High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New "Histo3D" System.

3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.

Pathogenesis of Anorectal Malformations in Retinoic Acid Receptor Knockout Mice Studied by HREM.

Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses.

Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM.

Super-resolution fluorescence microscopy allows imaging macromolecular complexes down to the nanoscopic scale and thus is a great tool to combine and integrate cellular imaging in the native cellular environment with structural analysis by X-ray crystallography or high-resolution cryo electron microscopy or tomography. Here we describe practical aspects of SMLM imaging by dSTORM, from the initial sample preparation using mounting media, antibodies and fluorescent markers, the experimental setup for data acquisition including multi-color colocalization and 3D data acquisition, and finally tips and clues on advanced data processing that includes image reconstruction and data segmentation using 2D or 3D clustering methods. This approach opens the path toward multi-resolution integration in cellular structural biology.

Neutrophils recruited by leukotriene B4 induce features of plaque destabilization during endotoxaemia.

Aims: Both leukotrienes and neutrophils have been linked to plaque destabilization. Despite being evoked, the role of leukotriene B4 (LTB4) in neutrophil recruitment to plaques and the concomitant effects of these two actors on plaque stability remain to be proven. Since both actors are elicited during endotoxaemia, a condition associated with the risk of cardiovascular events, we investigated whether endotoxaemia promotes LTB4-mediated neutrophil infiltration in plaques and explored the roles of LTB4 and neutrophils in plaque destabilization. Methods and results: Endotoxaemia induced by repeated peritoneal endotoxin injections at a non-lethal dose (1.5 mg/kg, 5 days) in chow-fed aged Apoe-/- mice (over 45 weeks old) resulted in neutrophil infiltration and activation in plaques. Subsequently to neutrophil invasion, plaques exhibited increased features of vulnerability: reduced collagen content, expanded necrotic cores, and thinned fibrous caps. These plaque features were reproduced by direct deposition of isolated neutrophils onto murine atheromatous carotid arteries in an in vivo assay. In endotoxemic mice, plaques produced increased amounts of LTB4. Genomic or pharmacological impairments of this production reduced neutrophil infiltration, collagenolysis, and apoptosis of smooth muscle cells in plaques of endotoxemic mice. Furthermore, conditioned media of human culprit plaques (CPs) contained more LTB4 than non-CPs and levels of LTB4 correlated to both neutrophil activation markers and endotoxin releases in CPs. Conclusion: These results show that the increased neutrophil recruitment elicited by LTB4 contributes to increase features of plaque destabilization in endotoxemic contexts and point out LTB4 as a potential therapeutic target in atherosclerosis.

3DClusterViSu: 3D clustering analysis of super-resolution microscopy data by 3D Voronoi tessellations.

Motivation: Single-molecule localization microscopy (SMLM) can play an important role in integrated structural biology approaches to identify, localize and determine the 3D structure of cellular structures. While many tools exist for the 3D analysis and visualization of crystal or cryo-EM structures little exists for 3D SMLM data, which can provide unique insights but are particularly challenging to analyze in three dimensions especially in a dense cellular context. Results: We developed 3DClusterViSu, a method based on 3D Voronoi tessellations that allows local density estimation, segmentation and quantification of 3D SMLM data and visualization of protein clusters within a 3D tool. We show its robust performance on microtubules and histone proteins H2B and CENP-A with distinct spatial distributions. 3DClusterViSu will favor multi-scale and multi-resolution synergies to allow integrating molecular and cellular levels in the analysis of macromolecular complexes. Availability and impementation: 3DClusterViSu is available under http://cbi-dev.igbmc.fr/cbi/voronoi3D. Supplementary information: Supplementary figures are available at Bioinformatics online.

Microtome-integrated microscope system for high sensitivity tracking of in-resin fluorescence in blocks and ultrathin sections for correlative microscopy.

Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context.

SharpViSu: integrated analysis and segmentation of super-resolution microscopy data.

UNLABELLED: We introduce SharpViSu, an interactive open-source software with a graphical user interface, which allows performing processing steps for localization data in an integrated manner. This includes common features and new tools such as correction of chromatic aberrations, drift correction based on iterative cross-correlation calculations, selection of localization events, reconstruction of 2D and 3D datasets in different representations, estimation of resolution by Fourier ring correlation, clustering analysis based on Voronoi diagrams and Ripley's functions. SharpViSu is optimized to work with eventlist tables exported from most popular localization software. We show applications of these on single and double-labelled super-resolution data. AVAILABILITY AND IMPLEMENTATION: SharpViSu is available as open source code and as compiled stand-alone application under https://github.com/andronovl/SharpViSu CONTACT: klaholz@igbmc.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy.

Super-resolution microscopy (PALM, STORM etc.) provides a plethora of fluorescent signals in dense cellular environments which can be difficult to interpret. Here we describe ClusterViSu, a method for image reconstruction, visualization and quantification of labelled protein clusters, based on Voronoi tessellation of the individual fluorescence events. The general applicability of this clustering approach for the segmentation of super-resolution microscopy data, including for co-localization, is illustrated on a series of important biological objects such as chromatin complexes, RNA polymerase, nuclear pore complexes and microtubules.

A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.

In vivo visualization of delta opioid receptors upon physiological activation uncovers a distinct internalization profile.

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation. CONCLUSIONS/SIGNIFICANCE: On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.

Lung deposition and toxicological responses evoked by multi-walled carbon nanotubes dispersed in a synthetic lung surfactant in the mouse.

In the present work, we elaborated a synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (MWCNT). MWCNT were dispersed in surfactant, saline or saline containing DPPC, BSA, Pluronic(®) F68 or sodium dodecyl sulfate, for comparison. Dispersions were characterized visually, and by light microscopy, dynamic light scattering and transmission electronic microscopy (TEM). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon single or repeated administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-ß1 and collagen, and by histology. We found that the elaborated surfactant is more effective in dispersing MWCNT when compared to the other agents, while being biocompatible. Surfactant-dispersed MWCNT distributed all throughout the mouse airways upon single and repeated administrations and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice that received a single administration of MWCNT showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC) and interleukin (IL)-17 in BALF when compared to controls. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-ß1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. Altogether, the elaborated lung surfactant could be a valuable tool to further study the toxicological impact of pristine MWCNT in laboratory animals.

Vinexinß, an atypical "sensor" of retinoic acid receptor gamma signaling: union and sequestration, separation, and phosphorylation.

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinß, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinß interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinß represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinß does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinß. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.

Live-cell one- and two-photon uncaging of a far-red emitting acridinone fluorophore.

Total synthesis and photophysical properties of PENB-DDAO, a photoactivatable 1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO) derivative of a far-red emitting fluorophore, are described. The photoremovable group of the DDAO phenolic function comprises a donor/acceptor biphenyl platform which allows an efficient (> or = 95%) and rapid (< 15 micros time-range) release of the fluorescent signal and displays remarkable two-photon uncaging cross sections (delta(a) x Phi(u) = 3.7 GM at 740 nm). PENB-DDAO is cell permeable as demonstrated by the triggering of cytoplasmic red fluorescent signal in HeLa cells after one-photon irradiation (lambda(exc) around 360 nm) or by the generation of a red fluorescent signal in a delineated area of a single cell after two-photon photoactivation (lambda(exc) = 770 nm).

Disruption of the interaction between transcriptional intermediary factor 1{beta} and heterochromatin protein 1 leads to a switch from DNA hyper- to hypomethylation and H3K9 to H3K27 trimethylation on the MEST promoter correlating with gene reactivation.

Here, we identified the imprinted mesoderm-specific transcript (MEST) gene as an endogenous TIF1beta primary target gene and demonstrated that transcriptional intermediary factor (TIF) 1beta, through its interaction with heterochromatin protein (HP) 1, is essential in establishing and maintaining a local heterochromatin-like structure on MEST promoter region characterized by H3K9 trimethylation and hypoacetylation, H4K20 trimethylation, DNA hypermethylation, and enrichment in HP1 that correlates with preferential association to foci of pericentromeric heterochromatin and transcriptional repression. On disruption of the interaction between TIF1beta and HP1, TIF1beta is released from the promoter region, and there is a switch from DNA hypermethylation and histone H3K9 trimethylation to DNA hypomethylation and histone H3K27 trimethylation correlating with rapid reactivation of MEST expression. Interestingly, we provide evidence that the imprinted MEST allele DNA methylation is insensitive to TIF1beta loss of function, whereas the nonimprinted allele is regulated through a distinct TIF1beta-DNA methylation mechanism.

Differential between protein and mRNA expression of CCR7 and SSTR5 receptors in Crohn's disease patients.

Crohn's disease (CD) is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 +/- 71 times in CD peripheral blood mononuclear cells (PBMCs). Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 +/- 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

Early development of the lower deciduous dentition and oral vestibule in human embryos.

The aim of this work was to investigate the early development of the deciduous dentition and oral vestibule in the human embryonic lower jaw. Histological sections and three-dimensional reconstructions from prenatal weeks 6-9 were used. A continuous anlage for the oral vestibule did not exist in the mandible. In contrast to the upper jaw, where we previously observed that the dental and vestibular epithelia developed separately, two dento-vestibular bulges differentiated in the incisor region of the mandible. The lingual parts of each bulge were found to give rise to the respective central and lateral incisors, whereas the labial parts differentiated into the vestibular epithelium. In the canine and molar areas, the dental and vestibular epithelia originated separately. Later, the segments of the vestibular epithelium fused into the labial vestibular ridge, giving rise to the lower oral vestibule in the lip region. In the cheek region, the oral vestibule was found to originate in the mucosal inflection between the developing jaw and the cheek. A similar heterogeneous developmental base for the oral vestibule was also observed in the upper jaw. There is thus no general scheme for the early development of the dental and vestibular epithelia that applies to both the upper and lower jaws, and to both their anterior and posterior regions.

Quantitative ultrasonic tissue characterization as a new tool for continuous monitoring of chronic liver remodelling in mice.

BACKGROUND/AIM: Recognition of the limitations of liver biopsies has led to the need for non-invasive tests to assess liver fibrosis from intensity and kinetic point of views. The aim of the present study was to evaluate non-invasive ultrasonic tissue characterization for the continuous monitoring of this process in mice. METHODS: Twelve-week-old male and female C57Bl6/J mice were submitted to repetitive carbon-tetrachloride (CCl4) intraperitoneal injections during 8 weeks or analysed 28 days after common bile duct ligation (BDL). The extent and kinetic of the disease progression were followed by the measurement of ultrasound backscatter intensity. This was compared with histological and blood parameter analysis. RESULTS: CCl4 induced a progressive increase in in vivo liver tissue backscatter intensity in both males and females. This increase was mainly correlated with interstitial fibrosis and, to a lower extent, with nuclear surface of the hepatocytes. A similar result was found after BDL. CONCLUSIONS: These data demonstrate for the first time in a systematic study that ultrasound tissue characterization can be used as a reliable tool to follow liver remodelling in mice continuously.

Vascular wall-produced prostaglandin E2 exacerbates arterial thrombosis and atherothrombosis through platelet EP3 receptors.

Prostanoids, bioactive lipids derived from arachidonic acid (AA), are important for vascular homeostasis. Among them, prostaglandin E2 (PGE2) enhances aggregation of platelets submaximally stimulated in vitro. This results from activation of EP3, one of the four PGE2 receptors, which decreases the threshold at which agonists activate platelets to aggregate. Although PGE2 altered venous thrombosis induced by administration of AA, its role in pathophysiopathological conditions has remained speculative. We report that arterial walls subjected to inflammatory stimuli produce PGE2. In several models, we show that PGE2 produced by the arterial wall facilitates arterial thrombosis. Next, we detected PGE2 in mouse atherosclerotic plaques. We demonstrate that this plaque-produced PGE2 is not altered and is still able to activate EP3. In addition, we present evidence that PGE2 can leave the plaque and activate EP3 on blood platelets. Consistent with these findings, we observed that atherothrombosis induced in vivo by mechanical rupture of the plaque was drastically decreased when platelets lacked EP3. In conclusion, PGE2 facilitates the initiation of arterial thrombosis and, hence, contributes to atherothrombosis. Inhibition of the platelet EP3 receptor should improve prevention of atherothrombosis.

Origin and developmental fate of vestigial tooth primordia in the upper diastema of the field vole (Microtus agrestis, Rodentia).

OBJECTIVE: Odontogenesis in voles is a convenient model to test hypotheses on tooth development generated from investigations in the mouse. Similar to other rodents, the functional dentition of the vole includes a toothless diastema. At its mesial end, a vestigial tooth bud has been found in the upper jaw of vole embryos. The aim of this study was to analyse the developmental dynamics of vestigial tooth structures in the upper diastema of the field vole and to compare it with the situation in the mouse. DESIGN: The development of odontogenic structures in the upper diastema of the field vole was investigated using serial histological sections and three-dimensional (3D) computer-aided reconstruction. RESULTS: A transient continuous dental lamina in the upper diastema of the field vole extended mesially to the first molar primordium, but was not continuous with the dental lamina in the incisor region. At its mesial limit, a large vestigial tooth primordium was regularly present. A further distinct vestigial bud was located mesially to the first molar primordium. The segmentation of the dental lamina suggested a potential to give rise to further vestiges in the upper diastema of the vole. CONCLUSIONS: In the prospective diastema of the vole exists as in the mouse a continuous dental lamina. Beside the prominent vestigial tooth bud in the mesial diastema, a further large bud was transiently located in front of the molars. The incorporation of dental epithelium into the first upper molar (M(1)) primordium in the vole differs from that in the mouse.