www.sciencemag.org/content/351/6275/849/suppl/DC1

Supplementary Materials for

Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling

W. Todd Farmer, Therese Abrahamsson, Sabrina Chierzi, Christopher Lui, Cristian

Zaelzer, Emma V. Jones, Blandine Ponroy Bally, Gary G. Chen, Jean-Francois Théroux, Jimmy Peng, Charles W. Bourque, Frédéric Charron, Carl Ernst, P. Jesper Sjöström,

Keith K. Murai*

*Corresponding author. E-mail: keith.murai@mcgill.ca

Published 19 February 2016, Science 351, 849 (2016) DOI: 10.1126/science.aab3103

This PDF file includes

Materials and Methods Figs. S1 to S25 Tables S1 to S4 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/351/6275/849/suppl/DC1)

Table S5 as an Excel table.

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Materials and Methods Animals and Cre recombination induction procedures

Experiments were done according to policies set forth by the Canadian Council on Animal Care and the Montreal General Hospital Facility Animal Care Committee. Animals were maintained in standard housing conditions. Both males and females were used for experiments. Littermate pairs were used whenever possible. The solution for in vivo activation of Cre recombinase was prepared by dissolving tamoxifen (Sigma) in 100% EtOH at 200 mg/ml by gentle heating. Once the tamoxifen was in solution it was quickly diluted 1:10 in corn oil (Sigma). The resulting 20 mg/ml solution was used for intraperitoneal (IP) injections of adult mice.

Shh reporter animals were bred by crossing Shh+/CreERT2 mice (31) with Rosa26mTmG/mTmG mice (32) to generate Shh+/CreERT2;Rosa26+/mTmG mice. Tamoxifen was given IP 5 weeks+ of age at 1 mg/injection, 1 or 2 injections/day for 5 days. Non-induced control animals received an equivalent number of injections of vehicle without tamoxifen. Animals were collected 2-4 weeks after the last dose of tamoxifen. Using the Rosa26Tom reporter line (Ai9)(33) produced the same results as the RosamTmG line. No recombination was seen in animals given vehicle alone or animals lacking CreERT2. As ShhCreERT2 is a Shh null allele, Shh+/CreERT2 mice were used as Shh heterozygous null animals (Shh+/-).

To produce conditional Smo loss-of-function mice, GlastCreERT2/CreERT2;Smo+/c animals were crossed with Smo+/c;Rosa26mTmG/mTmG to generate Glast+/CreER;Smo+/+;Rosa26+/mTmG and Glast+/CreER;Smoc/c;Rosa26+/mTmG experimental mice (19, 20). Tamoxifen was given IP at 5 weeks of age at 1 mg/injection, 2 injections/day for 5 days. Brains were collected 30 days after the final injection.

To generate genetically-induced Smo gain-of-function mice, GlastCreERT2/CreERT2;Rosa26Tom/Tom mice were crossed with Rosa26SmoM2/Tom mice to generate Glast+/CreER;Rosa26Tom/Tom and Glast+/CreER;Rosa26SmoM2/Tom mice (23). At 5 weeks of age, animals were given 1mg/injection of tamoxifen twice a day for 5 days. Brains were collected 14 days after the final injection. For early postnatal development studies, an intragastric injection of 50 ug of tamoxifen was given at postnatal day 2. Brains were collected at the indicated time points.

To produce conditional Shh loss-of-function mice, CaMKII-Cre +/tg;Shh+/c animals were crossed with Shh+/c;Rosa26Tom/Tom to give both CaMKII-Cre+/tg;Shh+/+;Rosa26+/Tom and CaMKII-Cre+/tg;Shhc/c;Rosa26+/Tom experimental mice (34, 35). Brains were collected at 8 weeks of age. Stereotaxic Virus Injections

Mice were anesthetized with isoflurane and mounted on a stereotaxic apparatus (Stoelting). An incision was made on the skin of the head to expose the bone. Using a microdrill, a square fragment (4-9 mm2) was removed from the bone above the cerebellum or motor/somato-sensory cortex. Two injections of Cre-expressing adenovirus suspension (ADV-005, Cell Biosystems, 1:5 in PBS, 0.5-1.5 ul total volume) were stereotaxically injected into the brain at a speed of 0.1 ul/min using a Hamilton syringe

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connected to a glass pipette driven by an automatic injector (Harvard Apparatus). The following coordinates were used: 0.0-0.5 mm anterior-posterior, 1.5-2.85 mm medial-lateral, 0.2-0.4 mm dorsal-ventral (cortex); 5.8 – 7.0 mm anterior-posterior, 0.5-2.5 mm medial-lateral, 0.2-0.7 mm dorsal-ventral (cerebellum). The glass pipette was left in the injection site for at least 5 minutes after the injector was stopped. Following injections, the skin was sutured and antibiotic powder was spread over the stitches. After recovering from anesthesia mice were returned to their cages. Analgesia was provided according to university standard operating procedures.

Immunofluorescence

Mice were transcardially perfused with 4% paraformaldehyde (PFA), brains were removed and incubated in 4% PFA overnight. After post-fixing, the brains were equilibrated in 30% sucrose before being snap frozen in OCT media in a bath of 100% EtOH and dry ice. Embedded brains were cryosectioned at 30-40 µm to produce free-floating sections. For immunostaining, sections were permeabilized with 1% Triton-X100 in PBS for ~15 minutes before blocking with 10% normal donkey serum (NDS) in PBS with 0.3% Triton-X100 for 1-2 hours. All antibody incubations and washes were performed in 1%NDS, 0.3% Triton-X100 PBS. Primary antibodies were applied for 18 – 72 hours at 4 °C. After washing three times, fluorescent secondary antibodies (Invitrogen, Jackson 1:1000) were applied for 1-2 hours before washing three times. For Patched 2 immunolabeling, sections were incubated in pre-heated 10 mM Sodium Citrate (pH 8.5) for 30 minutes then allowed to cool to room temperature before starting immunolabeling procedures. Sections were mounted with SlowFade Gold (Life Technologies) prior to imaging. See Table S1 for antibodies used.

DAB Immunolabeling

To visualize Shh protein in the adult brain, 10 µm cyrosections of 4% PFA perfused brains were collected on superfrost slides. Slides were incubated for 20 minutes in methanol solution with 3% H2O2, then incubated in citrate buffer (10mM citric acid, 0.05% Tween 20, pH 6.0) for 1 hour in a 95°C water bath. Slides were blocked with 10% goat serum in PBS with 0.1% Triton X-100 for 1 hour. All subsequent antibody incubations and washes were performed in 1% goat serum, 0.1% Triton X-100 PBS. Slides were incubated in rabbit anti-Shh monoclonal antibody (Genentech 95.9, 1:200; a kind gift from Suzie Scales) overnight at 4°C. After 3 washes, slides were incubated in goat anti-rabbit biotinylated secondary antibody (Vector Labs BA-1000, 1:200) for 1 hour. After 3 washes, slides were incubated in Vectastain Elite ABC kit (Vector Labs, PK-6100) in PBS for 30 minutes. After 3 washes in PBS, slides were reacted with 1 ml DAB (Sigma D6190) for 1-5 minutes before being washed with water and mounted in Mowiol. Slides were imaged in brightfield on a Leica DM4000 microscope. Note that the Genentech 95.9 antibody has been previously validated, showing no signal in Shh mutant neural tubes (36).

Image Acquisition and Colocalization Analysis

Images were acquired using an Olympus FV-1000 laser scanning microscope and Fluoview software (Olympus). To measure relative amounts of protein in astrocytes, a modified (compatible with the ImageJ ROI Manager) Intensity Colocalization Analysis

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(ICA) plugin for ImageJ was used to correlate the signal of antibody staining to the signal of the genetically expressed reporter in the astrocyte of interest (10). All ROIs were selected manually in blinded images. ROIs consisted of AQP4+ fragments of BG palisades (Fig. 1D, S3D, S12A-B), virus infected regions of cerebellar cortex molecular layer (Fig. 2B-C, S5), tdTomato+ astrocytes in the cerebellar granule cell layer (Fig. 3B-D, S9) or tdTomato+ protoplasmic astrocytes of the cerebral cortex, hippocampal regions CA1 and dentate gyrus (Fig. 4A-B, S15, S18, S19, S20, S22). Due to the low expression of SmoM2-YFP and therefore low signal-to-noise ratio, ROI-based background subtraction was used to exclude background staining of YFP in the experiments using the SmoM2-YFP allele. The resulting data was parsed, analyzed and plotted using Python and R. The average Pearson’s Correlation Coefficient of each animal was used for the bar graphs and statistics shown. Values are reported as mean ± SEM. Statistical tests were performed using the unpaired Student’s t-test for pairwise comparison (Fig. 2C) or one-way ANOVA with Tukey’s multiple comparison test.

Quantitative reverse transcriptase PCR (qRT-PCR)

For qRT-PCR, brains were quickly removed and dissected. Whole cerebellum, hippocampus and cortex (containing motor and sensory cortices) were separately snap frozen in cryotubes in a bath of 100% EtOH and dry ice. Total RNA was extracted using the Qiagen RNeasy Lipid Tissue Kit. First strand synthesis was performed using Qiagen QuantiTect Reverse Transcription Kit with random primers. Quantitative PCR was performed using Sybr Green Master Mix (Applied Biosystems Systems) on a StepOne Plus thermocycler (Applied Biosystems). Relative levels of mRNA were calculated using the CT method with GAPDH as the internal control. The RT-PCR primer sets used can be found in Table S2.

Cerebellar Primary Astrocyte Cultures and Immunofluorescence

For cerebellar glia cultures, 2-3 cerebella were dissected from P5-6 mice and dissociated by treatment with papain (0.1% papain, 0.02% BSA in Neurobasal-A medium for 15 minutes at 37°C) followed by trituration using a 1 ml pipette tip in Neurobasal-A medium supplemented with 2% B27, 1 mM Glutamax and 1% penicillin/streptomycin and 10% horse serum (Invitrogen). Glia were plated onto poly-L-lysine-coated coverslips ((0.1 mg/ml); Sigma) in 12-well dishes containing 1 ml of warmed Neurobasal-A density of 150,000 cells/cm2. Half of the media was replaced with fresh media the following day and every three days thereafter. For visualization of surface Smoothened, Glast, and Patched 2 levels, we used a protocol described previously (11). 14-day old cultures were chilled on ice, washed once with ice-cold PBS, and fixed with 4% paraformaldehyde/0.1 M phosphate buffer for 10 min. Non-permeabilized cells were blocked in 5% BSA/PBS (one hour) and incubated with an antibody against the extracellular portion of Smoothened (1:50, Santa Cruz sc-6366), Patched 2 (1:200, Santa Cruz sc-9672), or GLAST (1:200, Abcam ab-416) overnight at 4 degrees. After three washes in PBS, primary antibodies were revealed with a one hour incubation (room temperature) with Alexa Fluor secondary antibodies (1:300, 5% BSA/PBS) and confocal microscopy.

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Hippocampal Astrocyte Culture and qRT-PCR Dissociated astrocytes were prepared from P3 mice pups as previously described

(Jones et al, 2012). The astrocytes were grown on poly-D-lysine (0.1 mg/ml, Sigma) coated dishes in Minimal Essential Medium containing Earle’s salts and L-glutamine supplemented with 10% horse serum, 0.6% glucose, and 1% penicillin/ streptomycin (Invitrogen) at 37 °C and 5% CO2. Cells were treated with the sonic hedgehog antagonists cyclopamine, 5 uM, (Santa Cruz) and PF-5274857, 10 nM, (abcam) for 48 hours. Drugs were dissolved in 1% DMSO. Corresponding vehicle controls consisted of 1% DMSO. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). qRT-PCR was performed as described as above.

Electrophysiology

For preparing acute cerebellar slices, mice were anesthetized with isoflurane and the brains were quickly removed and submerged in ice-cold solution containing (in mM): 85 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 4 MgCl2, 25 glucose, and 75 sucrose, saturated with 95% O2 and 5% CO2. 200 µm thick parasagittal slices of the cerebellar vermis were cut using a vibratome (Camden Instruments).

To ensure optimal quality, hippocampal slices were obtained after cardiac perfusion. Tom/Tom and SmoM2/Tom mice were anesthetized with isoflurane and perfused through the left cardiac ventricle with an ice-cold solution containing (in mM): 50 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 10 MgCl2, 25 glucose, and 111 sucrose, saturated with 95% O2 and 5% CO2. After perfusion, the brains were quickly removed and 300 µm thick coronal slices were cut. The cutting solution had a lowered CaCl2 concentration of 0.5 mM.

Slices were incubated at 32 °C for 15-30 minutes, after which they were allowed to cool to room temperature. The external solution contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 25 glucose. For hippocampal slices, the solution was adjusted to ~320 mOsm to minimize the difference between internal and external osmolality (39), whereas for cerebellar slices the solution was adjusted to standard ~338 mOsm (40).

For cell visualization and electrophysiological recordings, labeled cerebellar Bergmann glia or astrocytes in the stratum radiatum were identified using a custom-built 2-photon laser scanning microscopy system (Buchanan et al., 2012). A MaiTai BB Titanium:Sapphire laser (Spectraphysics) was tuned to 840 nm to visualize GFP/Alexa488/Alexa594 fluorescence, or to 870 nm to visualize tdTomato labeling. Selected Bergmann glia or hippocampal astrocytes were patched using infrared Dodt-contrast and a CCD camera (Watec). Bergmann glia were voltage-clamped at 75 mV, and astrocytes at 80 mV using a BVC-700A amplifier (Dagan) with glass patch-electrodes of tip resistances between 4 and 5 M . Recordings were made at 32-34°C. Pipettes were backfilled with intracellular solution containing (in mM): 115 K-Gluconate, 10 HEPES, 5 KCl, 0.3 Na-GTP, 4 Mg-ATP, 10 Na-Phosphocreatine, 0.02 Alexa Fluor 594, adjusted to ~310 mOsm and pH 7.3. For recordings of hippocampal astrocytes 1 mM MgCl2 was added to and Alexa Fluor 594 was omitted from the solution. Series resistance and cell capacitance were compensated in recordings from astrocytes but not Bergmann glia. Liquid junction potential (-12 mV) was not corrected for. Currents were

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digitized at 10 kHz using PCI-6229 boards (National Instruments). Acquisition and analysis were carried out with custom-made IGOR Pro software (Wavemetrics).

To elicit AMPA currents from Bergmann glia, a custom-built photolysis system was incorporated into the 2-photon laser scanning system. A 120 mW, 405 nm violet solid-state laser (Amazon.ca) was gated with a Uniblitz LS3ZM2/VCM-D1 shutter (Vincent Associates) to a pulse duration of 10 ms, and was combined with the Ti:Sa 2-photon imaging beam using a Semrock FF665-Di02 dichroic mirror so that uncaging and imaging beams were controlled using the same pair of 6215H 3 mm galvanometric mirrors (Cambridge Technologies). Violet laser power was always at max, and was measured to ~11 mW at the objective back aperture using a Thorlabs power meter (PM100A/S121C). The caged compound (N)-1-(2-Nitrophenyl) ethylcarboxy-(S)- -1-(2-nitrophenyl) ethylcarboxyamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (NPEC-AMPA, Tocris) was dissolved at 1 mM in external recording solution (see above). To keep pH at 7.3, this solution was modified to include 20 mM HEPES buffer. NPEC-AMPA was locally applied via a glass puff pipette with a tip resistance of ~2 M . To ensure that the puff pipette did not clog, 20 M Alexa Fluor 488 was added to visualize local perfusion plume. The puff pipette was typically positioned ~50 m from the cell soma, as illustrated in Figure 2D. During uncaging pulses, the galvanometric mirrors were parked in the centre of the perfusion plume and in the middle of the arborisation of the recorded cell. Amplitudes of uncaging-evoked responses were measured in voltage clamp. These responses rapidly diminished in amplitude with each uncaging pulse, presumably due to AMPA receptor desensitization, so only the first and largest response was used for statistical comparisons. After a few minutes wait, responses typically recovered to their initial amplitude.

To activate Kir-currents in hippocampal astrocytes, voltages were repeatedly stepped from 160 to 0 mV, in 20 mV increments. An initial step of 0 mV was applied to inactivate non-Kir K+ -currents (see Figure 4). 100 M BaCl2 was bath applied to the slice approximately six minutes after the start of the recording. The current amplitude was measured as the difference between the baseline level before the initial voltage step, and the mean amplitude over a 10 ms window starting 10 ms after onset of the hyperpolarizing voltage step. The Ba2+ sensitive current was obtained by subtracting the current after Ba2+ application from the current before application in 2-4 consecutive sweeps. The rectification index (RI) of Ba2+ sensitive currents was determined by calculating the ratio of currents at 40 mV and 120 mV. Data was automatically classified using in-house agglomerative single-linkage hierarchical clustering software running in IGOR Pro. A 25% linkage threshold, as normalized to the greatest separation in the data set, was used as a best-cut selection criterion for the number of found clusters. The linkage percentage units do thus not directly correspond to percentage units on the x-axis because dendrograms were normalized to the greatest separation. The fuzzy c-means clustering procedure provided in IGOR Pro was also used and always gave the same classification of data points.

Values are reported as mean ± SEM. Statistical tests were performed using the unpaired Student’s t-test and the Wilcoxon-Mann-Whitney two-sample rank test.

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Single cell capture Mice were anesthetized with isoflurane and decapitated. Brains were removed and

immersed in chilled (2°C–4°C) and oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) (pH 7.35) comprising (in mM) 120 NaCl, 3 KCl, 1.23 NaH2PO4, 26 NaHCO3, 1.5 MgCl2, 2 CaCl2, and 10 glucose; osmolality adjusted to 305 mOsm with mannitol or water. 400 µm thick slices from the cerebellum were cut using a Leica VT1200 and transferred to oxygenated ACSF at 33°C (heated water bath) where they were left to recover for 1 hour. After recovery, they were transferred to the perfusion chamber for cell capture and perfused at a rate of 1 to 2 mL per minute using oxygenated ACSF 33°C (TC-344B Warner Instruments Corporation). Single-cell collection was done using autoclaved capillaries pulled to micropipettes with resistances between 1 to 2 M and filled with 1.0 µl of a solution comprising sterile PBS and RNaseOUT (40 U/µl; Invitrogen-Life Technologies; 10:0.5). Positive pressure was applied to the micropipette using a syringe connected by tubing. Cells were chosen by morphology and positive fluorescence. Upon contact, negative pressure (suction) was applied ensuring the entrance of fluorescence material inside the micropipette, once enough material was inside the micropipette the cell was lifted and completely suctioned inside. The content was then expelled by positive pressure into a 200 µl microcentrifuge tube stored on dry ice. 10 to 12 cells were collected for each sample (N=3 for each condition). All the solutions were filtered (0.2 mm filter) prior to use. All materials and surfaces were treated with DNAzap and RNaseZap (Ambion) prior to use. Once collection was done, samples were stored at -80°C.

RNA sequencing

Single cell capture samples spiked with ERCC controls (Ambion) were processed with the SMART-Seq UltraLow Input RNA Kit (Clontech, V4), followed by the Nextera XT DNA Sample Preparation kit, with indexing (Illumina). Sample quality was assessed using the 2200 TapeStation (Agilent), and sequenced at 6 libraries per lane on an Illumina 2500 HiSeq for 100bp paired-end reads at the McGill University and Genome Quebec Innovation Center. All libraries passed an initial quality control step using the FASTQC pipeline. The Fast-X toolkit was used to trim the first and last 10 bases of each read. TruSeq specific paired-end adapters and low-quality stretches were removed using Trimmomatic, while poly-A tails were trimmed using PrinSeq. Surviving paired and orphaned reads were separately aligned using TopHat to a modified version of the mouse genome (GRCm38 with the addition of the tdTomato and YFP gene sequences to more accurately reflect the experimental design). The number of reads mapping to each known mouse gene was computed using Bedtools' multiCov application. HTseq was used to count pairs of reads mapping to specific genes. The results from these two methods were coherent with one another. These raw read counts were normalized using the DESeq2 package which scaled each sample's counts based on their relative sequenced library size. Differential expression analyses using DESeq2 were then carried out to identify significant (p-value < 0.05) genes between Bergman glia (TOM/TOM) and Velate astrocytes (TOM/TOM)(see Table S5 for gene list). Expression levels for these genes were used to generate hierarchical clusters and heat maps from Bergman glia (TOM/TOM), Velate astrocytes (TOM/TOM), and Velate astrocytes (TOM/SmoM2) in

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order to identify potential shifts in astrocytes' expression patterns in the presence of SmoM2.

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Fig. S1. Smo and Ptch2 are expressed on the surface of cultured cerebellar astrocytes. (A-B) Cell surface immunofluorescence performed on non-permeabilized astrocyte cultures. (A) Smoothened (Smo, magenta) and Patched2 (Ptch2, green) are expressed in clusters along the surface of cerebellar astrocytes. Note the complimentary surface expression of Smo and Ptch2 (inset, upper right). Smo and Ptch2 are also detectable in a complimentary pattern on primary cilia on a subset of cultured astrocytes (inset, lower left). (B) Smo and GLAST are co-localized on the surface of cultured cerebellar astrocytes. Inset shows high magnification image of the dashed box. Scale bar, 30 m.

Surface Smoothened Surface GLAST Surface Smoothened

Surface GLAST

Surface SmoothenedSurface Patched2 Surface Patched2Surface Smoothened A

B

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E

F anti-Shh secondary-only control

A

B

GCL

ML

PCL

GCL

ML

PCL

Granule Cells in GCLCalbindin mGFP C

D

CreERT2Shh mTom mGFPlox-P lox-P

Rosa26 mGFPRosa26++TAM

G

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Fig. S2. Shh protein is produced by neurons in the mature cerebellar cortex. (A) Schematic showing the genetic reporter strategy to label Shh expressing cells in the mature brain. Tamoxifen-inducible Cre recombinase is expressed from the endogenous Shh locus. The Cre-dependent reporter allele expresses tdTomato before recombination and mGFP after recombination. Cells with an active Shh locus during Tamoxifen application will undergo Cre-mediated recombination and permanently express mGFP. Cre recombination was induced at >5 weeks. (B) A large number of granule neurons and their parallel fibers (green) are labeled upon Cre induction. Purkinje cells labeled with anti-Calbindin (magenta). (C) High magnification images of labeled granule cells (green) within the GCL. (D-E) Basket and stellate cell in the ML in the Shh reporter background coupled with a Cre-dependent tdTomato reporter line. (F) Immunoperoxidase labeling showing prominent expression of Shh in Purkinje cells, granule cells, and interneurons. (G) Immunolabeling in the absence of primary antibody shows no specific labeling. ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, B) 60 m, C) 20 m, D) 25 m, E) 20 m, F) 60 m

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0.00

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Exon1 Exon2ATG

lox-P lox-PSmo Smo

mTom mGFPlox-P lox-P

Rosa26

lox-PExon2

mGFPlox-P

Rosa26

+ +Tamoxifen

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Colocalization with mGFP (Pearson’s Coefficient)

AQP4

-1.0 -0.5 0.0 0.5 1.00

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Colocalization with mGFP (Pearson’s Coefficient)

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Fig. S3. Characterization of Smoothened (Smo) loss-of-function mice. (A) Schematic of the alleles used to induce conditional Smo loss of function and to visualize genetically recombined adult Bergman glia. Tamoxifen-inducible Cre is expressed from the GLAST locus. LoxP sites flank the ATG-containing Exon1 of Smo in the Smoc allele. The Rosa26 locus contains a Cre-dependent reporter cassette that expresses membrane-targeted tdTomato (mTom) before recombination and membrane-targeted EGFP (mGFP) after recombination. (B) Cre induction with tamoxifen (at 5 weeks) leads to a ~25% reduction of Smoothened mRNA levels in the Smoc/c cerebellum assayed by qRT-PCR one month after tamoxifen injection (n=10 Smo+/+, 7 Smo+/c, 6 Smoc/c mice). (C) Smo loss-of-function does not perturb motor learning performance on the accelerated rotarod in Smo conditional animals when compared to Smo WT (+/+) controls. Mice were trained 3 trials/day for 3 days for a total of 9 trials. Motor learning and coordination was measured by latency to fall in seconds. (D) Genetic loss-of-function of Smo in adult mice leads to a downregulation of GluA1 and an upregulation of AQP4 in BG palisades as assayed by colocalization analysis. Left, average Pearson’s Correlation Coefficient (PCC) of each animals (n = 6 pairs). Right, histograms showing the distribution of PCCs for individual palisades in the 6 pairs (n=1193 Smo+/+ ROIs, 2077 Smoc/c ROIs). Error bars denote ±SEM. One-way ANOVA with Bonferroni post-test (B), two-way ANOVA with Bonferroni post-test (C), and Student’s t-test (D). * p 0.05, ** p 0.01, *** p 0.001.

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Smoc

/c ;

mTo

m-m

GFP

mGluR1Calbindin CalbindinmGFPmGFP

Smo+

/+ ;

mTo

m-m

GFP

mGluR1mGFP

GLA

ST C

reER

T2G

LAST

Cre

ERT2

Fig. S4. No overt changes in cerebellar anatomy following genetic removal of Smo.Purkinje cells (Calbindin+) and glutamatergic synapses onto Purkinje cell dendrites (mGluR1+) remain present at high density after 1 month of loss of Smo in Bergmann glia (mGFP+). Scale bars, Low and high magnification, 20µm and 10µm, respectively.

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Smo+/+ ; mTom-mGFP Smoc/c ; mTom-mGFP

mG

FPG

LAST

mer

geG

LT1

Virally expressed Cre

Smo+/+ ; mTom-mGFP Smoc/c ; mTom-mGFP

mG

FPPt

ch2

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geVirally expressed CreA

Smo+/+ ; mTom-mGFP Smoc/c ; mTom-mGFP

mG

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luA4

mer

ge

Virally expressed CreB

Smo+/+ ; mTom-mGFP Smoc/c ; mTom-mGFPm

GFP

Kir4

.1m

erge

Virally expressed CreC D

Fig. S5. Loss of Smo in Bergmann Glia using a Cre-expressing virus results reduced expression of Ptch2 (A), GluA4 (B), GLAST (C), and Kir4.1 (D). Cells that have undergone Cre recombination express mGFP (green). GLT1 expression is not affected (C). See Figure 2 for quantification. Scale bar, 40 µm.

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Fig. S6. Smo loss-of-function does not disrupt Purkinje cell morphology. BG that have undergone Cre recombination express mGFP (green). Neither viral infection of BG (mGFP) nor loss of Smoothened affects the morphology of Purkinje cells as revealed by Calbindin staining. Scale bar, 20 µm.

mer

geC

albi

ndin

mG

FPSmo+/+ ; mTom-mGFP Smoc/c ; mTom-mGFP

Virally expressed Cre

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A

CreαCaMKII promoter

Exon1 Exon2lox-P lox-P

Shh Shhlox-P

+stop tdTomato

lox-P lox-PRosa26 Rosa26 tdTomato

Exon1

BShh+/+ ; Tom Shhc/c ; Tom

Tom

ato

Kir4

.1m

erge

GLA

ST

CaMKII Cre

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Fig. S7 Local Shh loss-of-function alters BG gene expression. (A) Schematic of the alleles used to drive Shh loss-of-function in mature PCs. Cre is expressed from a CamKII-Cre transgene. LoxP sites flank Exon2 of Shh. The Rosa26 locus contains a Cre-dependent reporter cassette that expresses tdTomato (Tom) after recombination. (B) Kir4.1 and GLAST downregulation in BG adjacent to PCs that have lost Shh (8 weeks, n=4 pairs). PCs that have undergone Cre recombination express tdTomato (red). In the BG adjacent to Shh- PCs, levels of Kir4.1 (green) and GLAST (blue) are drastically reduced (green). Scale bar, 30 m.

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Fig. S8. Loss of Shh from Purkinje cells does not perturb the expression of proteins common to both Bergmann glia and velate astrocytes. GLT1 (green) and Sox9 (blue), are not altered by Shh loss-of-function in PCs (n=4 pairs). PCs that have undergone Cre recombination express tdTomato (red). Scale bar, 30 µm.

Tom

ato

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geG

LT1

Sox9

Shh+/+ ; Tom Shhc/c ; TomCaMKII Cre

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SmoM2Tom

GluA1

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+ +TAM

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SmoM2/Tom, SmoM2(-)

SmoM2/Tom, SmoM2(+)

Tom

21

Fig. S9. Smo gain-of-function causes molecular changes to VAs in vivo. (A) Schematic of the alleles used to drive Hedgehog signaling in astrocytes throughout the brain. (B-D) In situ cell cytometry in Smoothened gain-of-function cerebellum shows that multiple BG-associated genes that are upregulated in VAs expressing SmoM2-YFP. Colocalization analysis from confocal images is shown. PCC of tdTomato versus SmoM2-YFP is along the y-axis. PCC of Tomato versus (B) GluA1 (n=464 Tom and n=350 Tom/SmoM2-YFP cells), (C) Kir4.1(n=271 Tom and n=160 Tom/SmoM2-YFP cells), and (D) GLAST(n=517 Tom and n=373 Tom/SmoM2-YFP cells) is along the x-axis (n=4 pairs of mice). The tdTomato/SmoM2-YFP PCC threshold (horizontal dashed line) is set so the tdTomato+ cells of Tom animals (blue) fall below this threshold. tdTomato+ cells of Tom/SmoM2 animals (red) were designated either SmoM2-YFP+ or SmoM2-YFP- based on this threshold. Individual color plots show the same data with the two genotypes separated. The shaded polygons outline the three populations of cells (Tom, SmoM2(-), and SmoM2(+)). The average (per animal) PCC of these proteins for each of the three populations are plotted in the bar graphs Figure 3.

22

Tom SmoM2-YFP Ki67C

ereb

ellu

mSmoM2/TomTom/Tom

Tom SmoM2-YFP Ki67C

orte

xC

A1 H

ippo

cam

pus

GLAST CreERT2

23

Fig. S10. Conditional activation of Shh signaling in vivo in the adult brain with SmoM2-YFP does not increase cell proliferation in cerebellum, cortex, or hippocampus. Confocal microscopy images show representative examples of Ki67 labeling of sections from different brain regions and genotypes. Note that rare, sparse Ki67 labeled cells (white) are detected in each section. The Ki67 signal was not found to be associated with SmoM2-YFP+ astrocytes (green). Scale bar, 100 m.

24

Fig. S11. Example of single cerebellar astrocyte collection by patch pipette.(A) Position of Bergmann glia cell soma prior to placement of the patch pipette. (B) Position of the patch pipette prior to the application of suction. (C) tdTomato+ material being aspirated into the patch pipette. (D) Absence of the Bergmann glia cell soma after the reretraction of the patch pipette. Arrowhead, Soma of Bergmann glia. Dashed bracket, position of patch pipette. Solid bracket, tdTomato+ material within the patch pipette. Hollow arrowhead, position of collected soma before collection. Scale bar, 20 µm.

PCL

ML patchpipette

A B C D

25

A

B

C

D

P8 P10 P13

GLA

ST

P7 (mGFP)

Pia

EGL

ML

PCL

GCL

Glu

A1

P7 P9 P13

ML

EGL

GCL

PCL

MLEGL

GCL

PCL

ML

EGL

GCL

PCL

P7 P9 P13 P15

4PQA

ML

EGL

GCL

PCL

ML

EGL

GCL

PCL

ML

EGL

GCL

PCL

ML

GCL

PCL

Fig. S12. Dynamic expression of GluA1, GLAST, and AQP4 in BG and VAs in the developing mouse cerebellum. (A) GluA1 is expressed at postnatal day 7 in radial glial cells and elaborating BG process-es in the developing molecular layer. GluA1 becomes enriched at postnatal day 9 and 13 as the molecular layer expands. (B) GLAST is expressed throughout early postnatal develop-ment in radial glia and BG in the molecular layer. GLAST expression starts to become reduced in the inner GCL around postnatal day 13. (C) AQP4 is expressed at postnatal day 7 in radial fibers and maturing BG. By postnatal day 9, AQP4 is strongly expressed in the developing molecular layer. However, by postnatal day 13, AQP4 expression is reduced in the molecular layer and becomes more restricted to astrocytic endfeet around blood vessels by postnatal day 15. AQP4 continues to be strongly expressed by VAs in the inner GCL. (D) mGFP expression in developing BG at postnatal day 7. mGFP expression was induced in GLAST CreERT2; mTom-mGFP mice with a single dose of tamoxifen at postnatal day 2. Tissue was recovered at postnatal day 7. EGL, external GCL; ML, molecular layer; PCL, Purkinje cell layer; GCL, inner GCL. Scale bars, A) 50 µm, B) 100 µm, C) 50 µm, D) 25 µm.

26

Fig. S13. Loss or activation of Shh signaling alters GluA1, GluA4, Kir4.1 and AQP4 expression in developing Bergmann glia. (A) Loss of Smo was induced in GLAST CreERT2; mTom-mGFP; Smoc/c mice with a single dose of tamoxifen at postnatal day 2. Tissue was recovered at postnatal day 15. Labeling for mGFP, GluA1, and AQP4 antibodies in both Smo+/+ and Smoc/c genotypes. Boxed area indicates a region of high AQP4 expression in the molecular layer. (B) Quanti-fication of changes in GluA1 and AQP4 expression in Smoc/c mice by colocalization analysis (n=3 pairs). (C) Activation of Shh signaling by expression of SmoM2 in VAs (arrowheads) increases GluA1 expression. (D) Activation of Shh signaling in VAs (arrow-heads) increases GluA4 and Kir4.1 expression. ML, molecular layer; GCL, granule cell layer. Error bars denote ±SEM. Student’s t-test. ** p ≤ 0.01, *** p ≤ 0.001. Scale bar, 50 µm.

A BmGFP GluA1 AQP4 Merge

ML

GCL

Smo+

/+ ;

mTo

m-m

GFP

Smoc

/c ;

mTo

m-m

GFP

GLA

ST C

reER

T2P15

Tom GluA4 Kir4.1

ML

GCL

Merge

Tom

/Tom

SmoM

2/To

mG

LAST

Cre

ERT2

P15

Tom

/Tom

SmoM

2/To

m

Tom GluA1 AQP4

ML

GCL

Merge

GLA

ST C

reER

T2

P15

GluA1

Smo+/+

Smoc/c

0.00

0.05

0.10

0.15 ***

Col

ocal

izat

ion

with

mG

FP

(Pea

rson

’s C

oeffi

cien

t)

AQP4

Smo+/+

Smoc/c

0.0

0.2

0.4

0.6

**

Col

ocal

izat

ion

with

mG

FP

(Pea

rson

’s C

oeffi

cien

t)

D

A

C

27

Fig. S14. Developmental activation of Shh signaling increases cell proliferation in the molecular and granule cell layers. (A) Activation of Shh signaling was induced in GLAST CreERT2; Rosa26Tom/Tom (Tom) and GLAST CreERT2; Rosa26SmoM2/Tom (SmoM2/Tom) mice with a single dose of tamoxifen at postnatal day 2. Tissue was recovered at postnatal day 15. Tissue was labeled for Ki67, a cell proliferation marker, and TO-PRO to counter-label cell nuclei. tdTomato, Ki67 double positive (Tom+/Ki67+) cells are labeled with arrows. (B) Quantification of the overall number of Ki67+ cells in the granule and molecular layers of Tom and SmoM-2/Tom mice. SmoM2 expression increases the average number of Ki67+ cells per animal (n=3 pairs). (C) Quantification of the number of Tom+/Ki67+ cells in the granule and molecular layers of Tom and SmoM2/Tom mice. SmoM2 expression increases the average number of Tom+/Ki67+ cells (n=3 pairs). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Error bars denote ±SEM. Student’s t-test. * p ≤ 0.05, *** p ≤ 0.001. Scale bar, 50 μm.

A

C

BTom Ki67 TO-PRO Merge

GCL

ML

PCL

Tom

/Tom

SmoM

2/To

m

GCL

ML

PCL

GLA

ST C

reER

T2

TomSmoM

20.0

0.1

0.2

0.3 ***

Ki67

+ ce

ll co

unt

(cell

s/100

µm2 )

TomSmoM

20.00

0.05

0.10

0.15 *

Tom

+/Ki

67+

cell c

ount

(c

ells

/100

µm2 )

28

Tom Tom/SmoM2To

mKi

r4.1

Tom

/Kir4

.1

Kir4.1 Tomato Kir4.1

SmoM

2/To

mTo

m/T

om

Tomato

MLGCL

GLA

ST C

reER

T2Cerebellum Hippocampus

Fig. 4Cortex Fig. S22

A

B

B

29

Fig. S15. Activating the Shh pathway in adult mice upregulates Kir4.1 expression throughout the brain. (A) Full sagittal sections of the mouse brain from GLAST CreERT2;Rosa26Tom/Tom (Tom) and GLAST CreERT2;Rosa26Tom/SmoM2 (Tom/SmoM2) mice. tdTomato reporter (magenta) and Kir4.1 (green) are shown. Tom control mice (left column) show the normal expression of Kir4.1 in the adult brain. Tom/Smo mice (right column) show widespread upregulation of Kir4.1in astrocytes throughout the brain. (B) Overview of Kir4.1 expression in the cerebellum of SmoM2 animals. Tomato/Kir4.1 double-positive VAs are not found in Rosa26Tom/Tom cerebella but are present in the cerebella of animal harboring the SmoM2 allele (arrowheads). Scale bars, 1 mm.

30

Dentate Gyrus

YFP+

YFP-

PCC(tdTom/Kir4.1)

PCC

(td

Tom

/Sm

oM2-

YFP)

TomSmoM2

TomSmoM2

A

CA1

YFP+

YFP-

PCC(tdTom/Kir4.1)

PCC

(td

Tom

/Sm

oM2-

YFP)

BSmoM2/Tom, SmoM2(-)SmoM2/Tom, SmoM2(+)Tom

SmoM2/Tom, SmoM2(-)SmoM2/Tom, SmoM2(+)Tom

Fig. S16. In situ cell cytometry in the hippocampus shows that Kir4.1 is upregulated in cells that express SmoM2-YFP. Colocalization analysis from confocal images is shown. Plots show Pearson’s Correlation Coefficient (PCC) for all cells in the study. PCC between tdTomato and Smo-YFP is shown along the y-axis. PCC of tdTom/Kir4.1 in the (A) Dentate gyrus (n=238 tdTom and n=158 SmoM2-YFP cells; n=4 mice) and (B) area CA1 of the hippocampus (n=271 tdTom and n=160 SmoM2-YFP cells; n=4 mice) are along the x-axis. To designate cells in the SmoM2-YFP animals (red) as SmoM2-YFP- or SmoM2-YFP+, the tdToma-to/SmoM2-YFP PCC threshold (horizontal dashed line) was set so that the tdTomato+ cells of animals lacking the SmoM2-YFP allele (blue) fall below this threshold. tdToma-to+ cells of animals harboring the SmoM2-YFP allele (red) were designated either SmoM2(-) or SmoM2(+) based on this threshold. Individual color plots show the same data with the two genotypes separated. The shaded polygons outline the three populations of cells (Tom, SmoM2(-), and SmoM2(+)). The average (per animal) PCC of Kir4.1 for each of the three populations are plotted in the bar graphs Figure 4.

31

-15-10-505

10

I m (n

A)

-160 -120 -80 -40 0Vhold (mV)

Fig. S17. Measurement of the rectification index (RI) of the Ba2+-sensitive Kir4.1 current in astrocytes. (A) 2-photon imaging maximum intensity projection of an RFP-labeled hippocampal astrocyte in stratum radiatum. (B) The RI of the Ba2+-sensitive component was measured as the ratio of currents at -40 mV and -120 mV (solid vertical lines), in this example RI(Ba)-40/-120 = 0.53. Dashed vertical line: Erev = -70.8 mV.

A

B

32

Gli1 Ptch1 Kir4.1 GFAP GLAST ALDH1L1 GLT1

0.0

0.5

1.0

1.5ControlCyclopamine (5uM)PF-5274857 (10nM)

Rel

ative

leve

ls of

mR

NA

****

*****

* *****

n.s.n.s. n.s. n.s.

Fig. S18. Pharmacological blockade of Hedgehog signaling reduces Kir4.1 expres-sion in culture hippocampal astrocytes.48hr application of the Smoothened antagonists Cyclopamine (5µM) or PF-5274857 (10nM) significantly reduced the levels of Gli1, Ptch, and Kir4.1 mRNAs in cultured hippocampal astrocytes while not affecting the levels of GFAP, GLAST, ALDH1L1, or GLT1 (n=3 experiments). Error bars denote SEM. One-way ANOVA with Tukey's multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001, n.s. not significant.

33

GLAST CA1

Tom SmoM2/Tom0.0

0.1

0.2

0.3

0.4n.s.

n.s.GLAST Dentate Gyrus

Tom SmoM2/Tom0.0

0.1

0.2

0.3

0.4

Colo

caliz

atio

n wi

th T

om(P

ears

on’s

Coef

ficie

nt)

SmoM2 (+)SmoM2 (-)

n.s.n.s.

Tom SmoM2-YFP GLAST

Tom

/ To

mSm

oM2

/ Tom

GLA

ST C

reER

T2

Fig. S19. Smo gain-of-function in adult mice does not alter GLAST in the hippocam-pus. (A) Images of GLAST immunostaining of hippocampal area CA1. SmoM2-YFP+ cells (arrowheads) do not have altered GLAST. (B) Colocalization analysis of cells in the dentate gyrus and CA1. Graphs show the average PCC per animal (n=4 pairs) for the three cell populations (Tom, SmoM2(-), and SmoM2(+)). Error bars denote SEM. One-way ANOVA with Tukey’s multiple comparison test. n.s. not significant. Scale bar, 50 μm.

A

B

34

B

Tom SmoM2-YFP Kir4.1

Tom

/ To

mSm

oM2

/ Tom

GLA

ST C

reER

T2

GluA1A

-0.2

-0.1

0.0

0.1

0.2GluA1 CA1

n.s.n.s.

Tom SmoM2/Tom

Fig. S20. Smo gain-of-function in adult mice does not alter GluA1 expression in astrocytes in the hippocampus. (A) Images of GluA1 immunostaining of hippocampal area CA1. SmoM2-YFP+ cells (arrowheads) do not have altered GluA1. (B) Colocalization analysis of cells in the dentate gyrus and CA1. Graphs show the average PCC per animal (n=3 pairs) for the three cell populations (Tom, SmoM2(-), and SmoM2(+)). Error bars denote SEM. One-way ANOVA with Tukey’s multiple comparison test. n.s. not significant. Scale bar, 50 μm.

-0.2

-0.1

0.0

0.1

0.2

n.s.n.s.

GluA1 Dentate Gyrus

Tom SmoM2/TomColo

caliz

atio

n wi

th T

om(P

ears

on’s

Coef

ficie

nt)

SmoM2 (+)SmoM2 (-)

35

Fig. S21. Smo gain-of-function in adult mice does not alter GluA4 expression in astrocytes in the hippocampus. (A) Images of GluA4 immunostaining of hippocampal area CA1. SmoM2-YFP+ cells (arrowheads) do not have altered GluA4. (B) Colocalization analysis of cells in the dentate gyrus and CA1. Graphs show the average PCC per animal (n=3 pairs) for the three cell populations (Tom, SmoM2(-), and SmoM2(+)). Error bars denote SEM. One-way ANOVA with Tukey’s multiple comparison test. n.s. not significant. Scale bar, 50 μm.

B

-0.1

0.0

0.1

0.2

0.3

n.s.n.s.

Tom SmoM2/Tom

GluA4 CA1

-0.1

0.0

0.1

0.2

0.3GluA4 Dentate Gyrus

Tom SmoM2/TomColo

caliz

atio

n wi

th T

om(P

ears

on’s

Coef

ficie

nt)

SmoM2 (+)SmoM2 (-)

n.s.n.s.

Tom SmoM2-YFP Kir4.1

Tom

/ To

mSm

oM2

/ Tom

GLA

ST C

reER

T2

GluA4A

36

A B

C

Kir4.1 Cortex

Tom SmoM2/Tom0.0

0.2

0.4

0.6

0.8 ******

Col

ocal

izat

ion

with

Tom

(P

ears

on’s

Coe

ffici

ent)

SmoM2 (-) SmoM2 (+)Tom

Tom Kir4.1 Tom SmoM2-YFP Kir4.1

Tom

/ To

mSm

oM2

/ Tom

Tom(+), SmoM2(+), Kir4.1 (high) Tom(+), SmoM2(-), Kir4.1 (low)

E FD

0.0

0.5

1.0

1.5

2.0Shh+/+

Shh+/-

Gli1Shh Kir4.1 AQP4 ALDH1L1

rela

tive

mR

NA

GLAST

*** *** ** ***

Shh+/- Cerebral Cortex

n.s.n.s.

Gli1 Ptch1 Kir4.1 AQP4 GLAST ALDH1L10.0

0.5

1.0

1.5

2.0TomSmoM2

rela

tive

mR

NA

****

SmoM2 Cerebral Cortex

n.s.n.s.n.s.

Shh+/

Cre

ERT2

; R

osa2

6+/To

m

mGFP Kir4.1 mEGFP / Kir4.1

Smo+

/+

mTo

m-m

GFP

Smoc

/c ;

mTo

m-m

GFP

35

Fig. S22. Bi-directional control of astrocyte molecular heterogeneity in the cerebral cortex through Shh signaling.(A) Adult induction of Shh CreERT2; Rosa26 Tomato reporter mice reveals that Shh is expressed by neurons in the adult cerebral cortex. (B) Loss of Smo through viral-mediat-ed Cre expression in Smo conditional allele mice results in loss of Kir4.1 expression in cortex. (C-E) Activation of Shh signaling by expression of SmoM2 in protoplasmic astrocytes increases Kir4.1 expression. (C) Activation of Shh signaling by expression of SmoM2-YFP causes a cell-autonomous increase in Kir4.1 expression in astrocytes throughout the cerebral cortex. High magnification images show both SmoM2(+) (white arrowheads) and SmoM2(-) cells (magenta arrowheads); only the SmoM2(+) cells upreg-ulate Kir4.1. (D) Quantification of the increase in Kir4.1 expression in the cerebral cortex by colocalization analysis (n=4 pairs). One-way ANOVA with Tukey’s multiple comparisons. (E) Expression of SmoM2 increases the mRNA levels of Gli1, Ptch1, and Kir4.1 while not affecting AQP4, GLAST, or ALDHL1 (n=12 Tom/Tom, 9 SmoM2/Tom animals). Student’s t-test. (F) Shh haploinsufficiency reduces the mRNA levels of Shh, Gli1, ALDHL1, and Kir4.1 while not affecting AQP4, GLAST (n= 10 Shh+/+, 8 Shh+/- mice). Student’s t-test. Error bars denote SEM. ** p ≤ 0.01, *** p ≤ 0.001. n.s. not significant. Scale bars, A) and B) 100 µm¬¬, D) 100 µm¬¬, 20 µm.

38

Cerebral CortexYFP+

YFP-

PCC(Tom/Kir4.1)

PCC

(T

om/S

moM

2-YF

P)

TomSmoM2

SmoM2/Tom, SmoM2(-)

SmoM2/Tom, SmoM2(+)

Tom

Fig. S23. In situ cell cytometry in the cerebral cortex shows that Kir4.1 is upregulat-ed in astrocytes that express SmoM2-YFP.Colocalization analysis from confocal images is shown. Plots show PCC for all cells in the study. PCC between Tomato and SmoM2-YFP is shown along the y-axis. PCC of Tom/Kir4.1 in the Cerebral Cortex (n=350 Tom and n=326 SmoM2-YFP cells; n=4 pairs of mice) is along the x-axis. To designate cells in the SmoM2-YFP animals (red) as SmoM2-YFP+ or SmoM2-YFP-, the Tomato/SmoM2-YFP PCC threshold (horizontal dashed line) was set so that the tdTomato-positive cells of animals lacking the SmoM2-YFP allele (Tom, blue) fall below this threshold. Tomato+ cells of animals harboring the SmoM2-YFP were designated either SmoM2(-) or SmoM2(+) based on this threshold. Individual color plots show the same data with the two genotypes separated. The shaded polygons outline the three populations of cells (Tom, SmoM2(-), and SmoM-2(+)). The average (per animal) PCC of Kir4.1 for each of the three populations are plotted in the bar graphs Figure S19.

39

mGFP

GLT1

GLAST

Merge

Virally expressed CreSmoc/c ; mTom-mGFP

Fig. S24. Conditional loss Smo through viral Cre expression does not alter GLAST or GLT expression in the cerebral cortex. Cells that have undergone Cre recombination are mGFP+ (green). Neither GLAST (blue) nor GLT1 (red) are changed upon Cre recombination. Scale bar, 50µm.

40

GLAST Cerebral Cortex

Tom SmoM2/Tom0.0

0.1

0.2

0.3

0.4n.s.

n.s.

Colo

caliz

atio

n wi

th T

om(P

ears

on’s

Coef

ficie

nt)

SmoM2 (+)SmoM2 (-)

Fig. S25. Increasing Smo signaling in adult mice does not alter GLAST in the cere-bral cortex. Colocalization analysis of GLAST and Tom expression in control astrocytes and astro-cytes expressing SmoM2 in the cerebral cortex is shown. The average GLAST/TOM PCC is the same in cells from Tom control animals and SmoM2-YFP+ and SmoM2-YFP- cells from the animals with the SmoM2-YFP allele. Error bars denote SEM. One-way ANOVA with Tukey’s multiple comparison test. n.s. not significant.

41

Antibody name, vendor, catalog number, and concentration used of the antibodies used for immuno labeling of tissue sections. Note that the Ptch2 antibody was used with Sodium Citrate antigen retrieval.

Table S1. List of antibodies used in this study.

Antibody Vendor Cat. # [Conc.] NotesGFP abcam ab13970 1:1000 - GFAP SySy 172 004 1:500 - GFAP Millipore AB5535 1:500 - GLAST abcam ab416 1:500 - GLT1 Millipore AB1783 1:500 - GluA1 Millipore AB1504 1:500 - GluA4 Millipore AB1508 1:250 - Ptch2 SCBT sc-9671 1:100 Antigen Retrieval AQP4 SCBT sc-9888 1:250 - Kir4.1 Alomone AGP-012 1:500 - Kir4.1 Millipore AB5818 1:500 - Smo SCBT sc-6366 1:100 -

42

Table. S2. List of RT-PCR primers used in this study. Forward Reverse

GAPDH TTGAAGTCGCAGGAGACAACC TTGAAGTCGCAGGAGACAACC

Shh CCCAATTACAACCCCGACATC GTCACTCGCAGCTTCACTC

Smo TCAGCATGTCACCAAGATGG AAACGCTTCTCTAACTCTGGG

Gli1 GAGCCTGAGTCTGTGTATGAG CAATGGCACACGAATTCCTTC

Ptch1 CTGCCTGTCCTCTTATCCTTC AGACCCATTGTTCGTGTGAC

Ptch2 CCTACTTGGCATCACTTTCAATG AAACACTCACCCATACGCTC

Kir4.1 CCGAGAAGCTCAAGTTGGAG AGCACTGGAAGAGGAAAAGG

GLAST TCCTCTACTTCCTGGTAACCC TCCACACCATTGTTCTCTTCC

GLT1 GATCACTGCTCTGGGAACTG TTAATGGTTGCTCCGACTGG

AQP4 AAGATCAGCATCGCTAAGTCC AGCGGTGAGGTTTCCATG

Aldh1L1 CATCCAGACCTTCCGATACTTC ACAATACCACAGACCCCAAC

Target

43

The numbers are mean ± SEM. Comparisons were made using Wilcox-on-Mann-Whitney two-sample rank test and Student’s t-test, as indicated.

Electrophysiological property Smo +/+ Smo c/c P-value (WMW test)

P-value (t-test)

Resting membrane potential (mV) -79 ± 0.7 -78 ± 0.8 0.35 0.28

Input resistance (M ) 9.6 ± 2 8.6 ± 2 0.78 0.64

Series resistance (M ) 12 ± 1 11 ± 1 0.69 0.75

Membrane current (nA) 0.29 ± 0.1 0.37 ± 0.1 0.40 0.58

EPSC peak latency (s) 1.3 ± 0.2 1.5 ± 0.2 0.35 0.36

EPSC amplitude (nA) 1.05± 0.3 0.31 ± 0.06 0.040 0.044

Table S3. Electrophysiological properties of the Smo+/+ (n = 7) and Smo c/c (n = 8) Bergmann glia cells in Figure 2E.

44

The numbers are mean ± SEM. Comparisons were made using Kruskal-Wallis one-way analysis of variance test and single factor ANOVA, as indicated. For input resistance, Wilcoxon-Mann-Whitney two-sample rank test additionally gave Tom vs SmoM2 (-) p: 0.64; Tom vs SmoM2 (+) p: 0.043; SmoM2 (-) vs SmoM2 (+) p: 0.029 and the Student’s t-test gave, Tom vs SmoM2 (-) p: 0.98; Tom vs SmoM2 (+) p: 0.029; SmoM2 (-) vs SmoM2 (+) p: 0.0067.

Electrophysiological property Tom SmoM2 (-) SmoM2 (+) P-value

(KW test) P-value (ANOVA)

Resting membrane potential (mV) -78 ± 0.8 -79 ± 0.6 -79 ± 0.7 0.50 0.50

Input resistance (M ) 4.7 ± 0.4 4.7 ± 0.2 3.3 ± 0.4 0.046 0.019

Series resistance (M ) 13 ± 1 13 ± 1 13 ± 1 0.86 0.96

Membrane current (nA) -1.03 ± 1 1.18 ± 1 0.88 ± 1 0.38 0.39

Table S4. Electrophysiological properties of the Tom (n = 10), SmoM2 (-) (n = 6) and SmoM2 (+) (n = 8) hippocampal astrocytes in figure 4.

45

Table S5. Genes differentially expressed between individually isolated Bergmann glia and velate astrocytes. As an Excel table.

46

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