Astrocyte morphogenesis requires self-recognition

Abstract

Self-recognition is a fundamental cellular process across evolution and forms the basis of neuronal self-avoidance1-4. Clustered protocadherins (Pcdh), comprising a large family of isoform-specific homophilic recognition molecules, play a pivotal role in neuronal self-avoidance required for mammalian brain development5-7. The probabilistic expression of different Pcdh isoforms confers unique identities upon neurons and forms the basis for neuronal processes to discriminate between self and non-self5,6,8. Whether this self-recognition mechanism exists in astrocytes, the other predominant cell type of the brain, remains unknown. Here, we report that a specific isoform in the Pcdhγ cluster, γC3, is highly enriched in human and murine astrocytes. Through genetic manipulation, we demonstrate that γC3 acts autonomously to regulate astrocyte morphogenesis in the mouse visual cortex. To determine if γC3 proteins act by promoting recognition between processes of the same astrocyte, we generated pairs of γC3 chimeric proteins capable of heterophilic binding to each other, but incapable of homophilic binding. Co-expressing complementary heterophilic binding isoform pairs in the same γC3 null astrocyte restored normal morphology. By contrast, chimeric γC3 proteins individually expressed in single γC3 null mutant astrocytes did not. These data establish that self-recognition is essential for astrocyte development in the mammalian brain and that, by contrast to neuronal self-recognition, a single Pcdh isoform is both necessary and sufficient for this process.

Figure 1. γC3 is the predominant Pcdh isoform in mouse and human astrocytes.

a, The mouse Pcdh gene cluster contains exons encoding 58 extracellular and transmembrane domains. The α and γ proteins share common exons encoding a terminal segment of the cytoplasmic domain. Each β comprises a distinct C-terminal encoded segment. b, RNA-Seq transcriptional profiling of Pcdh genes in neurons, oligodendrocytes, and microglia at P7 (upper panels) and at three stages of development in astrocytes (lower panels). Note the difference in the scale on the Y-axis between the upper and lower panels. These data are from databases produced by the Barres lab (Zhang et al. 2014 and Clarke et al. 2018 available at https://brainrnaseq.org/). c, Expression of Pcdh genes in astrocytes from different regions of the mouse central nervous system assessed from sequences purified by the Ribo-Tag method (see Endo et al., 2022) (upper panel). Human astrocytes immunopanned from fetal (18–18.5 weeks of gestation) and adult human brains (data from Zhang et al. 2016) (lower panel). Heatmap shows the log2 FPKM values of the Pcdh cluster gene.

Figure 2. Astrocyte morphology is disrupted in γC3 KO mice.

a, AAV vectors expressing Lck-GFP, controlled by an astrocyte-specific promoter were retro-orbitally injected into P1 neonates (see Methods). b, Sparse labeling of astrocytes across all cortical layers (P14 mouse cortex). c,Comparison of astrocyte morphology in P8 WT and γC3 KO mutants labeled with AAV expressing a myristoylated GFP (Lck-GFP) which localizes to the cytoplasmic face of the plasma membrane. Quantification of astrocyte volumes (see Methods). WT: L2/3, n=7; L4, n=8; and L5/6, n=49 astrocytes each from three mice. γC3KO mice: L2/3, n=6; L4, n=8;and L5/6, n=75 astrocytes each from three mice. d, Astrocytes in P21 WT and γC3KO animals. WT: L2/3, n=10; L4, n=23; and L5/6, n=35 astrocytes each from six mice. γC3KO: L2/3 n=56; L4, n=16; and L5/6 n=17 astrocytes each from six mice. e, Changes in astrocyte volume (see Methods) from L6 at P21. f, Astrocytes in L6 showed reduced volumes from P8 to P21 in γC3KO mice.P8: WT (n=48 astrocytes from three mice) and γC3KO (n=75 astrocytes from three mice); P14: WT (n=29 astrocytes from three mice) and γC3KO (n=21 astrocytes from three mice); P21: WT (n=35 astrocytes from six mice) and γC3KO (n=21 astrocytes from six mice). Error bars, s.e.m. Scale bars 10 μm. ***p<0.001.

Figure 3. Replacement of the Pcdhγ cluster with a single isoform rescues astrocyte morphology.

a, Strategy to determine if a single isoform alone is sufficient for normal astrocyte morphology (see text). b,c, Astrocytes lacking the entire Pcdhγ complex exhibit marked defects in morphology. The expression of γC3 only in astrocytes (see Methods) substantially rescues astrocyte morphological defects, while the γA1 isoform provides partial rescue. See Methods for genetic scheme. Control: n=41 astrocytes from three mice; Pcdhγ-KO, n=50 cells from three mice; Pcdhγ-KO; γC3, n=40 cells from three mice; Pcdhγ-KO; γA1, n=59 cells from five mice. Error bars, s.e.m. Scale bars, 10 μm. ***p<0.001.

Figure 4. γC3 homophilic recognition specificity is required for astrocyte morphology.

a, Schematic of proteins tested for rescue in WT and γC3 null mice. b, Summary of AUC experiments on the EC1-EC4 WT and mutant proteins. c, Structure-based design of mutations disrupting homophilic binding. Unsatisfied buried charges (red spheres) disrupt homophilic binding. d,e, Rescue experiments using AAV to drive different γC3 constructs under the control of the astrocyte-specific GfaABC₁D promoter in WT and γC3 KO mutants. WT, n=32 cells from three animals; γC3 KO-control, n=55 cells from six animals; γC3 KO-γC3FL, n=14 cells from six animals; γC3 KO-γC3L87E, n=33 cells from six animals; and γC3 KO-γC3L342E, n=25 cells from five animals. Error bars, s.e.m. Scale bars 10 μm. ***p<0.001.

Figure 5. Complementary chimeras expressed in astrocytes only, rescue the morphology defect in γC3 null mutant astrocytes.

a, Schematic showing protein constructs used in rescue experiments (see text). b, K_Ds for EC1-EC4 fragments of chimeras shown in A. c, Astrocytes transduced with virus-expressed chimeras under the control of the astrocyte-specific GfaABC₁D promoter were identified by staining using antibodies to the epitope tags (arrows). Weak signal to noise was often observed with ant.i-HA antibody. Astrocyte morphology was visualized using anti-Myc staining to visualize AAV.Lck-smMyc. d, Quantification of astrocyte volume in different genotypes as indicated. WT, n=32 cells in three mice; γC3 KO-control, n=55 cells in six mice; M1, n=26 cells in five mice; M6, n=25 cells in five mice; M1+M6, n=19 cells in five mice; M3, n=31 cells in five mice; M8, n=20 cells in five mice; M3+M8: n=16 cells in five mice. Error bars, s.e.m. e, The schematic shows that complementary pairs of chimeras (M1+M6 or M3+M8) enable heterophilic binding within the same astrocytes. Scale bars, 10 μm. ***p<0.001.