The discovery of olfactory bulb glomeruli was reported by the neuroanatomist Camillo Golgi in 1875, but the number of glomeruli in a mammalian olfactory bulb remains unknown. The 1141 intact odorant receptor genes in the mouse genome are key determinants of the coalescence of axons of olfactory sensory neurons into glomeruli at recognizable positions in the olfactory bulb. Here, we devised a methodology for quantifying the numbers, sizes, and shapes of the glomeruli of mouse olfactory bulbs. Using serial two-photon tomography, we generated complete image stacks of 12 mouse olfactory bulbs labeled immunohistochemically as whole mounts with antibodies against the glomerular marker VGLUT2. The mouse ages were postnatal days 14, 21, and 56. We manually segmented 33,137 glomeruli based on the VGLUT2-immunoreactive signal. This manual segmentation resulted in complete empirical counts of the heterogeneously-sized glomerular 3D objects instead of providing only statistical estimates. We counted a median of 2851 glomeruli per olfactory bulb at postnatal day 56, corresponding to 2.50 glomeruli per intact odorant receptor gene per olfactory bulb. For a 3D object of any shape, the effective diameter is defined as the diameter of a sphere that possesses the equivalent volume. We found that the effective diameters of the glomeruli are well modeled by a Gaussian distribution, with a median of 77.54 μm at postnatal day 56. By quantifying glomerular shape with aspect-length ratios and sphericity, we demonstrate that glomeruli do not conform to spherical shapes. We propose the descriptor "tuberiform" to encompass the diversity of glomerular shapes.

Several deep learning-based object segmentation models have been proposed for biomedical image analysis, each with different strengths and weaknesses. Here, we propose a weighted consensus method for the fusion of object segmentations. For each object, the semi-supervised learning method follows the credibilities to reach a consensus on the determination of the correct segmentation from different algorithms. The user can train the weights of different algorithms in the consensus network to predict a new fused segmentation that combines the advantages of the individual object segmentation algorithms. We apply this consensus model to the segmentation of glomeruli in the mouse olfactory bulb with improved performance over a single-model approach. This weighted consensus deep learning method can also be applied to the fusion of other object segmentations. We develop a widget in Napari that supports a user-friendly GUI for the convenient application of this human-in-the-loop method.

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years’ experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay’s ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1–2 weeks. This protocol is an update to Nat. Protoc. 11, 1757–1774 (2016): https://doi.org/10.1038/nprot.2016.105 We provide an updated protocol for image-based profiling with Cell Painting. A detailed procedure, with standardized conditions for the assay, is presented, along with a comprehensive description of parameters to be considered when optimizing the assay.

Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery: https://registry.opendata.aws/cellpainting-gallery . A portal to visualize a subset of the data is available at https://phenaid.ardigen.com/jumpcpexplorer/ .

On Dec 1, 2020, we reported a successful case of double-lung transplantation from a SARS-CoV-2 seropositive donor 105 days after the onset of mild COVID-19.1Ceulemans LJ Van Slambrouck J De Leyn P et al.Successful double-lung transplantation from a donor previously infected with SARS-CoV-2.Lancet Respir Med. 2021; 9: 315-318Summary Full Text Full Text PDF PubMed Scopus (42) Google Scholar Although repeated quantitative (q)RT-PCR analyses of donor nasopharyngeal swabs were negative, this technique detected RNA of the SARS-CoV-2 N gene (delta Ct 35) from a biopsy of the right lung taken during organ procurement. Viral culture of this biopsy was negative and donor-to-recipient transmission did not occur. Complementary orthogonal methods were needed to corroborate and interpret the qRT-PCR results. Therefore, we did ultrasensitive single-molecule fluorescence RNA in-situ hybridisation with RNAscope technology on formalin-fixed paraffin-embedded sections of the same lung biopsy (appendix p 1), and compared the results with those of a lung biopsy from a deceased patient with acute COVID-19 (figure A and B; appendix p 2). We stained 14 slides of the donor lung biopsy, each containing one 5 μm section, as follows: five slides with a probe for the N gene; five slides with a probe for the S gene; and four slides with probes for N and S. A probe for the basigin gene, which has been proposed to encode an alternative host recipient for SARS-CoV-2, served as a positive control on the ten slides stained for N or S only.2Wang K Chen W Zhang Z et al.CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells.Signal Transduct Target Ther. 2020; 5: 283Crossref PubMed Scopus (650) Google Scholar We identified characteristic RNAscope puncta in three out of nine slides for the N probe, and in six out of nine slides for the S probe (figure C and D). These puncta appeared to be located in clumps of sloughed-off material, and no cells or cell nuclei could be discerned in this debris-like tissue. To our knowledge, this is the first report of long-term (>100 days) persistence of SARS-CoV-2 RNA in lung tissue of an immunocompetent patient after convalescing from COVID-19. The debris-like tissue that contained SARS-CoV-2 RNA might be composed of degenerated endothelial cells that had detached from vessel walls, dysmorphic syncytial elements of pneumocytes, or dead neutrophilic plugs in the interstitium.3Bussani R Schneider E Zentilin L et al.Persistence of viral RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced COVID-19 pathology.EBioMed. 2020; 61103104Summary Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 4Schurink B Roos E Radonic T et al.Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study.Lancet Microbe. 2020; 1: e290-e299Summary Full Text Full Text PDF PubMed Scopus (357) Google Scholar We speculate that this debris-like tissue might shield SARS-CoV-2 RNA from degradation. Data on sputum, nasopharyngeal swabs, and bronchoalveolar lavage fluid indicate that prolonged detection of SARS-CoV-2 RNA is rare and limited to a few weeks.5Wölfel VM Corman W Guggemos M et al.Virological assessment of hospitalized patients with COVID-2019.Nature. 2020; 581: 465-469Crossref PubMed Scopus (4681) Google Scholar By contrast, SARS-CoV-2 RNA persisted in the lung parenchyma for 105 days after the onset of a mild course of COVID-19. Nonetheless, at the time of writing, 11 months after transplantation, the recipient is in good health. Our data show that the persistence of SARS-CoV-2 RNA in this donor lung tissue has been inconsequential. LJC is supported by a Katholieke Universiteit Leuven University Chair funded by Medtronic and a post-doctoral grant from University Hospitals Leuven (KOOR-UZ Leuven). LVG is supported by a post-doctoral grant from University Hospitals Leuven (KOOR-UZ Leuven). AV is supported by a fundamental research grant from the Research Foundation Flanders (FWO). DVR is supported by a grant from the Broere Charitable Foundation. RV and JW are supported as senior clinical research fellows by the FWO Belgium. EW is supported by Stichting tegen Kanker (basic and clinical oncology research). All other authors declare no competing interests. Download .pdf (.34 MB) Help with pdf files Supplementary appendix Successful double-lung transplantation from a donor previously infected with SARS-CoV-2In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its resulting respiratory disease, COVID-19, emerged.1 As the pandemic continues, with more than 62 million people with documented SARS-CoV-2 infection, a growing number of potential organ donors will have been infected. Since SARS-CoV-2 resides primarily in the respiratory tract, lung transplantation has the highest concern in terms of donor-derived viral transmission and impaired graft quality. Therefore, graft acceptance for lung transplantation should carefully be balanced against the longer waiting time for the recipient. Full-Text PDF

The olfactory system serves a critical function as a danger detection system to trigger defense responses essential for survival. The cellular and molecular mechanisms that drive such defenses in mammals are incompletely understood. Here, we have discovered an ultrasensitive olfactory sensor for the highly poisonous bacterial metabolite hydrogen sulfide (H2S) in mice. An atypical class of sensory neurons in the main olfactory epithelium, the type B cells, is activated by both H2S and low O2. These two stimuli trigger, respectively, Cnga2- and Trpc2-signaling pathways, which operate in separate subcellular compartments, the cilia and the dendritic knob. This activation drives essential defensive responses: elevation of the stress hormone ACTH, stress-related self-grooming behavior, and conditioned place avoidance. Our findings identify a previously unknown signaling paradigm in mammalian olfaction and define type B cells as chemosensory neurons that integrate distinct danger inputs from the external environment with appropriate defense outputs.

A mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) typically expresses one allele of one odorant receptor (OR) gene. It is widely thought that the great majority of the 1,141 intact mouse OR genes are expressed in one of four MOE zones (or bands or stripes), which are largely non-overlapping. Here, we develop a multiplex method to map, in 3D and MOE-wide, the expression areas of multiple OR genes in individual, non-genetically modified mice by three-color fluorescence in situ hybridization, semi-automated image segmentation, and 3D reconstruction. We classify the expression areas of 68 OR genes into 9 zones. These zones are highly overlapping and strikingly complex when viewed in 3D reconstructions. There could well be more zones. We propose that zones reflect distinct OSN types that are each restricted in their choice to a subset of the OR gene repertoire.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Extraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm lineage. Platelet-derived growth factor receptor alpha (PDGFRA) is thought to be essential for the derivation and maintenance of mouse XEN cell lines. Here, we have re-evaluated this requirement for PDGFRA. We derived multiple PDGFRA-deficient XEN cell lines from postimplantation and preimplantation embryos of a PDGFRA-GFP knockout strain. We also converted PDGFRA-deficient embryonic stem cell lines into XEN cell lines chemically by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of our 12 PDGFRA-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, PDGFRA is not essential for the derivation and maintenance of XEN cell lines.

Olfactory dysfunction is common in Parkinson's disease and is an early symptom, but its pathogenesis remains poorly understood. Hindering progress in our mechanistic understanding of olfactory dysfunction in Parkinson's disease is the paucity of literature about the human olfactory bulb, both from normal and Parkinson's disease cases. Qualitatively it is well established that the neat arrangement of the glomerular array seen in the mouse olfactory bulb is missing in humans. But rigorous quantitative approaches to describe and compare the thousands of glomeruli in the human olfactory bulb are not available. Here we report a quantitative approach to describe the glomerular component of the human olfactory bulb, and its application to draw statistical comparisons between olfactory bulbs from normal and Parkinson's disease cases. We subjected horizontal 10 µm sections of olfactory bulbs from six normal and five Parkinson's disease cases to fluorescence immunohistochemistry with antibodies against vesicular glutamate transporter-2 and neural cell adhesion molecule. We scanned the immunostained sections with a fluorescence slide scanner, segmented the glomeruli, and generated 3D reconstructions of whole olfactory bulbs. We document the occurrence of atypical glomerular morphologies and glomerular-like structures deep in the olfactory bulb, both in normal and Parkinson's disease cases. We define a novel and objective parameter: the global glomerular voxel volume, which is the total volume of all voxels that are classified immunohistochemically as glomerular. We find that the global glomerular voxel volume in Parkinson's disease cases is half that of normal cases. The distribution of glomerular voxels along the dorsal-ventral dimension of the olfactory bulb in these series of horizontal sections is significantly altered in Parkinson's disease cases: whereas most glomerular voxels reside within the ventral half of olfactory bulbs from normal cases, glomerular voxels are more evenly spread among the ventral and dorsal halves of olfactory bulbs from Parkinson's disease cases. These quantitative whole-olfactory bulb analyses indicate a predominantly ventral deficit in the glomerular component in Parkinson's disease, consistent with the olfactory vector hypothesis for the pathogenesis of this neurodegenerative disease. The distribution of serine 129-phosphorylated α-synuclein immunoreactive voxels correlates with that of glomerular voxels. The higher the serine 129-phosphorylated α-synuclein load of an olfactory bulb from a Parkinson's disease case, the lower the global glomerular voxel volume. Our rigorous quantitative approach to the whole olfactory bulb will help understand the anatomy and histology of the normal human olfactory bulb and its pathological alterations in Parkinson's disease.

Abstract It is known since 1996 that mouse odorant receptors (ORs) are involved in determining the positions of the sites of coalescence of axons of olfactory sensory neurons (OSNs)—the thousands of glomeruli in the olfactory bulb. But the molecular and cellular mechanisms of OR-mediated axonal coalescence into glomeruli remain unclear. A model was proposed in 2006–2009 whereby OR-derived cAMP signals, rather than direct action of OR molecules, determine the target destinations (glomeruli) of OSNs in the bulb. This model hypothesizes that OR-derived cAMP signals determine the expression levels of neuropilin 1 (Nrp1) in OSN axon termini; that levels of Nrp1 in glomeruli form a gradient from anterior-low to posterior-high throughout the bulb; and that these Nrp1 levels mechanistically determine anterior-posterior patterning of glomeruli. Here, we describe the first independent evaluation of the Nrp1 model since it was formulated a decade ago. We tested the model for the well-characterized mouse OR M71 using our gene-targeted mouse strains, which are publicly available. In contradiction to the model, we observed a variety of configurations for the M71 glomeruli in the conditional Nrp1 knockout. We then reassessed the model for the original OR transgene with which the model was developed, using the same publicly available mouse strains. We discovered that glomerular positions do not undergo the simple anterior shift that has been reported in the conditional Nrp1 knockout for this OR transgene. Taken together, our findings do not support the Nrp1 model for the anterior-posterior patterning of glomerular positions in the olfactory bulb.

Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5.

Significance In the mouse, olfactory sensory neurons choose one of ∼1,100 odorant receptor genes for expression. The receptor determines not only to what odorants the neuron responds but also in which glomerulus of the olfactory bulb its axon terminates. The positions of the ∼3,600 glomeruli are traditionally described as stereotyped. Using an automated method that cuts sections off brain samples and fluorescently images the block face, we quantitatively assess the positions for six types of glomeruli. The level of variability is more substantial than typically acknowledged, and some types of glomeruli are more variable in their position than others. Our results quantify the level of precision that is delivered by the mechanisms of axon wiring in the mouse olfactory system.

Ca2+ regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca2+ release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca2+ regulation are located in the tubular system, a system that is important for Ca2+ regulation and excitation–contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca2+ indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3μm and 4.1μm including pixel uncertainty with a mean distance of 2.52±0.10μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images.