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

Abstract

Astrocytes are a highly abundant glial cell type that perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogenous 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 brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: Myocilin (Myoc). We show that Myoc+ astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from rodents 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.

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., Table S1) 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., Table S1) 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 Tables S1,2) 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 Table S3). (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 Fig S1) 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.

(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 Fig 3). (c) Myoc+ astrocytes can also be observed just ventral to the hippocampus, lining the interventricular foramen and third ventricle. (d) Myoc+ astrocytes also separate the thalamus from the cortex, with one layer on the thalamic and one layer on the cortical side. Unlike in the cortex, in the thalamus, Myoc+ astrocytes can also be found wrapping penetrating vessels. (e, f) Example images of Myoc+ astrocytes in the adult spinal cord showing that Myoc+ astrocytes also form the spinal glia limitans superficialis by wrapping the entire tissue (see quantification in Fig 3) but are not present in the central canal.

Figure 3 |. Myoc+ astrocytes of the glia limitans superficialis emerge in early postnatal development and are likely instructed locally.

(a,b) 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). (c) Example in situ hybridization at P4 shows that during early postnatal development, Myoc+ astrocytes first emerge just ventral to the hippocampus and that the first Myoc+ GLS astrocytes mature at the cortical midline. 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 4 |. 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’) 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. (c, c’) Parenchymal astrocytes in the same brain show a typical process-bearing, bushy astrocyte morphology. (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’) Example images of GLS astrocytes on the cortical surface show densely-packed, process-extending cell bodies.

Figure 5 |. 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 Tables S1,3). (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,d’) 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 from six human brain tissues with identifiable surfaces.