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Summary 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., Highlights • PDGFRA is thought to be essential for extraembryonic endoderm stem (XEN) cells • We derived multiple XEN cell lines from a PDGFRA-GFP knockout strain • We chemically converted PDGFRA-deficient ESC lines into XEN cell lines • Thus, PDGFRA is not essential for the derivation and maintenance of XEN cells, 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 has been thought to be essential for the derivation and maintenance of mouse XEN cell lines. Mombaerts and colleagues have re-evaluated this requirement and conclude that it is not the case.

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.

Summary: 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. : Zapiec and Mombaerts report a multiplex method to map the expression areas of odorant receptor genes in individual, non-genetically modified mice. They classify the expression areas of 68 odorant receptor genes into 9 zones. These zones are highly overlapping and strikingly complex when viewed in 3D reconstructions. Keywords: olfaction, zone, spatial organization, expression pattern, olfactory sensory neuron, axonal wiring, glomerulus, olfactory bulb, 3D reconstruction, gene targeting

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.

Olfactory dysfunction is common in Parkinson’s disease. Zapiec et al. devise a rigorous quantitative approach to describe the glomerular component of the entire human olfactory bulb. The global glomerular voxel volume in Parkinson's disease is half that of controls, with differences seen predominantly in the ventral olfactory bulb., 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.

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.