Molecular, Cellular, and Developmental Organization of the Mouse Vomeronasal organ at Single Cell Resolution.

We have generated single cell transcriptomic atlases of vomeronasal organs (VNO) from juvenile and adult mice. Combined with spatial molecular imaging, we uncover a distinct, previously unidentified class of cells that express the vomeronasal receptors and a population of canonical olfactory sensory neurons in the VNO. High resolution trajectory and cluster analyses reveal the lineage relationship, spatial distribution of cell types, and a putative cascade of molecular events that specify the V1r, V2r, and OR lineages from a common stem cell population. The expression of vomeronasal and olfactory receptors follow power law distributions, but there is high variability in average expression levels between individual receptor and cell types. Substantial co-expression is found between receptors across clades, from different classes, and between olfactory and vomeronasal receptors, with nearly half from pairs located on the same chromosome. Interestingly, the expression of V2r, but not V1r, genes is associated with various transcription factors, suggesting distinct mechanisms of receptor choice associated with the two cell types. We identify association between transcription factors, surface axon guidance molecules, and individual VRs, thereby uncovering a molecular code that guides the specification of the vomeronasal circuitry. Our study provides a wealth of data on the development and organization of the accessory olfactory system at both cellular and molecular levels to enable a deeper understanding of vomeronasal system function.

Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones.

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings 1-5 . Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad) Hi Pdgfrα + mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cad Lo Albumin + hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs' reduction in the adult bone marrow (BM) 6,7 , depletion of Cxcl12 via Cdh2 CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

Compartmentalized ocular lymphatic system mediates eye-brain immunity.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2.

Spatial transcriptomics maps RNA molecules to the location in a tissue where they are expressed. Here we document the use of Slide-SeqV2 to visualize gene expression in the mouse olfactory bulb (OB). This approach relies on spatially identified beads to locate and quantify individual transcripts. The expression profiles associated with the beads are used to identify and localize individual cell types in an unbiased manner. We demonstrate the various cell types and subtypes with distinct spatial locations in the olfactory bulb that are identified using Slide-SeqV2.

A freeze-substitution approach with solvent-based glyoxal fixative to prevent distortion of ocular structures.

Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.

A review on histotechnology practices in COVID-19 pathology investigations.

COVID-19 disease in humans, caused by the novel SARS-CoV-2 virus, was first reported in the city of Wuhan, China in December 2019. This disease has quickly developed into a global pandemic, resulting in over 350,000 deaths worldwide and over 5 million confirmed infections in a matter of 6 months. Although the genome of this novel viral RNA was sequenced quickly and testing kits were manufactured to assist in combatting COVID-19, the diagnosis and treatment will remain relatively unsuccessful until the pathology of this disease is fully understood. Histotechnology plays an important role in understanding the pathology of many diseases, including COVID-19. The first postmortem biopsy of a COVID-19 patient was collected on 27 January 2020, and the pathology finding was published in mid-February 2020. Since then, more studies have been published in scientific literatures as the global outbreak continues. This mini-review summarizes the published articles in which histotechnology aspects were utilized with the intent to understand the pathology of COVID-19. In addition, it is anticipated there will be more molecular and immunohistochemical studies to further understand the mechanism of the disease in the near future.