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Bacterial meningitis is a major cause of morbidity and mortality, especially among infants and the elderly. Here we study mice to assess the response of each of the major meningeal cell types to early postnatalE. coliinfection using single nucleus RNA sequencing (snRNAseq), immunostaining, and genetic and pharamacologic perturbations of immune cells and immune signaling. Flat mounts of the dissected arachnoid and dura were used to facilitiate high-quality confocal imaging and quantification of cell abundances and morphologies. Upon infection, the major meningeal cell types – including endothelial cells (ECs), macrophages, and fibroblasts – exhibit distinctive changes in their transcriptomes. Additionally, ECs in the arachnoid redistribute CLDN5 and PECAM1, and arachnoid capillaries exhibit foci with reduced blood-brain barrier integrity. The vascular response to infection appears to be largely driven by TLR4 signaling, as determined by the nearly identical response induced by LPS administration and by the blunted response to infection inTlr4-/-mice.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmological diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting, constricted, and dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell-types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.