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Inhibitory input directs astrocyte morphogenesis through glial GABABR

Nature volume 617, pages 369–376 (2023)Cite this article

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Abstract

Communication between neurons and glia has an important role in establishing and maintaining higher-order brain function1. Astrocytes are endowed with complex morphologies, placing their peripheral processes in close proximity to neuronal synapses and directly contributing to their regulation of brain circuits2,3,4. Recent studies have shown that excitatory neuronal activity promotes oligodendrocyte differentiation5,6,7; whether inhibitory neurotransmission regulates astrocyte morphogenesis during development is unclear. Here we show that inhibitory neuron activity is necessary and sufficient for astrocyte morphogenesis. We found that input from inhibitory neurons functions through astrocytic GABAB receptor (GABABR) and that its deletion in astrocytes results in a loss of morphological complexity across a host of brain regions and disruption of circuit function. Expression of GABABR in developing astrocytes is regulated in a region-specific manner by SOX9 or NFIA and deletion of these transcription factors results in region-specific defects in astrocyte morphogenesis, which is conferred by interactions with transcription factors exhibiting region-restricted patterns of expression. Together, our studies identify input from inhibitory neurons and astrocytic GABABR as universal regulators of morphogenesis, while further revealing a combinatorial code of region-specific transcriptional dependencies for astrocyte development that is intertwined with activity-dependent processes.

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Fig. 1: Inhibitory neuron activity regulates astrocyte morphogenesis.
Fig. 2: Gabbr1 is required for astrocyte morphogenesis.
Fig. 3: Gabbr1 regulates astrocyte morphology through Ednrb.
Fig. 4: Region-specific regulation of Gabbr1 by SOX9 and NFIA.
Fig. 5: Regulation of astrocyte morphogenesis by region-specific mechanisms.

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Data availability

The bulk RNA-seq data from developing astrocytes and Ednrb-cKO astrocytes have been deposited at the NCBI GEO under accession number GSE198632. scRNA-seq data can be found at the NIH GEO (GSE198357, GSE198633). All other data in this Article are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

No custom code was used in this study. The R package limma eBayes function was used to define DEGs. The Bioconductor SVA/Combat package was used for batch correction.

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Acknowledgements

This work was supported by US National Institutes of Health grants NS071153, AG071687 and NS096096 to B.D. We thank the David and Eula Wintermann Foundation for support; and B. Bettler for providing the Gabbr1-floxed mouse line. scRNA-seq studies were performed at the Single Cell Genomics Core at BCM partially supported by NIH shared instrument grants (S10OD023469, S10OD025240) and P30EY002520. This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574) and the assistance of J. M. Sederstrom. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number P50HD103555 for use of the Microscopy Core facilities and the Animal Phenotyping & Preclinical Endpoints Core facilities.

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Authors and Affiliations

  1. Center for Cancer Neuroscience, Baylor College of Medicine, Houston, TX, USA

    Yi-Ting Cheng, Estefania Luna-Figueroa, Junsung Woo, Hsiao-Chi Chen, Zhung-Fu Lee, Akdes Serin Harmanci & Benjamin Deneen

  2. Program in Developmental Biology, Baylor College of Medicine, Houston, TX, USA

    Yi-Ting Cheng & Benjamin Deneen

  3. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

    Estefania Luna-Figueroa, Junsung Woo, Akdes Serin Harmanci & Benjamin Deneen

  4. Cancer Cell Biology Graduate Program, Baylor College of Medicine, Houston, TX, USA

    Hsiao-Chi Chen & Benjamin Deneen

  5. Development, Disease, Models and Therapeutics Graduate Program, Baylor College of Medicine, Houston, TX, USA

    Zhung-Fu Lee & Benjamin Deneen

  6. Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA

    Benjamin Deneen

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Contributions

Y.-T.C. and B.D. conceived the project and designed the experiments. Y.-T.C., J.W., Z.-F.L., H.-C.C. and E.L.-F. performed the experiments. J.W. performed the electrophysiology studies. Y.-T.C. and A.S.H. designed and performed the bioinformatics analyses. Y.-T.C. and B.D. wrote the manuscript.

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Correspondence to Benjamin Deneen.

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Extended data figures and tables

Extended Data Fig. 1 Normalization of astrocyte morphology in the adult after developmental activation of inhibitory neurons.

a-b. Analysis of SOX9 (e) and Ki67(f) expression within Aldh1l1-GFP astrocytes at P21 after activation of inhibitory neurons (or control); quantification was derived from n = 3 pairs of animals (a, 20,24 images; GLME; b, 12, 11 images; GLME). c. CNO only treatment of Aldh1l1-GFP mice from P7-P21 and analysis of astrocyte morphology at P21. n = 3 animals (39 cells; GLME model with Sidak’s multiple comparisons test). d. Heatmap depicting expression of GABA receptor subunits in developing astrocytes from the cortex (CX), hippocampus (HC), or olfactory bulb (OB) at P1, P7, and P14. See Extended Data Figure 2. d. Example of gating strategy and percentage of GFP+ astrocytes FACS isolated from P1 animal. e. Heatmap depicting expression of GABA receptor subunits in developing astrocytes from the cortex (CX), hippocampus (HC), or olfactory bulb (OB) at P1, P7, and P14. See Extended Data Figure 2. f. Schematic of DREADD-based activation of inhibitory neurons in post-natal Aldh1l1-GFP mice and experimental timeline. g. Imaging of P60 Aldh1l1-GFP astrocytes after hM3Dq activation of inhibitory neurons; quantification of morphological complexity using Sholl analysis, branch number, and total processes at P21; n = 3 pairs of animals (26,35 cells; upper, GLME model with Sidak’s multiple comparisons test; bottom, GLME model). Scale bars, 20 μm (a–c), 30 μm (g). Data represent mean ± s.d. (a–c, g upper), median, minimum value, maximum value and IQR (g bottom).

Source Data

Extended Data Fig. 2 Transcriptomic RNA-Sequencing analysis of developing astrocytes in the cortex, hippocampus, and olfactory bulb at P1, P7, and P14.

a. Heatmap depicting the expression of neuron-specific and astrocyte-specific genes from P1, P7, and P14 FACS isolated Aldh1l1-GFP astrocytes from the listed brain regions. b. Aldh1l1-GFP astrocytes from the cortex at P1, P7, P14. Principal component (PC) analysis against top 2,000 variable genes across the region and timepoints examined from 3-4 animals in each group. c. Heatmap depicting differential patterns of gene expression in developing astrocytes across brain regions and timepoints. d. Gene Ontology (GO) analysis of the common and region-specific patterns of gene expression. e–f. Ald1l1-CreER; ROSA-LSL-tdTomato mouse line treated with tamoxifen at P1, harvested at P28. Co-immunostaining of tdTomato labelled cells with Sox9, Olig2, NeuN, and Iba1 demonstrating astrocyte-specific activity of Aldh1l1-CreER line. n = 4 animals. Scale bars, 10 μm (b), 40 μm (e–h).

Source Data

Extended Data Fig. 3 Analysis of astrocyte development in the Gabbr1-cKO mouse line.

a. RNA-Scope imaging of Gabbr1 within Aldh1l1-GFP astrocytes from control and Gabbr1-cKO mouse lines; quantification derived from n = 3 pairs of animals (control: OB 18, CX 18, HC 16; cKO: OB 17, CX 19, HC 17 cells; LME model, ***P = 0.00020, ****P = 3.07e-06, **P = 0.0030). Dashed circle denotes astrocyte with Gabbr1. b. Antibody staining for SOX9 in Aldh1l1-GFP astrocytes from cortex of Gabbr1-cKO and control; quantification is derived from n = 3 pairs of animals (35 images; GLME model). c. Pulse-chase EdU-labelling and antibody staining at P28 from all brain regions analysed; quantification is derived from n = 3 pairs of animals (Gabbr1 control: OB 9, CX 9, HC 9, BS 9, CB 9; Gabbr1 cKO: OB 8, CX 9, HC 9, BS 9, CB 9 images; GLME model). d-e. Imaging of Aldh1l1-GFP astrocytes from the brain stem and cerebellum at P28; quantification of morphological complexity was derived from n = 3 pairs of animals (Gabbr1 control: BS 28, CB 29; Gabbr1 cKO: BS 32, CB 29 cells; GLME model with Sidak’s multiple comparisons test, *P = 0.0179, 0.0167). f. Quantitative analysis of branch points and process length from all brain regions analysed; n = 25-38 cells from 3 pairs of animals (Gabbr1 control: OB 38, CX 30, HC 29, BS 28, CB 25; Gabbr1 cKO: OB 33, CX 30, HC 29, BS 32, CB 29 cells; two way ANOVA, **P = 0.0014, *P = 0.0174, P = 0.9040, *P = 0.0132, P = 0.7126, **P = 0.0054, ****P < 0.0001, **P = 0.0066, P = 0.3763). Scale bars, 30 μm (d), 20 μm (c). Data represent mean ± s.d. (b-c, e-g median, minimum value, maximum value and IQR (a).

Source Data

Extended Data Fig. 4 Analysis of Ca2+ activity and sparsely labelled astrocytes in Gabbr1-cKO astrocytes.

a. Schematic describing the experimental timeline and mouse lines rendering astrocyte-specific knockout of Gabbr1 for sparse labelling experiments. b-c. Imaging and quantification of sparsely labelled, tdTomato-expressing astrocytes from Gabbr1-cKO and control mice from the cortex (b) and hippocampus (c); n = 3 pairs of animals (Gabbr1 control: CX 32, HC 30; Gabbr1 cKO: CX 36, HC 38 cells; b,c upper, GLME model with Sidak’s multiple comparisons test, *P = 0.0213, **P = 0.0012; b,c bottom, GLME model, ***P = 0.00043, **P = 0.0027, ***P = 0.00042, ****P < 0.0001). d. Imaging of GCaMP6s activity in control and Gabbr1-cKO astrocytes from the cortex at P28; quantification is derived from n = 3 pairs of animals (24,33 cells; GLME model, P = 0.6361, 0.2239). e. Imaging of GCaMP6s activity in the presence of TTX and baclofen; quantification derived from n = 40 cells from 3 pairs of animals (two-tailed Wilcoxon matched-pairs signed rank test, *P = 0.022, P = 0.89, ***P = 0.0006, P = 0.32). f. Two-photon, slice imaging of GCaMP6s activity in control and Gabbr1-cKO astrocytes from the cortex at P28. Quantification of Ca2+ activity in astrocytic microdomains in the Gabbr1-cKO and control animals, quantification is derived from n = 3 pairs of animals (19, 30 cells; GLME model). Scale bars, 30 μm (b-c), 20 μm (d-f). Data represent mean ± s.d. (b-c, upper), median, minimum value, maximum value and IQR (b-c, lower, d, and f).

Source Data

Extended Data Fig. 5 RNA-Seq of Gabbr1-cKO astrocytes and single cell RNA-Seq analysis of Gabbr1-cKO cortex.

a. Serut analysis of single cell RNA-Seq (scRNA-Seq) from Gabbr1-cKO and controls from P28 cortex. b. Quantification of cell clusters identifying CNS cell types from scRNA-Seq data. c. CellChat interaction diagram illustrating astrocyte interactions with neurons in the cortex from P28 Gabbr1-cKO mice; width of coloured arrow indicates scale of interaction. See Extended Data Figure 4. d-e. KEGG pathway analysis of neurons from Gabbr1-cKO scRNA-Seq (d, analysed by Enrichr) and dot plot of differentially expressed genes from KEGG (e, analysed by Seurat FindMarkers). f-h. Dot plot summaries demonstrating CellChat interaction profiles and expression patterns of key astrocyte-neuron interaction pathways.

Extended Data Fig. 6 Analysis of cortical neurons in the Gabbr1-cKO mouse line.

a. Antibody staining for BRN2 (Layers II/II). b. CTIP2 (Layers V). c. FOXP2 (Layers VI) layer-specific neuronal markers in the P28 cortex from Gabbr1-cKO and control; quantification is derived from n = 11-12 images from 3 pairs of animals (control 12, cKO 11 images; GLME model). d. Schematic of synaptic markers and cortical layers. e-f. Antibody staining for makers of excitatory synapses Vglut1–PSD95 (e) and Vglut2–PSD95 (f) in layer I of the cortex from Gabbr1-cKO or control mice at P28 (n =3 pairs of animals; GLME model, *P = 0.0490). g. Antibody staining for markers of inhibitory synapses VGAT–Gephyrin at P28; quantification is derived from 3 pairs of animals (GLME model). h-k. Representative traces of action potential in layer II/III excitatory neurons upon varying injected current in Gabbr1-cKO and control (h). Summary data of action potential firing (i; two way ANOVA). Summary data of resting membrane potential (j; two-tailed unpaired Welch’s t-test) and threshold (k; two-tailed unpaired Welch’s t-test) from 3 pairs of animals (n = 13, 12 cells). l-o. Representative traces of action potential in layer II/III inhibitory neurons upon varying injected current in Gabbr1-cKO and control (l). Summary data of action potential firing (m; two way ANOWA). Summary data of resting membrane potential (n; two-tailed unpaired Welch’s t-test, **P = 0.0091) and threshold (o; two-tailed unpaired Welch’s t-test) from 3 pairs of animals (n = 12, 15 cells). Scale bars, 100 μm (a-c), 3 μm (e-f). Data represent mean ± s.d. (a-c, e-g), mean ± s.e.m. (i-k, m-o).

Source Data

Extended Data Fig. 7 Loss of astrocytic Gabbr1 disrupts cortical circuit function.

a. Schematic of viral labelling of inhibitory neurons and experimental timeline. b-e. Representative traces of spontaneous EPSCs and IPSCs from excitatory and inhibitory neurons from cortex of Gabbr1-cKO and controls. Associated cumulative and bar plots demonstrate quantification of sEPSC and sIPSC from 3 pairs of animals (b, n = 13, 15 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.8207, **P = 0.003; c, n = 15, 12 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.1995, 0.5888; d, n = 13, 15 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.7856, 0.0504; e, n = 11, 9 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.2299, 0.3796). f. Experimental timeline for behavioural analysis. g. 3-chamber social interaction and pre-pulse inhibition studies on Gabbr1-cKO and control mice from 10 animals in control group and 11 animals in cKO group. (left, GLME model with Sidak’s multiple comparisons test, *P = 0.015; right, two-tailed Mann-Whitney test, *P = 0.043). h-m. Summary of behavioural assays conducted on Gabbr1-cKO and control animals including open field (h), elevated plus maze (i), rotarod (j), parallel foot fall (k), contextual fear conditioning (l), and cued fear conditioning (m) on Gabbr1-cKO and control mice from 10 animals in control group and 11 animals in cKO group (two-tailed Mann-Whitney test, ***P = 0.0004) Data represent mean ± s.e.m. (b-e), s.d. (g-m).

Source Data

Extended Data Fig. 8 Transcriptomic analysis of Gabbr1-cKO astrocytes an.

a. Volcano plots from RNA-Seq analysis of control and Gabb1-cKO astrocytes from cortex, hippocampus, and OB. b. Table of the number of differentially expressed genes (DEGs) from each region. c. Gene Ontology (GO) analysis of DEGs performed with Enrichr. d. Quantification of EDNRB expression in virally labelled astrocytes from the P28 cortex of mice where it has been knocked out using guideRNAs in the ROSA-LSL-Cas9-EGFP mouse line; quantification is derived from n = 3 pairs of animals (37, 38 cells; LME model, **P = 0.0068). e. Imaging of virally labelled astrocytes from the P28 cortex of Ednrb-cKO mice where Ednrb has been knocked out using guideRNAs in the ROSA-LSL-Cas9-EGFP mouse line; quantification of morphological complexity via Sholl analysis was derived from n = 3 animals (f, Ednrb-cKO: 22 mcherry-Cas9-EGFP+ cells, 49 mcherry+Cas9-EGFP+ cells; GLME model with Sidak’s multiple comparisons test, *P = 0.0307; GLME model, P = 0.06, **P = 0.008). Data represent mean ± s.d. (e left), median, minimum value, maximum value and IQR (d, e right).

Source Data

Extended Data Fig. 9 Analysis of astrocyte development in the Sox9-cKO and Nfia-cKO mouse lines.

a. Homer transcription factor motif analysis on differentially expressed genes (DEGs) from P1 and P14 timepoints from astrocytes isolated from the cortex, hippocampus, and olfactory bulb. b. Analysis of NFIA expression in Aldh1l1-GFP astrocytes from the Nfia-cKO and control at P7 in the cortex, hippocampus, and olfactory bulb; quantification of knockout efficiency was derived from 3 pairs of animals (two-way ANOVA, ****P < 0.0001). c. Analysis of SOX9 expression in Aldh1l1-GFP astrocytes from the Sox9-cKO and control at P7 in the cortex, hippocampus, and olfactory bulb; quantification of knockout efficiency was derived from 3 pairs of animals (two-way ANOVA, ****P < 0.0001). d. Analysis of NFIA expression in Aldh1l1-GFP astrocytes from the Nfia-cKO and control at P28 in the cortex, hippocampus, cerebellum, and olfactory bulb; quantification of knockout efficiency was derived from 3 pairs of animals (two-way ANOVA, ****P < 0.0001). e. Analysis of SOX9 expression in Aldh1l1-GFP astrocytes from the Sox9-cKO and control at P28 in the cortex, hippocampus, cerebellum, and olfactory bulb; quantification of knockout efficiency was derived from 3 pairs of animals (two-way ANOVA, ****P < 0.0001, *P = 0.0205). f. AAV-based overexpression of NFIA in the developing cortex, analysis of Gabbr1 expression at P28 via RNAscope; n = 3 pairs of animals (19, 18 cells; LME model, *P = 0.023, ***P = 0.00034). g. AAV-based overexpression of SOX9 in the developing olfactory bulb, analysis of Gabbr1 expression at P28 via RNAscope; n = 3 pairs of animals (20,25 cells). h. Chromatin immunoprecipitation of NFIA from P28 cortex or SOX9 from P28 olfactory bulb (OB), followed by PCR detection of association with motif in proximal promoter region of Gabbr1. Scale bars, 50 μm (b–e), 10 μm (f–g). Data represent mean ± s.d. (b–e), median, minimum value, maximum value and IQR (f–g).

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Extended Data Fig. 10 Analysis of astrocyte morphogenesis in the Sox9-cKO and Nfia-cKO mouse lines.

a–b. Two-photon, slice imaging of spontaneous GCaMP6s activity in control and Nfia-cKO astrocytes from the cortex at P28 (a) or control and Sox9-cKO astrocytes from the olfactory bulb at P28 (b); quantification is derived from 3 pairs of animals (two-tailed Mann-Whitney test). c–d. Pulse-chase EdU-labelling and antibody staining at P28 from the cortex of Nfia-cKO (c) and olfactory bulb of Sox9-cKO (d); quantification is derived from 3 pairs of animals (two-tailed Mann-Whitney test). e–f. Quantification of the number of Aldh1l1-GFP astrocytes in the cortex of the Nfia-cKO (e) or olfactory bulb from Sox9-cKO (f) and associated controls; quantification is derived from 3 pairs of animals (two-tailed Mann-Whitney test). g–h. Imaging of Aldh1l1-GFP astrocytes from the hippocampus, brainstem, and cerebellum at P28 from the Nfia-cKO (g) or Sox9-cKO (h) and associated controls; quantification of morphological complexity via Sholl analysis was derived from n = 3 pairs of animals (g, Nfia control: HC 64, BS 28, CB 47; Nfia cKO: HC 65, BS 43, CB 55 cells; GLME model with Sidak’s multiple comparisons test, **P = 0.0015; h, Sox9 control: HC 32, BS 36, CB 32; Sox9 cKO: HC 24, BS 24, CB 37; GLME model with Sidak’s multiple comparisons test). i–j. Quantification of astrocytic branch number and process length from Nfia-cKO (i) or Sox9-cKO (j) across cortex, olfactory bulb, hippocampus, brainstem, and cerebellum; derived from n = 3 pairs of animals (i, Nfia control: OB 59, CX 43, HC 54, BS 27, CB 48; Nfia cKO: OB 50, CX 56, HC 59, BS 38, CB 54 cells; two-way ANOVA, **P = 0.0054, ****P <0.0001; j, Sox9 control: OB 29, CX 31, HC 32, BS 37, CB 33; Sox9 cKO: OB 40, CX 27, HC 33, BS 21, CB 39; two-way ANOVA, **P = 0.0025, ****P < 0.0001, ***P = 0.0002). Scale bars, 10 μm (a–b), 50 μm (c–f), 30 μm (g–h). Data represent mean ± s.d. (a–f,i–j).

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Extended Data Fig. 11 Electrophysiological and behavioural analysis of the Nfia-cKO mouse line.

a. Schematic of synaptic markers and cortical layers. b–c. Antibody staining for makers of excitatory synapses Vglut1–PSD95 (b) and Vglut2–PSD95 (c) in layer I of the cortex from Nfia-cKO or control mice at P28, quantification is derived from 3 pairs of animals (GLME model). d. Antibody staining for markers of inhibitory synapses VGAT–Gephyrin at P28; quantification is derived from 3 pairs of animals (GLME model). e–f. Representative traces of spontaneous EPSCs and IPSCs from excitatory (e) and inhibitory (f) neurons from cortex of Nfia-cKO and controls. Associated cumulative and bar plots demonstrate quantification of amplitude and frequency of sEPSC and sIPSC from 3 pairs of animals (e, Kolmogorov-Smirnov test, **** P < 0.0001, *P = 0.0181, two-tailed Mann-Whitney test; f, Kolmogorov-Smirnov test, **P = 0.0026, ****P < 0.0001, two-tailed Mann-Whitney test, *P = 0.0149). g–h. Representative traces of action potential in layer II/III neurons upon varying injected current in Nfia-cKO and control. Summary data of action potential firing, resting membrane potential, and threshold from excitatory neurons (g) and inhibitory neurons (h); quantification is derived from at least 3 pairs of animals (two-way ANOVA and two-tailed Mann-Whitney test). i-j. 3-chamber social interaction and prepulse inhibition behavioural studies on Nfia-cKO and control mice from 11-13 animals in each group (i, n = 13 pairs of animals, two-way ANOVA, ***P = 0.0002; j, n = 13, 11 animals, two-tailed Mann-Whitney test, **P = 0.0050). k–p. Summary of behavioural tests from NFIA-cKO and control, including open field; n = 13, 11 animals (k), elevated plus maze; n = 10 animals (l), rotarod; n = 5, 8 animals (m), parallel footfall; n = 13, 11 animals (n), contextual fear conditioning; n = 12, 10 animals (o), and cued fear conditioning; n = 12, 10 animals (p) (two-tailed Mann-Whitney test, *P = 0.0265, 0.0136). Scale bars, 3 μm (b–d). Data represent mean ± s.d. (b–d, i, o–p), mean ± s.e.m. (e–h, j–n).

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Extended Data Fig. 12 Analysis of cortical neurons in the Nfia-cKO mouse line and co-immunoprecipitation with Lhx2 and NPAS3.

a. Antibody staining for BRN2 (Layers II/II), CTIP2 (Layers V), and FOXP2 (Layers VI) layer-specific neuronal markers in the P7 cortex from Nfia-cKO and control; quantification is derived from n = 3 pairs of animals (6 images, GLME model). b. CX-specific transcription factor DEGs increased between P7-P14. c. OB-specific transcription factor DEGs increased between P7-P14. d. Immunoprecipitation of LHX2 and immunoblot of LHX2 and NFIA from the P28 cortex; arrow heads label the proteins of interest. e. Immunoprecipitation of NPAS3 and immunoblot of NPAS3 and SOX9 from P28 cortex; arrow heads label the proteins of interest. f. Quantification of LHX2 expression in virally labelled astrocytes from the P28 cortex of mice where it has been knocked out using guideRNAs in the ROSA-LSL-Cas9-EGFP mouse line; quantification is derived from n = 3 pairs of animals (37, 39 cells; GLME model, ***P = 0.00099). g. Imaging of virally labelled astrocytes from the P28 cortex of Lhx2-cKO mice where Lhx2 has been knocked out using guideRNAs in the ROSA-LSL-Cas9-EGFP mouse line; quantification of morphological complexity via Sholl analysis was derived from n = 3 animals (Lhx2-cKO: 13 mcherry-Cas9-EGFP+ cells, 40 mcherry+Cas9-EGFP+ cells; GLME model with Sidak’s multiple comparisons test, **P = 0.0037; GLME model, **P = 0.006, 0.003). h. RNAscope for Gabbr1 expression in Cas9-EGFP cortical astrocytes lacking Lhx2 and controls at P28; quantitative analysis demonstrating reduction of Gabbr1 expression is derived from n = 3 pairs of animals (51,55 cells; GLME model, *P = 0.015; LME model, **P = 0.0003). Scale bars, 100 μm (a), 20 μm (m). Data represent mean ± s.d. (a, g right), median, minimum value, maximum value and IQR (f, g left, h).

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Supplementary information

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Supplementary Table 1

P1–P14 astrocyte transcriptomic data, showing Gene Ontology analysis across timepoints and regions.

Supplementary Table 2

RNA-seq transcriptomic data from Gabbr1-cKO and control astrocytes, showing DEGs between Gabbr1-cKO and controls.

Supplementary Table 3

scRNA-seq data from Gabbr1-cKO and control cortex, comparing DEGs.

Supplementary Table 4

P1–P14 astrocyte transcriptomic data, showing DEGs across timepoints and regions.

Supplementary Table 5

Transcription factor expression across the OB and cortex from P1 to P14.

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Cheng, YT., Luna-Figueroa, E., Woo, J. et al. Inhibitory input directs astrocyte morphogenesis through glial GABABR. Nature 617, 369–376 (2023). https://doi.org/10.1038/s41586-023-06010-x

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