Defining the molecular identity and morphology of glia limitans superficialis astrocytes in vertebrates

Abstract

Astrocytes are a highly abundant glial cell type and perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogeneous subtypes. The subtype identity and functions are, at least in part, associated with their anatomical location and can be highly restricted to strategically important anatomical domains. Here, we report that astrocytes forming the glia limitans superficialis, the outermost border of the brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: myocilin (Myoc). We show that glia limitans superficialis astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from zebrafish, rodents, and non-human primates to humans. Identification of this highly specialized astrocyte subtype will advance our understanding of CNS homeostasis and potentially be targeted for therapeutic intervention to combat peripheral inflammatory effects on the CNS.

Keywords: CP: Cell biology; CP: Neuroscience; astrocytes; glia limitans; meninges; scRNA-seq; spatial transcriptomics.

Figure 1:. Integrative sc/snRNA-seq and spatial transcriptomics uncovers Myoc+ glia limitans superficialis astrocytes.

(A) Reanalysis of sequencing-based spatial transcriptomics (from Castranio et al., Supp. Table 1) uncovered a continuous transcriptomic tissue wrapping the brain surface and delineating major brain regions defined by the marker Myocilin (Myoc). (B) sc/snRNAseq analysis from whole mouse brain (from Ximerakis et al., Supp. Table 1) shows that Myoc is selectively expressed in astrocytes. (C, D) Integration of 13 data sets (from Wheeler et al., Endo et al., Hasel et al., Zamboni et al., Zhou et al., Habib et al., MacLean et al., Mifflin et al., Milich et al. and Matson et al., see Supp. Tables 1,2) across four CNS regions (cortex, hippocampus, spinal cord and striatum) identifies a low number of Myoc+ astrocytes (average of 2.4% of all sequenced astrocytes across all regions) in the mouse brain and spinal cord. (E, F) Differential gene expression and GO term analysis of scRNA-seq data identifies the transcriptomic identity of Myoc+ astrocytes, indicating a white matter-like transcriptome and baseline reactivity. For a full list of genes enriched in Myoc+ astrocytes, see Supp. Table 3). (G, H) Similarity matrix (where are score of 1 describes maximum similarity) and multidimensional scaling plots show that Myoc+ astrocytes, while transcriptionally distinct, more closely resemble white matter astrocytes (see Supp. Fig. 1) when compared to grey matter astrocytes (reanalysis from Hasel et al.). GM = grey matter, WM = white matter, GLS = glia limitans superficialis. Differential gene expression for both spatial transcriptomics and scRNA-seq was performed using a Wilcoxon rank sum test with adjusted p-values (padj) of <0.05 being considered significant. The sagittal brain sections in a were downloaded from https://www.10xgenomics.com/resources/datasets.

Figure 2:. Myoc+ astrocytes occupy highly restricted anatomical domains and form the glia limitans superficialis in brain and spinal cord in postnatal development

(A) Example images of RNA in situ hybridization for Myoc and the astrocyte marker Slc1a3 show the highly region-restricted expression pattern of Myoc+ astrocytes in the adult mouse brain. (B) Myoc co-localizes with Slc1a3+ astrocytes on the cortical brain surface, thereby making up the glia limitans superficialis (see quantification in F). (C) Example images of Myoc+ astrocytes in the adult spinal cord showing that Myoc+ astrocytes also form the spina glia limitans superficialis by wrapping the entire tissue (see quantification in F) but are not present in the central canal (see also Supp. Fig. 1F). (D) RNA-Scope for Myoc and immunohistochemistry for the extracellular matrix protein laminin shows GLS cell bodies at the surface of the brain encased in laminin. Laminin covers the mouse brain surface and brain vasculature. (E, F) Example in situ hybridizations and quantifications for Myoc and the astrocyte marker Slc1a3 throughout late embryonic (E17) and early postnatal (P4, P8 and P12) development as well as quantification of adult (P60) Myoc+ glia limitans superficialis (GLS) astrocytes in cortex and spinal cord (n = 3-4 mice per condition). As expected, no Myoc+ astrocytes are found embryonically but start to emerge at P4, when 4.6% of all cortical GLS astrocytes are Myoc+. This drastically increases to 91.6% at P8 and 96.6% at P12 to ultimately reach 99.2% in adulthood. In the spinal cord, 99.8% of all GLS astrocytes are Myoc+. A small subpopulation of white matter astrocytes in the corpus collosum is also Myoc+. At no point in development do we observe Myoc+ astrocytes in the parenchyma, suggesting that they are instructed locally at the surface. Values in b represent adjusted p-values (see below). Boxplots represent the median with hinges showing first and third quartiles and whiskers showing the interquartile range. Statistical tests in b were performed using a one-way ANOVA and post-hoc Tukey HSD (Honestly Significant Difference) to calculate padj. A padj of < 0.05 was considered statistically significant.

Figure 3:. Glia limitans superficialis astrocyte cell bodies cover the cortical surface and show an atypical morphology with parenchymal processes.

(A) AdipoClear-cleared adult Aldh1l1^eGFP brains were imaged using light sheet microscopy. Example images from six animals show astrocyte cell bodies covering the cortical surface, with branched processes extending into the parenchyma. (B, B’) Example images of GLS astrocytes on the cortical surface show densely-packed, process-extending cell bodies. (C, C’) Top-down view of the cortical surface shows a superficial blood vessel surrounded by glia limitans superficialis (GLS) astrocyte cell bodies. Superficial blood vessels run in GLS ‘grooves’ and are covered in long astrocyte processes that can extend hundreds of micrometers. Color scale represents imaging depth, where 0 μm represents topmost brain structures. (D) Three-dimensional rendering of GLS astrocytes show flat cell bodies occupying the cortical surface and extending branched processes into the brain parenchyma. (E, E’) AdipoClear-cleared adult mouse brains stained for the astrocytic protein GFAP. GFAP+ cell bodies make up the mouse brain surface and have parenchymal processes. At regions where blood vessel penetrated the brain, astrocyte cell bodies tilt into the parenchyma. GLS astrocytes are then replaced by GFAP+ GLP astrocytes. (F) Top-down view of the GFAP stained mouse brain shows GFAP+ astrocyte cell bodies tiling the brain surface. (G, G’) Parenchymal astrocytes in the same brain show a typical process-bearing, bushy astrocyte morphology. (H) Transversal slice from serial electron microscopy of the zebrafish spinal cord at 6 dpf with GLS and neuropil astrocytes highlighted. (I) Cellular reconstructions from serial EM of the zebrafish cells highlighted in h demonstrating GLS have cell-bodies that are located on the edge of the spinal cord. (J) Cellular Reconstruction from serial EM of GLS (green) and neuropil (magenta) astrocytes in zebrafish in traversal cross sections and lateral views. Short arrow points to cell body of surface astrocytes, long arrow to neuropil astrocytes. CC = central canal. (K) Reconstruction from serial EM of three GLS astrocytes that tile the edge of the zebrafish spinal cord at 6 dpf. (L) Confocal images of Tg(uas:LifeAct-GFP) animals that were injected with gfap:gal4-TA-tdTomnls demonstrating GLS astrocytes with a nucleus (magenta) that lines the edge of the spinal cord and has a relatively simple morphology compared to neuropil astrocytes. Arrow points to surface astrocyte magnified in inset.

Figure 4:. MYOC+ astrocyte cell bodies make up the glia limitans superficialis in the human brain and have extensive parenchymal processes.

(A) Reanalysis of snRNA-seq data from postmortem human brain tissue from Sadick et al. and Siletti et al. identifies low numbers of MYOC+ astrocytes (0.13% of all astrocytes across all brain regions and data sets, see Supp. Tables 1,3). (B, C) snRNAseq and GO term analysis identifies the transcriptomic identity of human MYOC+ astrocytes. Similar to mouse, human MYOC+ astrocytes are enriched in GFAP, AQP4, and CLU and show indications of baseline reactivity, including enrichment for the transcription factor NFKB1. (D, E) Immunohistochemistry identifies GFAP+, MYOC+ astrocyte cell bodies on the brain surface of postmortem human brain tissue with processes that can reach deep into the human prefrontal cortex. Example images are from six human brain tissues with identifiable surfaces.