Abstract
Self-recognition is a fundamental cellular process across evolution and forms the basis of neuronal self-avoidance1,2,3,4. Clustered protocadherin (cPcdh) proteins, which comprise a large family of isoform-specific homophilic recognition molecules, have a pivotal role in the neuronal self-avoidance that is required for mammalian brain development5,6,7. The probabilistic expression of different cPcdh isoforms confers unique identities on neurons and forms the basis for neuronal processes to discriminate between self and non-self5,6,8. Whether this self-recognition mechanism also exists in astrocytes remains unknown. Here we report that γC3, a specific isoform in the Pcdhγ family, is enriched in human and mouse astrocytes. Using genetic manipulation, we demonstrate that γC3 acts autonomously to regulate astrocyte morphogenesis in the mouse visual cortex. To determine whether γC3 proteins act by promoting recognition between processes of the same astrocyte, we generated pairs of γC3 chimeric proteins that are capable of heterophilic binding to each other, but incapable of homophilic binding. Co-expression of 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 mediated by γC3 contributes to astrocyte development in the mammalian brain.
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Data availability
All data are available in the main text or the supplementary materials. Requests for further information, resources and reagents should be directed to and will be fulfilled by S.L.Z. Source data are provided with this paper.
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Acknowledgements
The authors would like to thank L. Tang, J. Trachtenberg, P. Katsamba and members of the Khakh and Zipursky laboratories for critical feedback; and S. Phillips for providing statistical analysis and consultation for our study. This work was supported by a grant from the W. M. Keck Foundation to S.L.Z., T32 Neurobehavioral Genetics (T32NS048004) training grant (J.H.L.) and NSF grant IOS-2321481 (B.H.). B.S.K. is supported by NS111583.
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Conceptualization: J.H.L., S.L.Z., A.P.S., L.S. and B.H. Data curation: J.H.L. and A.P.S. Formal analysis: J.H.L., A.P.S., G.A. and R.X. Funding acquisition: S.L.Z. and B.H. Investigation: J.H.L., S.L.Z., A.P.S., G.A. and K.M.G. Methodology: J.H.L., S.L.Z., B.S.K., J.A.W., A.P.S. and R.X. Project administration: J.H.L. and A.P.S. Resources: S.L.Z., B.S.K., J.A.W., L.S. and B.H. Software: J.H.L. and A.P.S. Supervision: S.L.Z., B.S.K., J.A.W., L.S. and B.H. Validation: J.H.L., G.A., S.M., F.B., K.M.G. and R.X. Visualization: J.H.L. and A.P.S. Writing, original draft: J.H.L., S.L.Z., A.P.S. and B.H. Writing, reviewing and editing: J.H.L., S.L.Z., B.S.K., J.A.W., A.P.S., L.S. and B.H.
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Extended data figures and tables
Extended Data Fig. 1 Efficient labeling of cortical astrocytes using AAV.
a, Sparse labeling of astrocytes across all cortical layers (P14 mouse brain). AAV.PhP.eB expressing Lck-GFP, under the control of an astrocyte-specific promoter (GfaABC1D), which were retro-orbitally injected into neonates at P1. b, Astrocytes were labeled from P8 to P21 in the visual cortex, and tissues were subsequently stained with GFP antibodies. Representative images were obtained from three mice and have been routinely observed in injections with similar results.
Extended Data Fig. 2 Identification of astrocyte γC3 gene expression in V1.
a, Low-magnification image illustrating γC3 expression in astrocytes across both upper (L2/3) and lower (L5/6) cortical layers. γC3 expression is also expressed in other cell types in these regions. Scale bar, 40 µm. Representative images are from experiments quantified in (b), which were repeated independently in three mice. b, Astrocyte cell areas were segmented based on the expression of the astrocyte-specific marker Slc1a3, detected using an RNAscope in situ hybridization probe. γC3 expression was detected using an isoform-specific RNA probe. Solid outlines indicate the cell boundaries of identified single astrocytes. γC3 RNA puncta per cell were quantified within Slc1a3-positive regions in layers 2/3 and 5/6 of V1 in WT mice at P21. N = 280 cells from three mice. Statistical comparisons were performed using the two-sided Wilcoxon rank-sum test, with nested analysis treating the animal as the unit of analysis. Error bars represent the standard error of the mean (s.e.m.). The probe specificity to γC3 is supported by two observations. First, each of the multiple probes (referred to as Z probes) in the probe set is assessed computationally for cross-reactivity with non-cognate sequences in the transcriptome. Second, signal amplification requires adjacent Z probes to bind simultaneously, which contributes additional stringency in signal detection. Scale bar, 10 µm.
Extended Data Fig. 3 Evaluating astrocyte morphology in the hippocampus CA1 using multiple morphological metrics.
a, To assess the morphological characteristics of astrocytes, we retro-orbitally injected WT or γC3 KO mice with AAVs expressing Lck-smV5, Lck-smMyc, and Lck-GFP under the control of the astrocyte-specific GfaABC1D promoter. The mice were harvested at P21 and subjected to immunostaining with anti-Myc, anti-V5, and anti-GFP antibodies. Roundness: Measures how closely the shape’s minor and major axes resemble a perfect circle. Circularity: Quantifies how similar the object’s area and perimeter are to a perfect circle. b, Representative images of single astrocytes, flattened in a confocal volume, obtained from the CA1 hippocampus of both WT and γC3 KO mice. Representative images were obtained from three mice. c, The results are summarized in plots representing various morphological parameters. Two-sided unpaired t-tests with Welch’s correction was used to compare WT and γC3KO groups. Apparent cell volume: WT, n = 19 astrocytes from three mice; γC3 KO, n = 19 astrocytes from three mice. Feret max, Feret min, aspect ratio, territory size, roundness, and circularity: WT, n = 30 astrocytes from three mice; γC3 KO, n = 24 astrocytes from three mice. Error bars, s.e.m. Scale bars, 10 µm. **p < 0.01; ***p < 0.001. Nested analysis was performed for all statistical comparisons to confirm the results, and details are provided in Supplementary Table 4.
Extended Data Fig. 4 Brain-wide multicolor labeling of astrocyte morphology.
a, To enhance the labeling of fine astrocytic processes, smFPs were targeted to the plasma membrane using the Lck domain and packaged into AAV.PhP.eb serotype, which was delivered via the retroorbital route into P1 mice. Brains were harvested at P21. b, Multicolor labeling of astrocytes in the brain. WT mice were injected with AAV expressing Lck-smV5, Lck-smMyc, and Lck-GFP driven by the astrocyte-specific GfaABC1D promoter. Stochastic multicolor labeling of astrocytes is observed throughout the brain, including the hippocampus, thalamus, and visual cortex. The right panel shows a 3D reconstruction of neighboring astrocytes in layer 6 of V1. Scale bars 1 mm (hippocampus), 40 µm (thalamus), 10 µm (V1). c, Astrocyte volumes were computed through surface reconstruction. The voxels inside each surface that overlap with each other were calculated and highlighted in yellow, generating a new surface from the overlapping regions. d,e, Astrocyte tiling index was calculated by dividing the overlapping volume between adjacent astrocytes by the volumes of a single astrocyte. Both WT and γC3 KO astrocytes exhibited minimal overlap with adjacent astrocytes. Two-sided unpaired t-tests with Welch’s correction was used to compare WT and γC3KO groups. WT, n = 44 astrocytes from three mice; γC3KO, n = 21 astrocytes from three mice. Error bars, s.e.m. Scale bars 10 µm. ****p < 0.0001. Nested analysis was performed for all statistical comparisons to confirm the results, and details are provided in Supplementary Table 4.
Extended Data Fig. 5 Astrocyte-specific Cre induction and gene recombination in Aldh1l1-Cre/ERT2 mice.
a, Schematic illustrating the crossing of Aldh1l1-Cre/ERT2 mice with ROSA26-LSL-Cas9-P2A-eGFP mice. Cre recombinase expression was induced by tamoxifen injection from P1 to P3, and V1 tissues were harvested at P21. b, Astrocytes were immunostained with an anti-Kir4.1 antibody, neurons with an anti-NeuN antibody, and GFP with an anti-GFP antibody. GFP co-localized with Kir4.1 staining, indicating astrocyte-specific Cre-mediated gene recombination, with no detectable GFP expression in neurons. Representative images were obtained from three mice. Scale bars: 10 µm.
Extended Data Fig. 6 Validation of astrocyte-specific Pcdhγ KO and rescue of γC3 in cortical astrocytes.
a, Diagrams of conditional Pcdhγ KO and Cre-inducible γC3 alleles. Each Pcdhγ protein is encoded by an mRNA comprising one of 22 variable exons (yellow) and the 3 constant “C” exons (blue). In the Pcdhgfcon3 KO allele, loxP sites flank the final constant exon, which is fused with GFP at the carboxy-terminus. Cre recombination results in loss of GFP-tagged Pcdhγ proteins. In the ROSA26-CAG::lox-Stop-lox-γC3-mCherry Cre-inducible mice, Cre-mediated excision of the stop codon leads to the expression of γC3 with mCherry fused to the carboxy-terminus. In animals carrying both alleles and astrocyte-specific Cre, GFP is lost. As mCherry sequences are incorporated into the 3’-end of the γC3 mRNA, in the absence of Cre, the “Stop” cassette leads to transcription termination. Thus, mCherry containing transcripts are only seen upon excision of the “Stop” cassette. b, c, In situ detection of Pcdhγ expression in visual cortex from whole-mount expanded tissues by EASI-FISH54. (b) Low-magnification view, (c) High-magnification view of single optical sections from whole-mount preparations of the visual cortex from the indicated genotypes (see Methods). Upper panel: Control, Pcdhγ/Pcdhγ (GFP + ). Middle panel: Pcdhγ-KO, Aldh1l1-Cre/ERT2; Pcdhγ/Pcdhγ (GFP-). Lower panel: Pcdhγ-KO; γC3, Aldh1l1-Cre/ERT2; Pcdhγ/Pcdhγ; γC3 (GFP- and mCherry + ). Astrocytes were labeled with Slc1a3 probes. d, Quantification of Cre-mediated recombination in astrocytes. RNA in situ hybridization confirmed efficient deletion of the Pcdhγ genes in conditional KO mice. In control mice, GFP-tagged RNA from Pcdhγ locus is expressed. In Pcdhγ-KO mice, the GFP-tagged RNA is removed from the Pcdhγ locus. In Pcdhγ-KO; γC3 mice, the GFP-tagged RNA is removed, and the γC3 RNA transcript expressed from the ROSA26 locus is tagged with mCherry sequence. Note control and Pcdhγ-KO do not contain the ROSA26-CAG::lox-Stop-lox-γC3-mCherry construct. Thus, the sparse mCherry puncta is non-specific hybridization. The Kruskal-Wallis test was used to compare the number of RNA puncta among the groups, followed by Dunn’s multiple comparison test for post-hoc pairwise comparisons. Specific p-values are provided in Supplementary Table 4. Control: n = 61 astrocytes from two mice; Pcdhγ-KO, n = 58 astrocytes from three mice; Pcdhγ-KO; γC3, n = 58 astrocytes from three mice. Error bars, s.e.m. Scale bars, 10 µm. ****p < 0.0001.
Extended Data Fig. 7 Expression of AAVs expressing γC3FL and γC3 homophilic binding mutants in vivo.
AAV.PhP.eB was used to express Lck-smMyc, γC3 full-length (γC3FL), γC3-L87E, and γC3-L342E mutants under an astrocyte-specific GfaABC1D promoter in γC3 KO mice. AAV.PhP.eB expressing γC3FL, γC3-L87E, and γC3-L342E, tagged with a C-terminal 3×V5, were retro-orbitally delivered into P1 mice. Astrocyte morphology was labeled by co-injecting AAV.GfaABC1D expressing Lck-smMyc and visualized with an anti-Myc antibody (green). Expression of γC3FL, γC3-L87E, and γC3-L342E was detected using an anti-V5 antibody (red). a, Expression of γC3FL. Representative images were obtained from five mice. b, Expression of γC3-L87E. Representative images were obtained from five mice. c, Expression of γC3-L342E. Representative images were obtained from five mice. Scale Bars 10 µm.
Extended Data Fig. 8 Design of heterophilic protocadherin chimera pairs that lost homophilic binding.
a, Contact between cell membranes (grey) of astrocyte sister branches (WT). γC3 molecules (blue) forming a trans-dimer (in curly brackets) are shown schematically with extracellular cadherin (EC) domains as ellipses. b,c, Homophilic-deficient cPcdh chimeras. d, A pair of chimeras (one from b and another from c) co-expressed in the same astrocyte. e, A pair of chimeras in D modified with mutations (asterisk, cyan) that enable heterophilic binding. f, Summary of AUC experiments of WT γC3, γC4, and γC5, parts of which were used for chimera design. Of note, AUC on γC5 was done in the context of the EC1-EC5 fragment, but only the EC1-EC4 trans-dimer is shown in the schematic. g, Summary of AUC experiments on the designed chimeras. See Methods for details on mutation (cyan) design. A sign with a circle and a line through it depicts inability to form dimers.
Extended Data Fig. 9 Trans-dimer models and their properties at the EC2-EC3 boundary.
a, Relative FoldX energies (in parenthesis in kcal/mol) of the EC1-EC4::EC4’-EC1’ trans-homodimers assuming all complexes form dimers identical in Cα backbone to γC4::γC4. The chimeras are color-coded based on the sequence composition: γC3 (blue), γC4 (green) or γC5 (red). All structures shown in ribbon representation with calcium atoms as green balls. b, Comparison of the amino acid properties at the EC2EC3 boundary of γC4 trans-dimer and γC3. Protein backbone is in ribbon representation. Residues are shown as sticks in the expanded view of the EC2EC3::EC3’EC2’ interface. Residues that differ in properties between γC4 and γC3 in a diamond-shaped area correspond to thicker sticks. Polar residues predicted to destabilize γC3 trans-dimer in the γC4-like orientation are underlined in cyan. c, Schematic representation of the expanded views shown in b for all WT and chimera proteins. H – hydrophobic boundary, PH – polar/hydrophobic non-complementary boundary.
Extended Data Fig. 10 Summary of astrocyte self-recognition and morphogenesis via γC3 and chimeric isoform binding.
Astrocyte morphology relies on self-recognition mediated by γC3. Binding between γC3 proteins is likely to activate intracellular signaling pathways which specify distinct morphological consequences. Chimeras which have lost homophilic binding (e.g. M1, M6, M3, and M8) do not promote normal morphogenesis on their own. By contrast, pairs of complementary chimeras (e.g. M1 + M6 and M3 + M8) which bind heterophilically promote normal morphogenesis when expressed in the same astrocyte. The precise mechanism by which γC3 regulates morphogenesis is unclear. Binding could activate repulsion15,48. The initial repulsive response may direct process extension away from sister branches and this would indirectly promote process outgrowth. Alternatively, transient binding between γC3 on opposing processes may directly promote the assembly of signaling complexes which could promote process outgrowth25,44 (see Discussion).
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Lee, J.H., Sergeeva, A.P., Ahlsén, G. et al. Astrocyte morphogenesis requires self-recognition. Nature 644, 164–172 (2025). https://doi.org/10.1038/s41586-025-09013-y
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DOI: https://doi.org/10.1038/s41586-025-09013-y
