzing by both analyzing both3D and 3D 0both 2Dand0 micro 3D2D andmicro-CT 2D imaging-CT micro imaging- CT imaging deviation(Bruker) softwarereporter(1SD) calculated on expression 2D sections from wasan from entire manuallyTsen54-lacZ dataset built w(asa by–definedc )analyzing and as wild a background typeboth brains 3D and ( (grayd –2Df color)). micro Sum; intervals of-CT mean imaging between and one standard1SD and modalitiesmodalities andmodalities histologicaland histological and sections.histological sections. The sections. regionsThe regions withThe regions withTsen54 Tsen54 -withlacZ - expressionlacZTsen54 expression-lacZ were expression were deter- deter- were deter- 2SDdeviation—blue;modalities (1SD) 2SD and calculated 3SD and— magenta;histological from an entireand sections. 3SD dataset and maximum wasThe definedregions—yellow. aswith a background Tsen54 The same-lacZ values (gray expression color); were applied intervals were for deter- between segmentation 1SD and of minedmined using usingmined the 2D the using and 2D 3Dtheand Allen2D 3D andAllen Brain 3D Brain AllenVirtual Virtual Brain Reference VirtualReference Atlas Reference Atlas tool (toolhttp://atlas.brain Atlas (http://atlas.brain tool (http://atlas.brain- - - the2SD—blue; micro-minedCT 2SD-derived and using 3SD—magenta; sections the 2D from and the and 3D wild 3SDAllen type and Brain brains. maximum—yellow. Virtual SD—standard Reference The deviation; same Atlas values Scaletool werebar(http://atlas.brain— applied1 mm. for segmentation- of map.org/map.org/ (accessedmap.org/ (accessed on (accessed 31/05/2021 on 31/05/2021 on)) 31/05/2021(Movie)) (Movies S5).)) s( TheMovieS5). anatomicalThes S 5).anatomical The structure anatomical structures characterized structures characterizeds characterized the micro-CT-derivedmap.org/ (accessed sections from on 31/05/2021 the wild type)) ( brains.Movie SD—standards S5). The anatomical deviation; structure Scale bar—1s characterized mm. by theby highest the highestby and the intermediatehighestand intermediate and intermediate levels levels of Tsen54 of levels Tsen54 expression of expressionTsen54 are expression summarized are summarized are summarized in Table in Table 1. in 1. Table 1. by the highest and intermediate levels of Tsen54 expression are summarized in Table 1. Table 1. Summary of the anatomical substructures characterized by a medium to high relative expression level of Tsen54-lacZ TableTable 1. Summary 1. TableSummary of 1. the Summary of anatomical the anatomical of the substructures anatomical substructures substructurescharacterized characterized characterizedby a medium by a medium toby high a medium to relativehigh relative to expression high expressionrelative level expression oflevel Tsen54 of Tsen54level- of- Tsen54- Table 1. Summarygene of the reporter. anatomical Anatomical substructures brain structurescharacterized and by substructures a medium to characterized high relative byexpression high and level intermediate of Tsen54 relative- expression lacZ genelacZ reporter.genelacZ reporter. gene Anatomical reporter. Anatomical bra Anatomicalin brastructuresin structures bra andin structures substructures and substructures and substructurescharacterized characterized characterizedby high by andhigh intermediate andby high intermediate and relativeintermediate relative expres- expres-relative expres- lacZ gene reporter.level Anatomical of Tsen54-lacZ brain structuresreporter gene and are substructures listed. The colorcharacterized code for theby high indicated and intermediate regions is extrapolated relative expres- from Allen Brain Atlas sion levelsion oflevel Tsen54sion of Tsen54level-lacZ of reporter-lacZ Tsen54 reporter -genelacZ reporteraregene listed. are gene listed. The are color The listed. colorcode The forcode colorthe for indicated codethe indicated for regionsthe indicated regions is extrapolated isregions extrapolated is from extrapolated Allenfrom Allen Brain from Brain Allen Brain sion level of Tsen54BrainExplorer-lacZ reporter 2 gene software. are listed. This colorThe color code code was appliedfor the indicated for virtual regions reconstruction is extrapolated of 3D Tsen54 from geneAllen expression Brain pattern in the Atlas AtlasBrainExplorer BrainExplorerAtlas BrainExplorer 2 software. 2 software. This 2 software. colorThis colorcode This wascode color applied was c odeapplied for was virtual forapplied virtual reconstruction for reconstruction virtual reconstruction of 3D of Tsen54 3D Tsen54 geneof 3D expressiongene Tsen54 expression gene pat- expression pat- pat- Atlas BrainExplorer 2 software. This color code was applied for virtual reconstruction of 3D Tsen54 gene expression pat- tern intern the in murine thetern murine murine inbrain the brain(Moviemurine brain (Movie (Movie S5);brain REL S5); (Movie S5);— RELrelative REL—relative S5);—relative REL expression— expressionrelative expression level expression oflevel level the of reporter of thelevel the reporter reporterof gene the reporteractivity.gene gene activity. activity. geneYellow activity.Yellow Yellow indicates indicates Yellow indicates areas indicates areasof areas of of areas subregions of tern in the murine brain (Movie S5); REL—relative expression level of the reporter gene activity. 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AnatomicalAnatomical AnatomicalRe- Re- Re-AnatomicalAnatomical Anatomical AnatomicalAnatomical Anatomical ColorColor Color Anatomical Re- AnatomicalAnatomical Region Anatomical StructureAnatomical Sub-Structure Color REL RELREL REL gion gion gion StructureStructure Structure Sub-SubStructure-StructureSub -Structure Codecode codeREL code gion Structure Sub-Structure code CorticalCortical plateCortical plate Cortical plate plateIsocortexIsocortex Isocortex Isocortex SecondarySecondary Secondary motorSecondary motor motorarea Layerarea motor area Layer Layer 1 area 1 Layer 1 Cortical plate Isocortex Secondary motor area Layer 1 CorticalCortical plateCortical plate plate IsocortexIsocortex Isocortex RetrosplenialRetrosplenialRetrosplenial area, area,dorsal dorsal partarea, part dorsal part Cortical plate Cortical plateIsocortex IsocortexRetrosplenial Retrosplenial area, dorsal area, dorsal part part CorticalCortical plateCortical plate plate IsocortexIsocortex Isocortex PerirhinalPerirhinal area,Perirhinal area,Layer Layer 1 area, 1 Layer 1 Cortical plate Isocortex Perirhinal area, Layer 1 DentateDentate gyrus,Dentate gyrus, Molecular Molecular gyrus, cell Molecular layer cell layer cell layer CorticalCortical plateCortical plate Hippocampal plateHippocampal Hippocampal formation formation formation Dentate gyrus, Molecular cell layer Cortical plate Hippocampal formation (temporal(temporal lobe)(temporal lobe) lobe) (temporal lobe) CorticalCortical plateCortical plate plateOlfactory Olfactory areaOlfactory area areaNucleus Nucleus of theNucleus of lateral the lateral of olfactory the olfactory lateral tract olfactory tract(NLOT) (NLOT) tract (NLOT) Cortical plate Olfactory area Nucleus of the lateral olfactory tract (NLOT) CorticalCortical plateCortical plate plateOlfactory Olfactory areaOlfactory area area AnteriorAnterior olfactoryAnterior olfactory nucleus olfactory nucleus (AON) nucleus (AON) (AON) Cortical plate Olfactory area Anterior olfactory nucleus (AON) CerebralCerebral nucleiCerebral nuclei nuclei StriatumStriatum Striatum Bed nucleusBed nucleus Bedof the nucleus of accessory the accessory of the olfactory accessory olfactory tract olfactory tract(BA) (BA) tract (BA) Cerebral nuclei Striatum Bed nucleus of the accessory olfactory tract (BA) InterbrainInterbrain Interbrain Hypothalamus HypothalamusHypothalamus LateralLateral mammilary Lateralmammilary nucleusmammilary nucleus (LM) nucleus (LM) (LM) Interbrain Hypothalamus Lateral mammilary nucleus (LM) InterbrainInterbrain Interbrain Hypothalamus HypothalamusHypothalamus LateralLateral preoptic Lateralpreoptic area preoptic (LPO)area (LPO) area (LPO) Interbrain Hypothalamus Lateral preoptic area (LPO) MedullaMedulla Medulla HindbrainHindbrain Hindbrain Medulla LateralLateral reticular Lateralreticular nucleus reticular nucleus (LRN) nucleus (LRN) (LRN) Hindbrain (Motor(Motor related) (Motorrelated) related) Lateral reticular nucleus (LRN) (Motor related) MedullaMedulla Medulla HindbrainHindbrain Hindbrain Medulla CuneateCuneate nucleusCuneate nucleus (CU) nucleus (CU) (CU) Hindbrain (Sensory(Sensory related)(Sensory related) related) Cuneate nucleus (CU) (Sensory related) MedullaMedulla Medulla HindbrainHindbrain Hindbrain Medulla SpinalSpinal nucleus nucleusSpinal of trigeminal nucleus of trigeminal of caudal trigeminal caudal part partcaudal part Hindbrain (Sensory(Sensory related)(Sensory related) related) Spinal nucleus of trigeminal caudal part (Sensory related) Pons Pons Pons HindbrainHindbrain Hindbrain Pons PontinePontine grayPontine gray gray Hindbrain (Motor(Motor related) (Motorrelated) related) Pontine gray (Motor related) CerebellumCerebellum Cerebellum Cerebral Cerebral cortexCerebral cortex cortex VermalVermal regions Vermalregions regions Cerebellum Cerebral cortex Vermal regions CerebellumCerebellum Cerebellum Cerebral Cerebral cortexCerebral cortex cortex HemisphericHemisphericHemispheric regions regions regions Cerebellum Cerebral cortex Hemispheric regions

3. Discussion3. Discussion3. Discussion 3. Discussion In thisIn manuscript, this manuscript,In this manuscript,we introduced we introduced we a introduced novel a novel methodology amethodology novel methodology for gene for geneexpression for expression gene analysis expression analysis analysis In this manuscript, we introduced a novel methodology for gene expression analysis of wholeof whole-mountof- mountwhole murine -murinemount brains. murinebrains. We validated brains.We validated We it byvalidated itstudying by studying it bythe studying expression the expression the of expression Tsen54 of Tsen54, a of, Tsen54a , a of whole-mount murine brains. We validated it by studying the expression of Tsen54, a gene genecausing causinggene the rarecausing the diseaserare the disease rarepontocerebellar diseasepontocerebellar pontocerebellar hypoplasia, hypoplasia, inhypoplasia, the in mouse the mouse in brain. the brain.mouse This meth-This brain. meth- This meth- gene causing the rare disease pontocerebellar hypoplasia, in the mouse brain. This meth- odologyodology is basedodology is based on isusing onbased using the on gene the using genepromoter the promoter gene of promoterinterest of interest driving of interest driving the lacZ drivingthe reporterlacZ the reporter lacZ and reporter and and odology is based on using the gene promoter of interest driving the lacZ reporter and convenconventionaltionalconven laboratory laboratorytional micro laboratory micro-CT X-CT -microray X imaging-ray-CT imaging X- rayequipment. imaging equipment. Weequipment. demonstrated We demonstrated We demonstrated that thethat the that the conventional laboratory micro-CT X-ray imaging equipment. We demonstrated that the non-solublenon-soluble nonprecipitate- solubleprecipitate product precipitate product of  -product gal/Xof -gal/X-gal of deposited -gal-gal/X deposited-gal in deposited situ in of situ the of in enzymatic the situ enzymatic of the reaction enzymatic reaction reaction non-soluble precipitate product of -gal/X-gal deposited in situ of the enzymatic reaction is a radiodenseis a radiodenseis a probe radiodense probe for X for- rayprobe X detection-ray for detection X-ray of detection- galof  activity.-gal of activity. - gal activity. is a radiodense probe for X-ray detection of -gal activity. A keyA feature key featureA of key this featureof micr this omicr of-CT thiso method-CT micr methodo -isCT that method is itthat allows it is allows that for itvolumetric forallows volumetric for localization volumetric localization oflocalization of of A key feature of this micro-CT method is that it allows for volumetric localization of the specificthe specific anatomicalthe specific anatomical structures anatomical structures and structures subregions and subregions and in subregions which in which the inreporter the which reporter genethe reporter geneis expressed. is expressed.gene is expressed. the specific anatomical structures and subregions in which the reporter gene is expressed. This Thisis possible is Thispossible because is possible because we becausecombined we combined we the combined X the-gal X staining-gal the staining X- galprotocol staining protocol with protocol withdehydration dehydration with dehydration by by by This is possible because we combined the X-gal staining protocol with dehydration by ethanolethanol and ethanol paraffiand paraffin andembeddingn paraffi embeddingn ofembedding the of mouse the mouse of brain. the brain.mouse The d The ehydrationbrain. dehydration The d process,ehydration process, by remov- process, by remov- by remov- ethanol and paraffin embedding of the mouse brain. The dehydration process, by remov- ing watering water molecules,ing molecules, water increases molecules, increases the increases tissue the tissue density the density tissue and densityrendersand renders andthe mouserenders the mouse brain the brainmouse visible visible brain to tovisible to ing water molecules, increases the tissue density and renders the mouse brain visible to Brain Sci. 2021Brain, 11 Sci., x FOR2021 ,PEER 11, x FORREVIEW PEER REVIEW 13 of 22 13 of 22 Brain Sci. 2021 , 11, x FOR PEER REVIEW 13 of 22 Brain Sci. 2021, 11, x FOR PEER REVIEW 13 of 22

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The and dehydration renders the process, mouse by brain remov- visible to ing waterhard molecules, X-ray imaging increases without the tissue the need density for and any renders additional the treatmentmouse brain with visible non-specific to contrasting agents. In addition, the paraffin embedding maintains the shape and correct morphology of both the whole sample and internal brain structures. By combining this sample preparation with X-gal staining, we obtained micro-CT images of murine brains exhibiting clear views of both anatomical brain substructures and radiodense lacZ gene expression sites. We managed to correlate them with the stereotaxic coordinate of the mouse brain atlas ([70] and https://mouse.brainmap.org (accessed on 31 May 2021)). We performed manual registration analysis of the Tsen54-lacZ expression using the reference Mouse Alan Brain Atlas. The process of registration can be substantially improved by applying advanced bioinformatics protocols based on machine learning algorithms, which will allow for automatic recognition of anatomic regions where the lacZ reporter gene Brain Sci. 2021, 11, 746 14 of 22

is expressed. Such program for automatic anatomical brain region registration has been recently presented [71]. The X-ray imaging modality does not damage the sample and allows a consecutive histological analysis. The samples after X-ray imaging can be sectioned and further in- vestigated by light microscopy. Thus, the application of micro-CT imaging inverts the sequence of steps usually performed to obtain 3D images from histological consecutive 2D sections because the brain samples are first imaged and then analyzed histologically. This combined imaging pipeline is optimized to obtain the most out of the functional data from biological samples. A very practical advantage of the method is based on the usage of the low-cost X-gal substrate for detection of β-gal activity and the procurability of lacZ reporter-expressing model organisms. Over the years, many lacZ reporter murine lines were engineered both in single laboratories and as part of global projects, such as the International Knockout Mouse Consortium (IKMC). In our studies, we took advantage of an available reporter/knockout Tsen54-lacZ murine line engineered by IKMC for the International Mouse Phenotyping Consortium (IMPC). The IMPC has the goal to determine the functions of every mammalian gene by systematic physiological and molecular phenotyping of 20,000 knockout mouse lines. These knockout alleles are engineered by replacing a critical gene exon with the lacZ reporter gene. Therefore, all knockout murine lines are lacZ reporter lines (https: //www.beta.mousephenotype.org/about-ikmc-strategies (accessed on 31 May 2021)) [72]. To date, about 5000 lacZ reporter mouse lines have been produced by the Consortium (www.mousephenotype.org (accessed on 31 May 2021)) and are available to the scientific community [73]. The data accumulated by the IMPC have demonstrated that lacZ is an excellent reporter system to study gene expression in ex vivo murine organs because it faithfully recapitulates the activity of the promoter that drives the lacZ gene. In addition, it has been demonstrated that endogenous lacZ activity in the mouse brain does not produce a detectable background in light imaging studies [54]. Additionally, the lacZ gene reporter can be delivered in a tissue-specific manner in the brains of wild type animals without the need for tissue-specific germ line reporter targeting by gene delivery methods, such as adenoviral infection and electroporation [74,75]. We performed our analysis of the murine brains using top-bench Bruker Skyscan 1172 micro-CT that has a resolution ranging from 5 to 20 µm/voxel. This resolution permits inspecting, in detail, the anatomical substructures in which the reporter gene is expressed but not high enough to identify the cell types expressing lacZ. This obstacle can be overcome by using more advanced micro-CT apparatuses, which support a substantial increase in imaging resolution such as nano-CT systems, propagation-based X-ray phase- contrast tomography and synchrotron-based X-ray microtomography. Several groups demonstrated the feasibility of these advanced X-ray-based technologies for the analysis of murine organs and, specifically, the mouse brain at cellular resolution, as such making the resolution of X-ray imaging methods comparable to histological analysis [39–42]. The application of these up-to-date machineries to our X-ray-based gene expression method would be highly beneficial to directly link gene expression patterns with cellular specificity in three dimensions. A feature that is unique to the X-ray imaging procedure, which gives it an unbiased strength for gene expression analysis, is that it is based on the detection of the density of an aggregated precipitate produced by the enzymatic β-gal/X-gal reaction. Thereby, it enables a way for both relative and absolute quantitative analysis of the promoter activity which drives the lacZ reporter. Hence, we introduced, here, a scheme aimed at the relative quantitative estimation of Tsen54-lacZ reporter gene expression. This analysis is based on comparison of grayscale values in different anatomical brain substructures within the same micro-CT dataset. This approach, however, does not allow for evaluation of gray values on an absolute scale and for direct comparison between different datasets because of the current limitations of scanning calibration methods. Yet, there is a huge potential to achieve the absolute quantification of the densities derived from the lacZ gene activity by Brain Sci. 2021, 11, 746 15 of 22

developing novel instrument calibration procedures. In our experiments, we conducted density-based calibration of images using water as a phantom, a common standard for normalization of datasets. For future studies, the calibration phantoms of the lacZ/X-gal product obtained after incubation with a known amount of the β-gal enzyme could be custom-built. With this calibration scheme in hand, the absolute quantification of lacZ activity within the samples and their comparison will be feasible. In addition, β-gal labeling can be combined with existing metal-based X-ray labels in order to achieve simultaneous cell type-specific labeling in the same volume image. This would not only empower X-ray analysis of soft tissues to a level analogous to cell-specific immune-fluorescence imaging but also would provide the option to perform an absolute quantitative analysis of the labeled targets. The data presented by us demonstrate that the β-gal reaction product has radiodense characteristics ex vivo. This knowledge can also lead to the development of novel β-gal- based molecular probes for in vivo X-ray-based imaging of gene expression. The main obstacle to use X-gal as a substrate for in vivo imaging is that X-gal does not diffuse inside living mammalian cells. Therefore, possible chemical modification of the X-gal molecule, which would lead to diffusion of the X-gal substrate across the cell membrane in live cells, will allow extending X-ray imaging of lacZ expression analysis to living organisms. This would make in vivo X-ray-based lacZ reporter analysis similar to quantitative biolumines- cence imaging with the addition of tomographic characteristics. This possibility would provide important knowledge of the modulation of gene activity in development and in response to environmental events in normal model organisms and in disease. Our micro-CT-based analysis demonstrated that the Tsen54-lacZ reporter is diffusely expressed in the cortex, with the highest expression observed in the retrosplenial cortex (RSC) which is important for a variety of cognitive tasks including memory, navigation and prospective thinking both in rodents and in mice [76,77]. This suggests that Tsen54 may play an important role in cognitive processes and, specifically, in the formation of spatial memory in humans and mice. High Tsen54 expression is observed in the temporal lobe of the dentate gyrus, the region which is essential for declarative memory (conscious memory for facts and events) and spatial memories in humans and in rodents [78]. Very specific high Tsen54 expression marks the lateral mammillary (LM) nucleus. The LM nucleus is part of the hypothalamic system of the interbrain. Lateral mammillary bodies are critical for generating signals controlling head direction and movements [79]. In the cortical plate of the brain, a high- expression pattern forms a fork-like structure at the rostral part of the murine brain, which is composed of several substructures: the anterior olfactory nucleus, the bed nucleus of the accessory olfactory (BA), the nucleus of the lateral olfactory tract (NLOT), the lateral preoptic area tract (LPO) and the vascular organ of the lamina terminals. Especially interesting is an expression pattern observed in the hindbrain. Particularly strong signals were detected in the pons, with the highest expression limited to the pontine gray area. These data suggest an important role for the Tsen54 gene specifically in development and functions of the pontine area and are also supported by the knowledge that in PCH patients, a variable degree of pons flattening is observed [59]. In the medulla, high expression in precerebellar nuclei: external cuneate, lateral reticular and spinal trigeminal, is observed. These pons and medulla substructures are important for proper signal exchange with the cerebellum, exerting fine control on balance, motor movements and motor learning [80]. All these results demonstrate that the function of the Tsen54 gene is strictly required especially in the regions critical for memory processing and body movement control. In summary, here, we introduced a novel method for 3D characterization of lacZ reporter gene expression by X-ray micro-CT imaging. The presented method provides research with an additional tool for relative comparison of gene expression within an entire organ. This method can be combined with histological and light microscopy analysis and could provide a quantitative dimension to the analysis of lacZ reporter gene activity. Brain Sci. 2021, 11, 746 16 of 22

4. Material and Methods 4.1. Ethics Statement After weaning, mice were housed by litters of the same sex, 3 to 5 per cage, and maintained in a temperature-controlled room at 21 ± 2 ◦C, on a 12-h light/dark cycle (lights on at 7 a.m. and off at 7 p.m.), with food and water available ad libitum in a specific pathogen-free facility. The experimental protocols and animal care procedures were reviewed and approved by the Ethical and Scientific Commission of Veterinary Health and Welfare Department of the Italian Ministry of Health (protocol approval reference: 118/2012-B). Approval is in agreement with the Italian laws and regulations according to the ethical and safety rules and guidelines for the use of animals in biomedical research in application of the relevant European Union’s directives (n. 86/609/EEC and 2010/63/EU).

4.2. Animals The Tsen54-lacZ reporter allele was engineered by the IKMC. The targeting strategy is deposited in the IMPC portal (https//www.mousephenotype.org (accessed on 31 May 2021)). In brief, knockout mice were generated from Tsen54tm1a(EUCOMM)Wtsi ES cells (https://www. mousephenotype.org/data/genes/MGI:1923515 (accessed on 31 May 2021)) and are depicted in Supplementary Figure S2. ES cells have a C57Bl/6N background. The ES cells were injected in blastocysts, and germ line transmission was obtained from chimeras. All animals were maintained only on C57Bl/6N background for all generations. To remove the neo selection cassette, the Tsen54Tm1a (EUCOMM)Wtsi animals were crossed with ROSA26Cre (MGI: 5285392; C57Bl/6N background) animals. As a result, heterozygous animals with the Tsen54Tm1b allele were produced. The Tsen54Tm1b allele is a knockout and lacZ reporter allele of the Tsen54 gene in which exon 6 is replaced by the lacZ reporter (Supplementary Figure S2). A total of 10 mouse brains were used for analyses. The brains were obtained from littermates (5 mice of Tsen54-lacZ and 5 wild type animals).

4.3. Processing for Micro-CT Prior to perfusion, adult animals (at least 2 months old) were anesthetized with 2.5% avertin solution (100 µL/10 g of body weight). The animals then were intracardially perfused with PBS (20 mL per animal at room temperature) followed by chilled 4% PFA (25 mL per animal). Brains were extracted from the skull and fixed for an additional 30 min with 4% PFA on ice. The brains were then incubated in wash buffer (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% of Nonidet-P40, 0.1 M sodium phosphate buffer pH 7.3) for 30 min on ice. Brains were stained with 1 mg/mL X-gal, freshly added from stock solution of 25 mg/mL X-gal (Sigma-Aldrich, B4252) in dimethylformamide, to wash buffer supplemented with 0.2% of potassium ferrocyanide (Sigma-Aldrich, P-9387) and 0.16% potassium ferricyanide (Sigma-Aldrich, P-8131) for 48 h at 37 ◦C. For staining in the presence of tetranitroblue tetrazolium chloride instead of potassium ferrocyanide and potassium ferricyanide, tetranitroblue tetrazolium chloride (Sigma cat: 87961) was added to wash buffer at a final concentration of 3.3 µg/mL. After staining, brains were rinsed in wash buffer and additionally fixed with 4% PFA, overnight in the cold room. The embedding was performed as described [81]. Briefly, samples were dehydrated by incubation in graded series of ethanol solution: 50% ethanol for 2 h, 70% ethanol overnight and 95% ethanol for 1 h. All steps were performed in the cold room. Dehydration of the brains was completed by incubation in 100% ethanol at room temperature for 2 h. Subsequently, the samples were transferred in xylene and incubated for 45 min. Then, the samples were transferred in xylene/paraffin mixture (1:1 ratio) and incubated at 56 ◦C for 45 min. After incubation was completed, samples were placed in fresh liquid paraffin and incubated overnight at 56 ◦C. The next morning, the paraffin was replaced with fresh paraffin, and samples were incubated for 1 h at 56 ◦C. Then, the brains were transferred in the embedding molds filed with liquid paraffin and left to polymerize for 1 h at room temperature. Brain Sci. 2021, 11, 746 17 of 22

4.4. Micro-CT Imaging Images were acquired using Skyscan 1172G (Bruker, Kontich-Belgium) with an L7901- 20 Microfocus X-ray Source (Hamamatsu). The paraffin-embedded brains were attached using Orthodontic Tray Wax (09246 Kerr) to the micro-CT imaging platform and placed vertically inside the instrument. The X-ray source was set at 39 kV and the current at 240 µA (9 W). Scans were performed with 650 and 700 projections at full rotation, with a total scan time of approximately 30 min. The following scanning parameters were applied: no filter, near camera position, dimension: 1000 × 666 (1 K), large pixel size, exposure time 60 ms, magnification 20 µm, rotation step 0.4◦ and partial width 88%. Single images (stored as TIFF-file) were reconstructed from projection images using NRecon Skyscan Reconstruction software (Brucker). The following reconstruction parameters were applied: smoothing set at 2, misalignment compensation at +/−2, ring artefact reduction at 6, beam-hardening correction at 40%. The reconstructed datasets were calibrated in Hounsfield units (HU), using water as a phantom and CTAN software. The calibration was performed as described in the Bruker manual (Bruker-micro-CT Method Note: HU calibration, 2005). After calibration, water HU = 0 and air HU = −1000 were implemented. The datasets were analyzed using analyzer software CTAN v 1.13 (Bruker) and CTVOL 2.0 and CTVOX (Bruker) software. To obtain volume rendering views and 3D movies, visualization software CTVOL v 2.0 was used. Multiple intensity projection analysis was performed using CTVOX (Bruker) software.

4.5. Light Microscopy X-gal/FeCN-stained brains, embedded in paraffin, were sectioned at 16 µm and stained with eosinY 1% aqueous solution (Bio-Optica 05-M10002). Images were obtained with the stereomicroscope MZ12 (Leica) equipped with a color camera.

4.6. XPS Analysis of β-Galactosidase Reaction Products In Vitro The reaction was performed in 50 µL volume of wash buffer with 0.2% of potassium fer- rocyanide (Sigma-Aldrich, P-9387) and 0.16% potassium ferricyanide (Sigma-Aldrich, P-8131) supplemented with 1 mg/mL of X-gal, freshly added from stock solution (25 mg/mL X-gal (Sigma-Aldrich, B4252) in dimethylformamide) with 0.16 units of β-galactosidase (Sigma cat: G4155) or without an enzyme as a control. Then, the entire volumes were deposited on the glass as a thin film and dried at room temperature. XPS measurements were carried out in a spectrometer Escalab MkII (VG Scientific Ltd., London, UK) by using an Al Kα source. The acquisition parameters were the following: analyzer pass energy = 40 eV, and large area lens mode A1 × 22 with an analysis diameter of about 10 mm.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/brainsci11060746/s1, Figure S1: MicroCT images of cerebellar lobe from wild type (A) and Tsen54-lacZ(B) brains stained with FeCH and imaged with the resolution of 5 µm/voxel. The 300 µm lines indicate the region from which paraffin background grey values calculations were performed. Three parallel 300 µm lines separated by 2 pixels were drown through molecular and granular layers. Three voxels were averaged and mean and standard deviation were calculated. The background grey values were subtracted from every averaged pixels. Distribution of grey scale intensity values were plotted in Figure3E; Figure S2: A.Schematic representation of the Tsen54tm1a allele. lacZ reporter gene is inserted into intronic locus, following exon5, replacing exons 6 of the Tsen54 gene. Cre-mediated recombination removes exon6 and neo targeting cassette from genomic Tsen54 gene locus and convert Tsen54tm1a allele into Tsen54tm1b allele. B. The Tsen54tm1b allele is a knockout of Tsen54 gene and is a lacZ reporter allele of Tsen54 gene expression. Ex- exon; En2A SA-splice acceptor site; IRES-internal ribosomal entry site; lacZ - bacterial β-galactosidase reporter gene; pA-poly A; hBactP- human β-actin promoter; neo-neomycin resistance gene; FRT- FLP recombination sites and LoxP–Cre recombination sites are indicated; Video S1: MicroCT derived RGB volume of the Tsen54-lacZ mouse brain stained with X-gal/FeCN method. The volume image was acquired using camera resolution of 1 K (image matrix of 1000 × 575 pixel with the 20 µm/voxel size). Screenshot of the transfer function editor windows of the CTVOX analyser (Bruker software) demonstrates Brain Sci. 2021, 11, 746 18 of 22

setting of the RGB transfer function curves for building a colour volume-rendered 3D model; colour coded for the tissue density function: red-blue-green; transparency level defined by the purple line (supplementary info); Video S2: MicroCT derived RGB movie of the wild type mouse brain stained with X-gal/FeCN method. The volume image was acquired using camera resolution of 1 K (image matrix of 1000 × 575 pixel with the 20 µm/voxel size). Screenshot of the transfer function editor windows of the CTVOX analyser (Bruker software) demonstrates setting of the RGB transfer function curves for building a colour volume-rendered 3D model; colour coded for the tissue density function: red-blue-green; transparency level defined by the purple line (supplementary info); Video S3: Segmantation analysis of the Tsen54-lacZ brain, whole-mount stained with X-gal/FeCN and imaged by microCT. MicroCT imaging was performed with the resolution of 20 m of voxel size. Calculated using CTAN (Bruker software) mean of Hounsfield units from the Tsen54-lacZ brain and one standard deviation (1SD) were defined as a background, while values outside of this range were colour coded: values from 1SD to 2SD-blue; from 2SD-3SD-magenta; from 3SD and up to maximum–yellow. The reconstruction of the segmented Tsen54-lacZ brain was performed using CTVOL analysis (Bruker software); Video S4: Segmentation analysis of the Tsen54-lacZ brain whole- mount stained with X-gal/tetrazolium protocol and imaged by microCT with the resolution of 20 µm of voxel size. Calculated using CTAN (Bruker software) mean of Hounsfield units of the Tsen54-lacZ brain and one standard deviation (1SD) were defined as a background, while values outside of this range were colour coded: values from 1SD to 2SD-blue; from 2SD-3SD-magenta; from 3SD and up to maximum–yellow. The reconstruction of the segmented Tsen54-lacZ brain was performed using CTVOL analysis (Bruker software); Video S5: Virtually reconstructed in three-dimension expression pattern of Tsen54 gene. The reconstruction was based on the microCT analysis of the Tsen54-lacZ mouse brain ex vivo and performed using A. Brain Atlas BrainExplorer2 software using Allen Mouse Common Coordinate Framework option. The selected for the 3D reconstruction brain structures are listed in Table1. Dentate gyrus was not included because expression was observed only in the temporal lobe of dentate gyrus. Cerebellum also was excluded from virtual reconstruction for the best visualization of the nuclei in medulla. Author Contributions: Conceptualization, O.E. and T.O.; methodology, T.O.; investigation, O.E.; T.O.; P.F.; F.C.; A.G.; S.P.; A.M.; S.K.; M.P.; resources, P.F.; F.C.; A.G.; M.P.; M.R.; F.S.; writing—original draft preparation, O.E.; T.O.; M.C.; G.P.T.-V.; writing—review and editing, O.E.; T.O.; M.C.; G.P.T.-V.; supervision, G.P.T.-V.; project administration, O.E.; funding acquisition, G.P.T.-V. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by Consiglio Nazionale delle Ricerche Progetto d’Interesse Startegico Invecchiamento (DSB.AD009 to G.P.T.-V.) and European Union Sixth Framework Pro- gramme and Seventh Framework Programme: EUCOMM, EUCOMMTOOLS and PHENOSCALE. Funding was also received from EMMA-Infrafrontier (European Mouse Mutant Archives INFRA- CT2008-227490 to G.P.T.-V.). The APC was funded by Institute of Biochemistry and Cell Biology (IBBC), CNR, Italy. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical and Scientific Commission of Veterinary Health and Welfare Department of the Italian Ministry of Health (protocol approval reference: 118/2012-B). Approval is in agreement with the Italian laws and regulations according to the ethical and safety rules and guidelines for the use of animals in biomedical research in application of the relevant European Union’s directives (n. 86/609/EEC and 2010/63/EU). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are grateful to S. Manghisi, A. Grop, E. Manghisi, G. D’Erasmo, M. Parmi- giani and S. Gozzi from European Mouse Model Archives (EMMA), Monterotondo, Italy, for excellent technical support in animal handling, sample preparation and data analysis. We are grateful to G. D. Tocchini-Valentini (IBBC, CNR, Monterotondo, Italy) for advice in X-ray-based imaging methods and to D. Marazziti (IBBC, CNR, Monterotondo, Italy) for critical reading of the manuscript. Special acknowledgments to A. Ferrara and T. Cuccurullo for secretarial work. Conflicts of Interest: The authors declare no conflict of interest. Brain Sci. 2021, 11, 746 19 of 22

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