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News Article | May 10, 2017
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HEK293 cells stably transfected with the STF plasmid encoding the firefly luciferase reporter under the control of a minimal promoter, and a concatemer of 7 LEF/TCF binding sites32, were obtained from J. Nathans. Mouse L cells stably transfected with the STF plasmid and a constitutively expressed Renilla luciferase (control reporter) were obtained from C. Kuo. L cells transfected with a mouse WNT3A expression vector to produce conditioned media were obtained from the ATCC. A375, SH-SY5Y and A549 cells were stably transfected with the BAR plasmid encoding the firefly luciferase reporter under the control of a minimal prompter and a concatemer of 12 TCF/LEF binding sites and a constitutively expressed Renilla luciferase (control reporter) using a lentiviral-based approach33. All reporter cell lines were cultured in complete DMEM medium (Gibco) supplemented with 10% FBS, 1% penicillin, streptomycin, and l-glutamine (Gibco), at 37 °C and 5% CO and cultured in the presence of antibiotics for selection of the transfected reporter plasmid. C3H10T1/2 cells were obtained from the ATCC. Human primary MSCs were obtained from Cell Applications, Inc. Mouse primary MSCs were obtained from Invitrogen. Cell lines have not been tested for mycoplasma contamination. The coding sequence of B12 containing a C-terminal 6×His-tag was cloned into the pET28 vector (Novagen) for bacterial cytoplasmic protein expression. Protein expression was performed in transformed BL21 cells, expression was induced with 0.7 mM IPTG at an OD   of 0.8 for 3–4 h. Cells were pelleted, lysed by sonication in lysis buffer (20 mM HEPES, pH 7.2, 300 mM NaCl, 20 mM imidazole), and soluble fraction was applied to Ni-NTA agarose (QIAGEN). After washing the resin with lysis buffer containing 500 mM NaCl, B12 was eluted with 300 mM imidazole, and subsequently purified on a Superdex 75 size-exclusion column (GE Healthcare) equilibrated in HBS (10 mM HEPES, pH 7.2, 150 nM NaCl). XWnt8 was purified from a stably transfected Drosophila S2 cell line co-expressing XWnt8 and mouse FZD8 CRD–Fc described previously4. Cells were cultured in complete Schneider’s medium (Thermo Fisher Scientific), containing 10% FBS and supplemented with 1% l-glutamine, penicillin and streptomycin (Gibco), and expanded in Insect-Xpress medium (Lonza). A complex of XWnt8 and FZD8 CRD–Fc was captured from the conditioned media on Protein A agarose beads (Sigma). After washing with 10 column volumes of HBS, XWnt8 was eluted with HBS containing 0.1% n-dodecyl-β-d-maltoside (DDM) and 500 mM NaCl, while the FZD8 CRD–Fc remained bound to the beads. All other proteins were expressed in High Five (Trichoplusia ni) cells (Invitrogen) using the baculovirus expression system. To produce the B12-based surrogate, the coding sequences of B12, a flexible linker peptide comprising of 0, 1, 2 or 3 GSGSG-linker repeats, followed by the C-terminal domain of human DKK1 (residues 177–266), and a C-terminal 6×His-tag, were cloned into the pAcGP67A vector (BD Biosciences). To clone the scFv-based surrogate ligand, the sequence of the Vantictumab was retrieved from the published patent, reformatted into a scFv, and cloned at the N terminus of the surrogate variant containing the GSGSG linker peptide. To produce recombinant FZD CRD for crystallization, surface plasmon resonance measurements, SEC-MALS experiments and functional assays, the CRDs of human FZD1 (residues 113–182), human FZD4 (residues 42–161), human FZD5 (residues 30–150), human FZD7 (residues 36–163), human FZD8 (residues 32–151) and human FZD10 (residues 30–150), containing a C-terminal 3C protease cleavage site (LEVLFQ/GP), a biotin acceptor peptide (BAP)-tag (GLNDIFEAQKIEWHE) and a 6×His-tag were cloned into the same vector. The human FZD8 CRD used for crystallization contained only a C-terminal 6×His-tag, in addition to a Asn49Gln mutation to mutate the N-linked glycosylation site. FZD1/FZD8 CRD for inhibition assay contained a C-terminal 3C protease cleavage site, Fc-tag (constant region of human IgG), and a 6×His-tag. Human DKK1 (residues 32–266) with a C-terminal BAP-tag and 6×His-tag, and the two furin-like repeats of human RSPO2 (residues 36–143) with a N-terminal Fc-tag and a C-terminal 6×His-tag, were cloned also into the pAcGP67A vector. All proteins were secreted from High Five insect cells grown in Insect-Xpress medium, and purified using Ni-NTA affinity purification, and size-exclusion chromatography equilibrated in HBS (10 mM HEPES, pH 7.3, 150 nM NaCl). Enzymatic biotinylation was performed in 50 mM bicine, pH 8.3, 10 mM ATP, 10 mM magnesium acetate, 0.5 mM d-biotin with recombinant glutathione S-transferase (GST)-tagged BirA ligase overnight at 4 °C, and proteins were subsequently re-purified on a Superdex 75 size-exclusion column to remove excess biotin. We attempted to mimic the native Wnt–FZD lipid–protein interaction with a de novo designed protein–protein binding interface. A 13-residue alanine helix was docked against the lipid-binding cleft using Foldit34. This structural element was grafted onto a diverse set of native helical proteins using the Rosetta Epigraft35 application to discover scaffolds with compatible, shape-complementary backbones. Prototype designs were selected by interface size and optimized using RosettaScripts36 to perform side-chain redesign. 50 selected designs were further manually designed to ensure charge complementarity and non-essential mutations were reverted to the wild-type amino acid identity to maximize stability. DNA was obtained from Gen9 and screened for binding via yeast surface display as previously described with 1 μM biotinylated FZD8 CRD pre-incubated with 025 µM SAPE (Life Technologies)37. A design based on the scaffold with PDB code 2QUP, a uncharacterized four-helix bundle protein from Bacillus halodurans, demonstrated binding activity under these conditions, whereas knockout mutants Ala52Arg and Ala53Asd made using the Kunkel method38 abrogated binding, verifying that the functional interface used the predicted residues. Wild-type scaffold 2QUP did not bind, confirming that activity was specifically due to design. To improve the affinity of the original design, a full-coverage site-saturation mutagenesis library was constructed for design based on the 2QUP scaffold via the Kunkel mutagenesis method38 using forward and reverse primers containing a ‘NNK’ degenerate codon and 21-bp flanking regions (IDT). A yeast library was transformed as previously described39 and sorted for three rounds, collecting the top 1% of binders using the BD Influx cell sorter. Naive and selected libraries were prepared and sequenced, and the data was processed as previously described37 using a Miseq (Illumina) according to manufacturer protocols. The most enriched 11 mutations were identified by comparison of the selected and unselected pools of binders and were combined in a degenerate library containing all enriched and wild-type amino acid identities at each of these positions. This combination library was assembled from the oligonucleotides (IDT) listed below for a final theoretical diversity of around 800 k distinct variants. This library was amplified, transformed, and selected to convergence over five rounds, yielding the optimized variant B12. The B12–FZD8 CRD(N49Q) complex was formed by mixing purified B12 and FZD8 CRD(N49Q) in stoichiometric quantities. The complex was then treated with 1:100 (w/w) carboxypeptidase A (Sigma) overnight at 4 °C, and purified on a Superdex 75 (GE Healthcare Life Sciences) size-exclusion column equilibrated in HBS. Purified complex was concentrated to around 15 mg ml−1 for crystallization trials. Crystals were grown by hanging-drop vapour diffusion at 295 K, by mixing equal volumes of the complex and reservoir solution containing 42–49% PEG 400, 0.1 M Tris, pH 7.8–8.2, 0.2 M NaCl, or 20% PEG 3000, 0.1 M sodium citrate, pH 5.5. While the PEG 400 condition is already a cryo-protectant, the crystals grown in the PEG 3000 condition were cryoprotected in reservoir solution supplemented with 20% glycerol before flash freezing in liquid nitrogen. Crystals grew in space groups P2 (PEG 400 condition) and P2 (PEG 3000 condition), respectively, with 2 and 4 complexes in the asymmetric units. Cell dimensions are listed in Supplementary Table 1. Data were collected at beamline 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. All data were indexed, integrated, and scaled with the XDS package40. The crystal structures in both space groups were solved by molecular replacement with the program PHASER41 using the structure of the FZD8 CRD (PDB code 1IJY) and the designed model of a minimal core of B12 as search models. Missing residues were manually build in COOT42 after initial rounds of refinement. Several residues at the N terminus (residues 1 to 16/17/20/21), at the C terminus (residues after 117) and several residues within loop regions were unstructured and could not been modelled. Furthermore, we observed that in both crystal forms, B12 underwent domain swapping, and one B12 molecule lent helix 3 and 2 to another B12, resulting in a closely packed B12 homodimer. The density of the loops connecting helixes 1 and 2, and 3 and 4 were clearly visible, and folded into helical turns. Yet, SEC-MALS experiments confirmed that B12 existed as a monomer in solution, and complexed FZD8 CRD with a 1:1 stoichiometry. PHENIX Refine43 was used to perform group coordinate refinement (rigid body refinement), followed by individual coordinate refinement using gradient-driven minimization applying stereo-chemical restraints, NCS restraints, and optimization of X-ray/stereochemistry weight, and individual B-factor refinement. Initial rounds of refinement were aided by restraints from the high-resolution mouse FZD8 CRD structure as a reference model. Real space refinement was performed in COOT into a likelihood-weighted SigmaA-weighted 2mF  − DF map calculated in PHENIX. The final model in the P2 space group was refined to 3.20 Å with R and R values of 0.2002 and 0.2476, respectively (Supplementary Table 1). The quality of the structure was validated with MolProbity44. 99.5% of residues are in the favoured region of the Ramachandran plot, and no residue in the disallowed region. The structure within the P2 space group was refined to 2.99 Å with R and R values of 0.2253 and 0.2499, respectively, with 99.2% of residues in the favoured region of the Ramachandran plot, and no residues in the disallowed region. See Supplementary Table 1 for data and refinement statistics. Structure figures were prepared with the program PYMOL. Binding measurements were performed by surface plasmon resonance on a BIAcore T100 (GE Healthcare) and all proteins were purified on SEC before experiments. Biotinylated FZD1 CRD, FZD5 CRD, FZD7 CRD and FZD8 CRD were coupled at a low density to streptavidin on a SA sensor chip (GE Healthcare). An unrelated biotinylated protein was captured at equivalent coupling density to the control flow cells. Increasing concentrations of B12 and scFv–DKK1c were flown over the chip in HBS-P (GE Healthcare) containing 10% glycerol and 0.05% BSA at 40 μl ml−1. The chip surface was regenerated after each injection with 2 M MgCl in HBS-P or 50% ethylene glycol in HBS-P (scFv–DKK1c measurements), or 4 M MgCl in HBS-P (B12 measurements) for 60 s. Curves were reference-subtracted and all data were analysed using the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model to determine the K values. To characterize the FZD-specificity of B12, the yeast display vector encoding B12 was transformed into EBY100 yeast. To induce the display of B12 on the yeast surface, cells were growing in SGCAA medium45, 46 for 2 days at 20 °C. 1 × 106 yeast cells per condition were washed with PBE (PBS, 0.5% BSA, 2 mM EDTA), and stained separately with 0.06–1,000 nM biotinylated FZD1/4/5/7/8/10 CRDs for 2 h at 4 °C. After washing twice with ice-cold PBE, bound FZD CRDs were labelled with 10 nM strepdavidin-Alexa647 for 20 min. Cells were fixed with 4% paraformaldehyde, and bound FZD CRD was analysing on an Accuri C6 flow cytometer. FZD8 fused to an N-terminal HaloTag47 and LRP6 fused to an N-terminal SNAP-tag48 were cloned into the pSEMS-26m vector (Covalys Biosciences) by cassette cloning49, 50. The template pSEMS-26m vectors had been coded with DNA sequences of the SNAP-tag or the HaloTag, respectively, together with an Igκ leader sequence (from the pDisplay vector, Invitrogen) as described previously50. The genes of full-length mouse Fzd8 or human LRP6 without the N-terminal signal sequences were inserted into pSEMS-26m via the XhoI and AscI or AscI and NotI, restriction sites, respectively. A plasmid encoding a model transmembrane protein, maltose-binding protein fused to a transmembrane domain, fused to an N-terminal HaloTag was prepared as described recently13. HeLa cells were cultivated at 37 °C, 5% CO in MEM Earle’s (Biochrom AG, FG0325) supplemented with 10% fetal calf serum and 1% nonessential amino acids. Cells were plated in 60-mm cell culture dishes to a density of 50% confluence and transfected via calcium phosphate precipitation49. 8–10 h after transfection, cells were washed twice with PBS and the medium was exchanged, supplied with 2 μM porcupine inhibitor IWP-2 for inhibiting maturation of endogenous Wnt in HeLa cells51. 24 h after transfection, cells were plated on glass coverslips pre-coated with PLL-PEG-RGD52 for reducing nonspecific binding of dyes during fluorescence labelling. After culturing for 12 h, coverslips were mounted into microscopy chambers for live-cell imaging. SNAP-tag and HaloTag were labelled by incubating cells with 50 nM benzylguanine-DY649 (SNAP-Surface 649, New England Biolabs) and 80 nM of HaloTag tetramethylrhodamine ligand (HTL-TMR, Promega) for 20 min at 37 °C. Under these conditions, effective degrees of labelling estimated from single molecule assays with a HaloTag–SNAP-tag fusion protein were ~40% for the SNAP-tag and ~25% for the HaloTag13. After washing three times with PBS, the chamber was refilled with MEM containing 2 μM IWP-2 for single-molecule fluorescence imaging. Single-molecule fluorescence imaging was carried out by using an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) illumination condenser (Olympus) and a back-illuminated EMCCD camera (iXon DU897D, 512 × 512 pixel from Andor Technology). A 561-nm diode solid state laser (CL-561-200, CrystaLaser) and a 642-nm laser diode (Luxx 642-140, Omicron) were coupled into the microscope for excitation. Laser lights were reflected by a quad-line dichroic beam splitter (Di R405/488/561/647, Semrock) and passed through a TIRF objective (UAPO 150×/1.45, Olympus). For simultaneous dual-colour detection, a DualView microimager (Optical Insight) equipped with a 640 DCXR dichroic beamsplitter (Chroma) in combination with bandpass filters FF01-585/40 and FF01 670/30 (Semrock), respectively, was mounted in front of the camera. The overlay of the two channels was calibrated by imaging fluorescent microbeads (TetraSpeck microspheres 0.1 μm, T7279, Invitrogen), which were used for calculating a transformation matrix. After channel alignment, the deviation between the channels was below 10 nm. For single-molecule imaging, typical excitation powers of 1 mW at 561 nm and 0.7 mW at 642 nm measured at the objective were used. Time series of 150–300 frames were recorded at 30 Hz (4.8–9.6 s). An oxygen scavenging system containing 0.5 mg ml−1 glucose oxidase, 40 mg ml−1 catalase, and 5% (w/v) glucose, together with 1 μM ascorbic acid and 1 μM methyl viologene, was added to minimize photobleaching53. Receptor dimerization was initiated by incubating with 100 nM Wnt proteins or surrogates. Images were acquired after 5 min incubation in the presence of the ligands. All live-cell imaging experiments were carried out at room temperature. A 2D Gaussian mask was used for localizing single emitters54, 55. For colocalization analysis to determine the heterodimerization fraction, particle coordinates from two channels were aligned by a projective transformation (cp2tform of type ‘projective’, MATLAB 2012a) according to the transformation matrix obtained from microbead calibration measurement. Particles colocalized within a distance of 150 nm were selected. Only co-localized particles, which could be tracked for at least 10 consecutive frames (that is, molecules co-locomoting for at least 0.32 s) were accepted as receptor heterodimers or hetero-oligomers, which has been previously found to be a robust criterion for protein dimerization13. The fraction of heterodimerization or hetero-oligomerization was determined as the number of co-locomotion trajectories with respect to the number of the receptor trajectories. Since the receptor expression level of FZD8 or LRP6 was variable in the transiently transfected cells, only cells with similar receptor expression levels were considered (less than three times the excess of one subunit over the other). The smaller number of trajectories of either FZD8 or LRP6 was regarded as the limiting factor and therefore taken as a reference for calculating the heterodimerized/hetero-oligomerized fraction. Oligomerization values were not corrected for the degree of labelling. Single-molecule trajectories were reconstructed using the multi-target tracing (MTT) algorithm56. The detected trajectories were evaluated with respect to their step length distribution to determine the diffusion coefficients. For a reliable quantification of local mobilities, we estimated diffusion constants from the displacements with three frames (96 ms). Step-length histograms were obtained from all single molecule trajectories and fitted by a two-component model of Brownian diffusion, thus taking into account the intrinsic heterogeneity of protein diffusion in the plasma membrane57, 58. A bimodal probability density function p(r) was used for a nonlinear least square fit of the step-length histogram: where is the percentage of the fraction, contains the diffusion coefficient of each fraction (nδt = 96 ms). Average diffusion coefficients were determined by weighting the diffusion coefficients with the corresponding fractions. Single-molecule intensity distribution of individual diffraction-limited spots was extracted from the first 50 images of the recorded time lapse image sequence, in which photobleaching of dyes was kept below 10%13. Oligomerization of receptors was evaluated by fitting the obtained single molecule intensity with a multi-component Gaussian distribution function59. To ensure a reliable analysis, monomeric receptors were first distinguished based on the observation that monomers diffused much faster than oligomers. Therefore, the characteristic intensity distribution of monomeric receptor subunits was obtained by tracking of the fast mobile fraction. Fractions of the monomer, dimer, trimer and higher oligomers were then de-convoluted from the single molecule intensity distribution, presuming that intensities of clusters were multiples of the monomer intensity distribution. Immortal cells were seeded in triplicate for each condition in 96-well plates, and stimulated with surrogates, XWnt8, WNT3A conditioned media, control proteins, or other treatments for 20–24 h. After washing cells with PBS, cells in each well were lysed in 30 μl passive lysis buffer (Promega). 10 μl per well of lysate was assayed using the Dual Luciferase Assay kit (Promega) and normalized to the Renilla luciferase signal driven constitutively by the human elongation factor-1 alpha promoter to account for cell variability. A375 BAR, SH-SY5Y BAR, L STF and HEK293 STF cells were plated at a density of 10,000–20,000 cells per well, and treatment was started after 24 h in fresh medium. A549 BAR cells were plated at a density of 5,000 cells per well in the presence of 2 μM IWP-2 (Calbiochem) to suppress endogenous Wnt secretion, and treatment was started after 48 h in fresh medium containing fresh IWP-2. To induce β-catenin accumulation, SH-SY5Y BAR cells were treated for 2 h with scFv–DKK1c, WNT3A conditioned media (positive control), B12 (negative control protein) and mock conditioned media (from untransfected L cells, negative control) at 37 °C, 5% CO . After, cells were washed twice with PBS. For β-catenin stabilization assay, cells were scraped into hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl , supplemented with protease inhibitors), incubated on ice for 10 min, and homogenized using a hypodermic needle. Sucrose and EDTA were added to final concentration of 0.25 M and 1 mM, respectively. For LRP6 phosphorylation assay, cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxylate, 1% Triton X-100), supplemented with protease inhibitor and phosphatase inhibitor for 1 h at 4 °C. Lysates were centrifuged at 12,000g for 1 h at 4 °C. Supernatants were then diluted into SDS sample buffer. For immunoblotting, samples were resolved on a 12% Mini-PROTEAN(R)TGX precast protein gel (Bio-Rad) and transferred to a PVDF membrane. The membranes were cut horizontally approx. at the 64 kDa mark of the SeeBlue plus 2 molecular mass marker (Invitrogen). Top half of the blot was incubated with anti-β-catenin primary antibody ((D10A8)XP, rabbit, Cell Signaling 8480), LRP6 antibody ((C47E12), rabbit, Cell Signaling 3395), and P-LRP6 (S1490) antibody (rabbit, Cell Signaling 2568), and the bottom part with the anti-α-tubulin primary antibody (mouse, DM1A, Sigma) in PBS containing 0.1% Tween-20 and 5% BSA overnight at 4 °C. Blots were then washed, incubated with the corresponding secondary antibodies in the same buffer, before washing and developing using the ECL prime western blotting detection reagent (GE Healthcare). To induce β-catenin accumulation, K562 and cells were stimulated for 0, 15, 30, 45, 60, 90 and 120 min with 10 nM scFv–DKK1c, recombinant Wnt3a (R&D Systems), B12 (negative control protein) or plain complete growth medium at 37 °C, 5% CO . After, cells were washed twice with PBS, fixed with 4% PFA for 10 min at room temperature, and permeabilized in 100% methanol for at least 30 min at −80 °C. The cells were than stained with Alexa-647 conjugated anti-β-catenin antibody (L54E2) (Cell Signaling Technology, 1:100–1: 50 dilution). Fluorescence was analysed on an Accuri C6 flow cytometer. Total RNA was isolated using either TRIZOL (Invitrogen) or RNeasy plus micro kit (QIAGEN) according to manufacturer’s protocols. A total of 2 μg RNA were used to generate cDNA using the RevertAid RT kit (Life Technologies) using oligo(dT)18 mRNA primers (Life Technologies) according to manufacturer’s protocol. 12 ng of cDNA per reaction were used. qPCR was performed using SYBR Green-based detection (Applied Biosystems) according to the manufacturer’s protocol on a StepOnePlus real-time PCR system (ThermoFisher Scientific). All primers were published, or validated by us. Transcript copy numbers were normalized to GAPDH for each sample, and fold induction compared to control was calculated. The following gene-specific validated primers were used: human FZD1: F: 5′-ATCCTGTGTGCTCCTCTTTTGG-3′, R: 5′-GATTGCTTTTCTCCTCTTCTTCAC-3′; human FZD2: F: 5′-CTGGGCGAGCGTGATTGT-3′, R: 5′-GTGGTGACAGTGAAGAAGGTGGAAG-3′; human FZD3: F: 5′-TCTGTATTTTGGGTTGGAAGCA-3′, R: 5′-CGGCTCTCATTCACTATCTCTTT-3′; human FZD4: F: 5′-TGGGCACTTTTTCGGTATTC-3′, R: 5′-TGCCCACCAACAAAGACATA-3′; human FZD5: F: 5′-CCATGATTCTTTAAGGTGAGCTG-3′, R: 5′-ACTTATTCAAGACACAACGATGG-3′; human FZD6: F: 5′-CGATAGCACAGCCTGCAATA-3′, R: 5′-ACGGTGCAAGCCTTATTTTG-3′; human FZD7: F: 5-TACCATAGTGAACGAAGAGGA-3′, R: 5′-TGTCAAAGGTGGGATAAAGG-3′; human FZD8: F: 5′-ACCCAGCCCCTTTTCCTCCATT-3′, R: 5′-GTCCACCCTCCTCAGCCAAC-3′; human FZD9: F: 5′-GCTGTGACTGGAATAAACCCC, R: 5′-GCTCTGCTTACAAGAAAGACTCC-3′; human FZD10: F: 5′-CTCTTCTCTGTGCTGTACACC, R: 5′-GTCTTGGAGGTCCAAATCCA-3′; mouse Fzd1: F: 5′-GCGACGTACTGAGCGGAGTG, R: 5′-TGATGGTGCGGATGCGGAAG-3′60; mouse Fzd2: F: 5′-CTCAAGGTGCCGTCCTATCTCAG, R: GCAGCACAACACCGACCATG-3′60; mouse Fzd3: F: 5′-GGTGTCCCGTGGCCTGAAG-3′, R: 5′-ACGTGCAGAAAGGAATAGCCAAG-3′60; mouse Fzd4: F: 5′-GACAACTTTCACGCCGCTCATC-3′, R: 5′-CAGGCAAACCCAAATTCTCTCAG-3′60; mouse Fzd5: F: 5′-AAGCTGCCTTCGGATGACTA-3′, R: 5′-TGCACAAGTTGCTGAACTCC-3′60; mouse Fzd6: F: 5′-TGTTGGTATCTCTGCGGTCTTCTG-3′, R: 5′-CTCGGCGGCTCTCACTGATG-3′60; mouse Fzd7: F: 5′-ATATCGCCTACAACCAGACCATCC-3′, R: 5′-AAGGAACGGCACGGAGGAATG-3′60; mouse Fzd8: F: 5′-GTTCAGTCATCAAGCAGCAAGGAG-3′, R: 5′-AAGGCAGGCGACAACGACG-3′60; mouse Fzd9: F: 5′-ATGAAGACGGGAGGCACCAATAC-3′, R: 5′-TAGCAGACAATGACGCAGGTGG-3′60; mouse Fzd10: F: 5′-ATCGGCACTTCCTTCATCCTGTC-3′, R: 5′-TCTTCCAGTAGTCCATGTTGAG-3′60; human AXIN2: F: 5′-CTCCCCACCTTGAATGAAGA-3′, R: 5′-TGGCTGGTGCAAAGACATAG-3′; human GAPDH: F: 5′-TGAAGGTCGGAGTCAACGGA-3′, R: 5′-CCATTGATGACAAGCTTCCCG-3′; mouse Gapdh: F: 5′-CCCCAATGTGTCCGTCGTG-3′, R: 5′-GCCTGCTTCACCACCTTCT-3′. Differentiation of C3H10T1/2, and human and mouse primary MSCs were performed essentially as described previously61. In brief, approximately 10,000 cells cm−2 were plated in normal culture medium (αMEM + FBS + penicillin/streptomycin), and allowed to adhere overnight. The following day, the medium was replaced with osteogenic medium (αMEM, 10% FBS, 1% penicillin/streptomycin, 50 μg ml−1 ascorbic acid, 10 mM β-glycerol phosphate (βGP), and replaced every other day. To determine alkaline phosphatase enzymatic activity, cells were fixed for 10 min with 10% formalin in PH7 PBS, before incubation in NBT-BCIP solution (1-Step(tm) NBT/BCIP Substrate Solution (Thermo Fisher Scientific, 34042) for 30 min. qPCR reactions were done with the SYBR method using the following primers: human ACTB F: 5′-GTTGTCGACGACGAGCG-3′, R: 5′-GCACAGAGCCTCGCCTT-3′; human ALPL: F: 5′-GATGTGGAGTATGAGAGTGACG-3′, R: 5′-GGTCAAGGGTCAGGAGTTC-3′; mouse Alpl: F: 5′-AAGGCTTCTTCTTGCTGGTG-3′, R: 5′-GCCTTACCCTCATGATGTCC-3′; mouse Actb: F: 5′-GGAATGGGTCAGAAGGACTC-3′, R: 5′-CATGTCGTCCCAGTTGGTAA-3′; mouse Col2a1 F: 5′-GTGGACGCTCAGGAGAAACA-3′, R: 5′-TGACATGTCGATGCCAGGAC-3′. P26N, normal adult human colon organoids, were established from a tumour-free colon segment of a patient diagnosed with CRC as described18, 62, 63. CFTR-derived colorectal organoids were obtained from a patient at Wilhelmina Children’s Hospital WKZ-UMCU. Informed consent for the generation and use of these organoids for experimentation was approved by the ethical committee at University Medical Center Utrecht (UMCU) (TcBio 14-008). Human stomach organoids, derived from normal corpus and pylorus, were from patients that underwent partial or total gastrectomy at the University Medical Centre Utrecht (UMCU) and were established as described19, 64, 65. Pancreas organoids were obtained from the healthy part of the pancreas of patients undergoing surgical resection of a tumour at the University Medical Centre Utrecht Hospital (UMC) and were established as described66, 67. The liver organoids were derived from freshly isolated normal liver tissue from a patient with metastatic CRC who presented at the UMC hospital (ethical approval code TCBio 14-007) and were established as described20, 68. For the performance of 3D cultures, Matrigel (BD Biosciences) was used and overlaid with a liquid medium consisting of DMEM/F12 advanced medium (Invitrogen), supplemented with additional factors as outlined below. 2% RSPO3-CM (produced via the r-PEX protein expression platform at U-Protein Express BV), WNT3A conditioned medium (50%, produced using stably transfected L cells in the presence of DMEM/F12 advanced medium supplemented with 10% FBS), and Wnt and Wnt/RSPO2 surrogates at different concentrations were added as indicated. Single-cell suspensions of normal human organoids were cultured in duplicate or triplicate in round-bottom 96-well plates to perform a cell viability test using Cell Titer-Glo 3D (Promega). In brief, organoids were trypsinized to single-cell suspension and plated in 100 μl medium in the presence of the different reagents. 3 μM IWP-2 was added to inhibit endogenous Wnt lipidation and secretion. After 12 days, 100 μl of Cell Titer-Glo 3D was added, plates were shaken for 5 min, incubated for an additional 25 min and centrifuged before luminescence measurement. All animal experiments were conducted in accordance with procedures approved by the IACUC at Stanford University. Experiments were not randomized, the investigators were not blinded, and all samples/data were included in the analysis. Group sample sizes were chosen based on (1) previous experiments, (2) performance of statistics analysis, and (3) logistical reasons with respect to full study size, to accommodate all groups. Adenoviruses (E1 and E3 deleted, replication deficient) were constructed to express scFv–DKK1c or scFv–DKK1c–RSPO2 with an N-terminal signal peptide and C-terminal 6×His-tag (Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2), respectively. Adenoviruses expressing mouse IgG2α Fc (Ad-Fc), human RSPO2–Fc fusion protein (Ad-RSPO2–Fc) and mouse WNT3A (Ad-Wnt3a) were constructed and described in the companion paper by Yan et al.26 The adenoviruses were cloned, purified by CsCl gradient, and titred as previously described69. Adult C57Bl/6J mice were purchased from Taconic Biosciences. Adult C57Bl/6J mice between 8–10 weeks old were injected intravenously with a single dose of adenovirus at between 1.2 × 107 p.f.u. to 6 × 108 p.f.u. per mouse in 0.1 ml PBS. Serum expression of Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2 were confirmed by immunoblotting using mouse anti-6×His (Abcam ab18184, 1:2,000) or rabbit anti-6×His (Abcam ab9108, 1:1,000), respectively. All experiments used n = 4 mice per group and repeated at least twice. qRT–PCR on liver samples were performed as following. Total cDNA was prepared from each liver sample using Direct-Zol RNA miniprep kit (Zymo Research) and iScript Reverse Transcription Supermix for RT-qPCR (BIO-RAD). Gene expression was analysed by -ΔΔC or fold change (2−ΔΔCt). Unpaired Student’s t-test (two tailed) was used to analyse statistical significance. Primers for mouse Axin2 and Cyp2f2 were previously published70. Additional primers used were listed as below: For the parabiosis experiment, age- and gender-matched C57Bl/6J mice were housed together for at least 2 weeks before surgery. At 2 days before surgery, the ‘donor’ mice were injected intravenously with a single dose of adenovirus at between 1.2 × 107 pfu to 6 × 108 pfu per mouse in 0.1 ml PBS and were separated from the ‘recipient’ mice until surgery. The parabiosis surgery was performed as described previously71. The establishment of shared circulation was confirmed at day 5 after surgery by presence of adenovirus-expressed proteins in the serum of both donors and recipients. Mouse livers were collected and fixed in 4% paraformaldehyde. 5 μm paraffin-embedded sections were stained with the following antibodies after citrate antigen retrieval and blocking with 10% normal goat serum: mouse anti-glutamine synthetase antibody (Millipore MAB302, 1:200), mouse anti-PCNA (BioLegend 307902, 1:200), and rabbit anti-HNF4α (Cell Signaling 3113S, 1:500). The immunostained tissue sections were analysed and images were captured on a Zeiss Axio-Imager Z1 with ApoTome attachment. Atomic structure factors and coordinates have been deposited to the Protein Data Bank (PDB) under accession numbers 5UN5 and 5UN6. All other data are available from the corresponding author upon reasonable request.


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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The accumulation assay was performed in triplicate in batches of ten samples, with each batch containing either tetracycline or ciprofloxacin as a positive control. E. coli MG1655 was used for these experiments as this strain has been only minimally altered from its K-12 progenitor33. For each replicate, 2.5 ml of an overnight culture of E. coli was diluted into 250 ml of fresh Luria Bertani (LB) broth (Lennox) and grown at 37 °C with shaking to an optical density (OD ) of 0.55. The bacteria were pelleted at 3,220 r.c.f. for 10 min at 4 °C and the supernatant was discarded. The pellets were re-suspended in 40 ml of phosphate buffered saline (PBS) and pelleted as before, and the supernatant was discarded. The pellets were re-suspended in 8.8 mL of fresh PBS and aliquoted into ten 1.5 ml eppendorf tubes (875 μl each). The number of colony-forming units (CFUs) was determined by a calibration curve. The samples were equilibrated at 37 °C with shaking for 5 min, compound was added (final concentration = 50 μM), and then samples were incubated at 37 °C with shaking for 10 min. A 10 min time point was chosen because it is longer than the predicted amount of time required to reach a steady-state concentration34, but short enough to minimize metabolic and growth changes (no changes in OD observed; CFUs were reduced by a factor of five after ciprofloxacin treatment for 10 min, but no other antibiotics had an effect). After incubation, 800 μl of the cultures were carefully layered on 700 μl of silicone oil (9:1 AR20/Sigma High Temperature, cooled to −78 °C). Bacteria were pelleted through the oil by centrifuging at 13,000 r.c.f. for 2 min at room temperature (supernatant remains above the oil); the supernatant and oil were then removed by pipetting. To lyse the samples, each pellet was dissolved in 200 μl of water, and then they were subjected to three freeze-thaw cycle of three minutes in liquid nitrogen followed by three minutes in a water bath at 65 °C. The lysates was pelleted at 13,000 r.c.f. for 2 min at room temperature and the supernatant was collected (180 μl). The debris was re-suspended in 100 μl of methanol and pelleted as before. The supernatants were removed and combined with the previous supernatants collected. Finally, remaining debris was removed by centrifuging at 20,000 r.c.f. for 10 min at room temperature. Supernatants were analysed by LC–MS/MS. Samples were analysed with the 5500 QTRAP LC/MS/MS system (AB Sciex) with a 1200 series HPLC system (Agilent Technologies) including a degasser, an autosampler, and a binary pump. The liquid chromatography separation was performed on an Agilent SB-Aq column (4.6 × 50 mm, 5 μm) (Agilent Technologies) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). The flow rate was 0.3 ml min−1. The linear gradient was as follows: 0–3 min, 100% mobile phase A; 10–15 min, 2% mobile phase A; 15.5–21 min, 100% mobile phase A. The autosampler was set at 5 °C. The injection volume was 15 μl. Mass spectra were acquired with both positive electrospray ionization at the ion spray voltage of 5,500 V and negative electrospray ionization at the ion spray voltage of −4,500 V. The source temperature was 450 °C. The curtain gas, ion source gas 1, and ion source gas 2 were 33, 50 and 65, respectively. Multiple reaction monitoring was used to quantify metabolites. Power analysis was not used to determine the number of replicates. Error bars represent the standard error of the mean of three biological replicates. All compounds evaluated in biological assays were ≥ 95% pure. Assays measuring permeabilization by colistin were performed as above, with the addition of 6.0 μM colistin sulfate immediately before the compound of interest was added. MRSA and P. aeruginosa isolates were from Cubist Pharmaceuticals. E. coli MG1655 was provided by C. Vanderpool (UIUC). E. coli clinical isolates were provided by L. Zechiedrich (Baylor School of Medicine). E. cloacae ATCC 29893 was provided by W. van der Donk (UIUC). A. baumannii isolates were provided by J. Quale. E. cloacae and K. pneumonia clinical isolates were provided by D. Hooper (Massachusetts General Hospital). BAA-2469 (ref. 35) was obtained from ATCC. Susceptibility testing was performed in biological triplicate, using the micro-dilution broth method as outlined by the Clinical and Laboratory Standards Institute. Müller–Hinton broth was used. Strains in Fig. 5: (1) ATCC 29213; (2) NRS3; (3) MG1655, (4) ELZ4017; (5) ELZ4045; (6) ELZ4346; (7) ELZ4081; (8) ELZ4072; (9) ELZ4239; (10) BAA-2469; (11) ATCC 19606; (12) KB304; (13) KB349; (14) WO22; (15) BD335; (16) IF101; (17) ATCC 27736; (18) 4-38; (19) 6-73; (20) ATCC 29893; (21) 1-60; (22) 3-46; (23) PAO1. The method used to compare the outer membrane proteins of E. coli BW25113 to outer membrane of the ∆ompR E. coli from the KEIO collection was adapted from ref. 36. Briefly, bacteria were grown to OD  = 1.0 at 37 °C in LB broth. 4 ml were centrifuged for 10 min at 2,350g at 4 °C, washed with 1 ml of 100 mM Tris-HCl, pH 8.0, with 20% sucrose and incubated on ice for 10 min. Cells were pelleted as before and taken up in 1 ml of 100 mM Tris-HCl, pH 8.0, 20% sucrose containing 10 mM sodium ethylenediaminetetraacetate (EDTA). Lysozyme was added to a final concentration of 100 mg ml−1 and incubated on ice for 10 min. MgSO was added to 20 mM final concentration and RNaseA and DNaseI were added to a final concentration of 10 mg ml−1. Cells were disrupted with five freeze–thaw cycles in dry ice/ethanol and a room temperature water bath. A sixth freezing sample was left to thaw on ice for 2 h. Membranes were pelleted for 25 min at 16,100g at 4 °C. The supernatants were discarded, and the pellet was washed and pelleted three times in 1 ml of 20 mM NaPO , pH 7 and 0.5% sarkosyl. The protein extracts were taken up in 60 ml of Laemmli sample buffer (80 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.02% bromophenol blue), boiled for 5 min and subjected to SDS–PAGE (12% polyacrylamide). Proteins were visualized by staining with Coomassie blue-G. As small molecules that traverse the outer membrane of Gram-negative bacteria predominately cross through porins, knocking out the major porins of E. coli would be expected to decrease compound accumulation. Although there are many porins present in E. coli, the two major non-specific porins are OmpF and OmpC, which are differentially expressed (based on the extracellular osmolarity) by the EnvZ/OmpR two-component regulatory system. A ∆ompR strain of E. coli from the KEIO knockout collection was therefore chosen to effectively knockout both OmpF and OmpC (see Extended Data Fig. 5a for validation of knockout). For the control high accumulators ciprofloxacin, tetracycline and chloramphenicol, a significant decrease in accumulation is observed in the ∆ompR strain of E. coli compared to the parental strain E. coli BW25113 (Extended Data Fig. 5b), consistent with previous data demonstrating that these antibiotics enter through the OmpF and OmpC porins37, 38, 39. Eight high-accumulating compounds from the test set were also evaluated, and accumulation decreased in the ∆ompR strain (Extended Data Fig. 5b), suggesting that these porins are a major gateway to small-molecule accumulation for the compounds tested. A limitation to measuring accumulation in whole cells is that no distinction is made between periplasmic and cytoplasmic accumulation. To reach the cytoplasm of E. coli, compounds must also diffuse through the inner membrane, whose filtering properties may be different from the filtering properties of the outer membrane28. To examine this, high-accumulating compounds and some of their derivatives were tested for accumulation in E. coli protoplasts, cells lacking the outer membrane and peptidoglycan. As shown in Extended Data Fig. 5c, minimal variation in accumulation was observed between compounds in this experiment, supporting the hypothesis that traversing the outer membrane is the main barrier to small-molecule accumulation in Gram-negative bacteria. The method for preparing protoplasts was adapted from Weiss40. 850 μl of an overnight culture of E. coli MG1655 was diluted into 85 ml of fresh LB broth and grown at 37 °C with shaking to an OD  = 1.0. The bacteria were pelleted at 3,220 r.c.f. for 10 min at 4 °C and the supernatant was discarded. The pellet was washed three times with 10 ml of 10 mM Tris HCl buffer (pH 8), and the pellet was resuspended in 30 ml of 10 mM Tris HCl (pH 8) containing 0.5 M sucrose. Potassium ethylenediaminetetraacetate (EDTA, 0.5 M, pH 8.0) was added slowly over a period of 20 min to a final concentration of 0.01 M. The bacteria were shaken at 130 r.p.m. for 20 min at 37 °C, and then collected as before. The supernatant was discarded and the pellets were washed two times with SMM buffer (0.5 M sucrose, 20 mM sodium maleate, 20 mM MgCl , pH 6.5). The bacteria were then resuspended in 30 ml of SMM buffer, 30 mg of lysozyme was added, and the bacteria were shaken at 130 r.p.m. for 1.5 h at 37 °C. The protoplasts were harvested by centrifuging at 2,000 r.c.f. for 20 min at 4 °C. The protoplast pellet was resuspended in 20 ml of SMM buffer, and protoplast formation was confirmed by diluting an aliquot in water and observing a threefold decrease on OD . To test accumulation, 500 μl aliquots containing 20 μM compound were shaken at 130 r.p.m. for 5 min at 37 °C. Samples were pelleted at 2,000 r.c.f. for 10 min at room temperature and the supernatants were discarded. The pellets were resuspended in 200 μl of water and incubated at room temperature for 5 min. The lysed protoplasts were pelleted by centrifuging at 21,130 r.c.f. for 10 min, and the supernatant was analysed by LC–MS/MS for compound concentration as before. Statistical significance of accumulation was determined by using a two sample Welch’s t-test (one-tailed test, assuming unequal variance) relative to the negative controls. Variance in accumulation across molecules was found to be unequal by Bartlett’s test. For all other experiments, statistical significance was determined using an unpaired Student’s t-test assuming equal variance as determined by an F-test. Chemical structure data for the MicroFormat library was obtained from the ChemBridge website. Chemical structure data for the ZINC Natural Products library was obtained from http://zinc.docking.org/browse/catalogs/natural-products. Chemical structure data for the MLSMR-NP library was obtained from PubChem. Compounds containing particular functional groups were identified and counted by substructure filtering with Open Babel, using the following SMARTS queries: primary aliphatic amine, [$([N;H2;X3][CX4]),$([N;H3;X4+][CX4])]; secondary aliphatic amine, [$([CX4][N;H1;X3][CX4]),$([CX4][N;H2;X4+][CX4])]; tertiary aliphatic amine, [NX3]([CX4])([CX4])[CX4]; carboxylic acid: [CX3]( = O)[OX1H0−,OX2H1]. Data sets of chemical structures were created and managed using Canvas (Version 2.6, Schrödinger, LLC). Initial structure preparation and 3D minimization was performed with LigPrep (Version 3.6, Schrödinger, LLC) using OPLS_2005 force fields. Tautomeric and protonation states were determined using Epik (Version 3.4, Schrödinger, LLC) at pH 7.4 (refs 41, 42). Generation of ensembles of conformations was performed using Conformational Search in MOE 2015.10 (ref. 43) using the LowModeMD method with default settings. Physiochemical descriptors (297, both 2D- and 3D-based) were calculated using MOE for each conformation. Descriptors were averaged (unweighted mean) across all conformations for each molecule. Data with descriptors were used to train a random forest classification prediction model using the R package caret. The random forest model offers many advantages for this application including resistance to over-fitting and the ability to measure descriptor importance44. Preprocessing of data removed descriptors with near-zero variance or high co-correlation with other descriptors. Source code for data analysis and model training can be found at https://github.com/HergenrotherLab/GramNegAccum. An additional approximation of three-dimensionality, average distance to the plane of best fit (PBF), was calculated using a custom Python program. The PBF algorithm45 was implemented in Python with SDfile I/O and structure representation being handled by libraries from Schrödinger. For each compound, the PBF algorithm determines the plane that best fits a set of 3D coordinates that represent the positions of all heavy atoms in the molecule using single value decomposition. The distance of each heavy atom to this plane is measured in angstroms and averaged. This Python program was incorporated into the Maestro GUI for convenient use. Source code can be found at https://github.com/HergenrotherLab/GramNegAccum. ClogD values were calculated using the online compound property calculation software FAFdrugs3 (http://fafdrugs3.mti.univ-paris-diderot.fr/index.html). Rotatable bonds: count of single bonds, not in a ring, bound to a non-terminal heavy atom. Excluded from the count are C–N amide bonds because of their high rotational energy barrier46. ClogD , the predicted octanol/water distribution coefficient at pH 7.4; globularity, inverse condition number of the covariance matrix of atomic coordinates (a value of 1 indicates a perfect sphere while a value of 0 indicates a two- or one-dimensional object); PBF, average distance in angstroms of each heavy atom in the molecule to the plane that best fits atomic coordinates; PMI 1, first diagonal element of diagonalized moment of inertia tensor47; PMI1/molecular weight, ratio of PMI 1 and molecular weight; vsurf_A, a vector pointing from the centre of the hydrophobic domain to the centre of the hydrophilic domain. The vector length is proportional to the strength of the amphiphilic moment27. Full experimental details and characterization data for all new compounds are included in the Supplementary Information. Although SMD does not directly provide the free energy landscape, SMD is frequently employed to map the pathway for long timescale processes and has been previously used to study OmpF48. The simulation model was constructed using CHARMM-GUI49, 50, 51 and comprised one OmpF monomer (PDB 3POX), 108 (90%) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipid molecules, 12 (10%) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG)52, and solvated with 8,234 water molecules in 150 mM NaCl (36 Na+ and 15 Cl−) for a total of 45,402 atoms. Hexagonal periodic boundary conditions were applied with a distance of 77.3 Å in the xy-direction and 92.8 Å in the z-direction. Electrostatic interactions were calculated using the particle-mesh Ewald (PME) method. Protein residues E296, D312, and D127 were protonated53, 54, 55. The simulations were performed at constant pressure (1 atm) and temperature (303 K) with a time step of 2 fs. Each small-molecule under investigation was manually placed directly above the pore. Restraints were initially applied to protein backbone and small-molecule analyte atoms and then removed to equilibrate the system. For SMD production simulations, each small-molecule analyte was pulled at the molecules centre of mass (5 kcal mol−1 Å−2) at a constant velocity (10 Å ns−1) along the z-axis for 4 ns. The all-atom CHARMM force field was used for protein and lipids56, 57. TIP3P was used for water58. All the molecular dynamics simulations were carried out using the NAMD 2.11 scalable molecular dynamics program and run on Stampede at TACC. CHARMM residue topology and parameter files for the small molecules were constructed using CGenFF59. SMD trajectories were analysed and visualized using VMD 1.9.2 and rendered using Pov-Ray 3.6 (ref. 60). Source code for data analysis and model training can be found at https://github.com/HergenrotherLab/GramNegAccum. The authors declare that all other data supporting the findings of this study are available within the paper and the Supplementary Information.


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Ntn1βgeo(ref. 7) and Dcc (ref. 18) knockout lines have previously been described and genotyped by PCR. The Ntn1 conditional knockout was created (Genoway) by inserting two loxP sites flanking the coding sequences containing both the principal ATG (based on Ntn1 cDNA sequence NM_008744) and the cryptic ATG (based on Ntn1 cDNA: BC141294) and the alternative promoter described in intron 3 (ref. 28). The targeting vector was constructed as follows: three fragments of 2.1 kb, 3.4 kb and 4.6 kb (respectively, the 5′, floxed and 3′ arms) were amplified by PCR using 129Sv/Pas ES DNA as a template and sequentially subcloned into the pCR4-TOPO vector (Invitrogen). These fragments were used for the construction of the targeting vector in which a FRT-flanked neomycin cassette was inserted in 5′ of the loxP-flanked region. The linearized construct was electroporated into 129Sv/Pas mouse embryonic stem (ES) cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot analysis both with a neomycin and a 5′ external probe. The positive ES cell clones were injected into C57BL/6J blastocysts and gave rise to male chimaeras with a significant ES cell contribution. Breeding was established with C57BL/6 mice expressing the Flp-recombinase, to produce the heterozygous Ntn1 conditional knockout line devoid of the neomycin cassette. To generate a null allele of Ntn1, Ntn1fl/fl mice were crossed to Krox20:cre mice, which express Cre recombinase in the male and female germline after sexual maturity29. To ablate netrin-1 expression in the floor plate we used the Shh:cre line30 (Jackson laboratories). In this line, the eGFP reporter was also inserted in the Shh locus. Lastly, we crossed Ntn1fl/fl mice to Foxg1:cre mice31 and Nes:cre mice22 (Jackson laboratories). The Ai9 RosatdTomato reporter line (RosaTom; Jackson Laboratories) was used to monitor Cre expression. Developing inferior olivary neurons were visualized by crossing the RosaTom line with the Ptf1a:creERT2 line15. They were also further crossed to Ntn1βgeo mice. All mice are kept in C57BL/6 background. The day of the vaginal plug was counted as embryonic day 0.5 (E0.5). Mice were anaesthetized with intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg). All animal procedures were carried out in accordance to institutional guidelines and approved by the UPMC Charles Darwin Ethics Committee. Embryos of either sex were used. Ptf1a:creERT2;RosaTom pregnant mice were intraperitoneally injected at E10 with 1 mg of tamoxifen (Sigma-Aldrich, T-5648) dissolved in corn oil (Sigma-Aldrich, C-8267). The embryos were collected at E11. Pregnant females were injected intraperitoneally with EdU (1 mg per 10 g body weight) and killed three hours later. The proliferating cells were visualized after immunohistochemistry using the Alexa Fluor 647 Click-iT EdU Imaging Kit (Invitrogen). Antisense riboprobes were labelled with digoxigenin-11-d-UTP (Roche Diagnostics) as described elsewhere12, by in vitro transcription of cDNA of mouse Ntn1 (ref. 7) or mouse Ntn1 exon 3. E12-E13 hindbrains fixed in 4% PFA in an open book configuration were injected using a glass micropipette with DiI crystals or small drops of DiI (Invitrogen) diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich). Samples were kept for 24 h at 37 °C in 4% PFA. Embryos were fixed by immersion in 4% PFA in 0.12 M phosphate buffer, pH 7.4 (PFA) overnight at 4 °C. Samples were cryoprotected in a solution of 10% sucrose in 0.12 M phosphate buffer (pH 7.2), frozen in isopentane at 50 °C and then cut at 20 μm with a cryostat (Leica Microsystems). Immunohistochemistry was performed on cryostat sections after blocking in 0.2% gelatin in PBS containing 0.5% Triton-X 100 (Sigma-Aldrich). Sections were then incubated overnight with the following primary antibodies: goat anti-human Robo3 (1:250, R&D Systems AF3076), goat anti-Dcc (1:500, Santa Cruz sc-6535), goat anti-Robo1 (1:500, R&D Systems AF1749), rat anti-mouse netrin-1 (1:500, R&D Systems MAB1109), mouse anti-nestin–Alexa488 (1:1000, Abcam ab197495), mouse anti-neurofilament (1:300, DSHB 2H3), goat anti-Foxp2 (1:1000, Santa Cruz sc-21069), rabbit anti-Foxp2 (1:1000, Abcam ab16046), rabbit anti-Sox2 (1:500, Abcam ab97959), rabbit anti-βgal (1:500, Cappel 55976), goat anti-human ALCAM (1:500, R&D Systems AF656), rabbit anti-GFP (1:800, Life Technologies A11122), rabbit anti-DsRed (1:500, Clontech 632496) followed by 2 h incubation in species-specific secondary antibodies directly conjugated to fluorophores (Cy-5, Cy-3, Alexa Fluor 647 from Jackson ImmunoResearch, or from Invitrogen). For netrin-1 immunostaining, an antigen retrieval treatment was performed on the sections before to process them for immunochemistry. The sections were boiled in citrate buffer (pH 6) during 9 min. Sections were counterstained with DAPI (1:1,000, Sigma-Aldrich). In the case of netrin-1 immunostaining on non-permeabilized tissue, the Triton was removed from all the steps. Slides were scanned with a Nanozoomer (Hamamatsu) and laser scanning confocal microscope (FV1000, Olympus). Brightness and contrast were adjusted using Adobe Photoshop. Whole-mount immunostaining and 3DISCO optical clearing procedure has been described previously32. 3D imaging was performed with an ultramicroscope using Inspector Pro software (LaVision BioTec). HEK-293T cells (from ATCC, not authenticated, tested for mycoplasma contamination with a negative result) were transfected with pCDNA3, pCDNA3-human NTN3, pCDNA3-human NTN1 or pCDNA3-mouse Ntn1 plasmids using Fugene HD transfection reagent (Promega) according to the manufacturer’s instructions. Cells were harvested and lysed 36 h after transfection. Cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Roche Diagnostics)) and incubated for 1 h at 4 °C. Floor plates were micro-dissected from hindbrains and spinal cords from Ntn1fl/fl, Shh:cre;Ntn1fl/fl and Ntn1−/− E11 embryos. Floor plates were lysed in RIPA buffer and incubated for 20 min at 4 °C. Protein content was determined by a BCA assay. 25 μg of total protein was loaded on a 10% Mini Protean TGX precast gel (Biorad) and blotted onto a nitrocellulose membrane (Biorad). Membranes were blocked with 5% dried milk and 3% of BSA in TBS-0.1% Tween (TBS-T) for 1 h at room temperature and incubated for 90 min at room temperature with primary antibodies: anti-actin (Sigma-Aldrich, A5060, rabbit polyclonal, 1:1,500), anti-HPRT (Abcam, ab109021, rabbit monoclonal EPR5299, 1:10,000), anti-Ntn1 (R&D Systems, MAB1109, rat monoclonal, 1:500), anti-NTN3 (Abcam, ab185200, rabbit polyclonal, 1:1,000) and anti-Slit2 (Abcam, ab134166, rabbit monoclonal, 1:400). After three washes in TBS-T, membranes were incubated with the appropriate HRP-conjugated secondary antibody (1:5,000, Jackson ImmunoResearch). Detection was performed using Pierce ECL Western Blotting Substrate (ThermoScientific). All data quantification was done by an observer blinded to the experimental conditions. We did not perform randomization into groups. No statistical methods were used to predetermine sample size. Data are presented as mean values ± s.e.m. Statistical significance was calculated using one-sided unpaired tests for non-parametric tendencies (Kruskal–Wallis and Mann–Whitney). For western blot, at least three independent cases were quantified from independent experiments using densitometric analysis (ImageJ) by normalizing phosphorylation signals to total protein levels. The control cases were normalized to 1 and for the mutants, data were presented as mean values ± s.e.m. (0.1133 ± 0.05 for Shh:cre; Ntn1fl/fl 1 for Ntn1fl/fl and 0 for Ntn1−/−). Differences were considered significant when P < 0.05. The thickness of hindbrain commissural bundles was quantified for each embryo on nine coronal sections. The sections were representative of three different hindbrain antero-posterior levels (three sections for each level). To minimize the developmental variations, mutant embryos and littermate controls were compared (except for Ntn1−/− embryos which were compared to wild-type embryos). The ratio of the commissural axon bundle size was normalized to controls. Six embryos of each genotype were quantified, from at least two different litters. Data are presented as mean ± s.e.m.: wild type: Dcc, 0.994 ± 0.052; Robo3, 1 ± 0.061; neurofilament, 1 ± 0.048; Ntn1βgeo/βgeo: Dcc, 0.416 ± 0.013; Robo3, 0.402 ± 0.007; neurofilament, 0.416 ± 0.01; Ntn1−/−: Dcc, 0.091 ± 0.008; Robo3, 0.061 ± 0.008; neurofilament, 0.104 ± 0.007; Shh:cre;Ntn1fl/fl: Dcc, 1.051 ± 0.011; Robo3, 1.084 ± 0.028; neurofilament, 1.063 ± 0.019; and Foxg1:cre;Ntn1fl/fl: Dcc, 0.411 ± 0.053; Robo3, 0.369 ± 0.056; neurofilament, 0.416 ± 0.050. Differences were considered significant when P < 0.05. Statistical analyses of the mean and variance were performed with Prism 7 (GraphPad Software). The data that support the findings of this study are available from the corresponding author upon reasonable request.


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The Cdh5(PAC)-CreERT2 transgenic mice (iECre) were a gift from R. H. Adams39. Krit1fl/fl and Ccm2fl/fl animals have been previously described40, 41. Tlr4fl/fl, Cd14−/−, Ai14 (R26-LSL-RFP), and R26-CreERT2 animals42, 43, 44, 45 were obtained from the Jackson Laboratories. The Slco1c1(BAC)-CreERT2 transgenic mice have been previously described18. All experimental animals were maintained on a mixed 129/SvJ, C57BL/6J, DBA/2J genetic background unless specifically described. C57BL/6J and timed pregnant Swiss Webster mice were purchased from Charles River Laboratories. Germ-free Swiss Webster mice were purchased from Taconic. Breeding pairs between two and ten months of age were used to generate the neonatal CCM mouse model pups. Mice were housed in a specific pathogen-free facility where cages were changed on a weekly basis; ventilated cages, bedding, food, and acidified water (pH 2.5–3.0) were autoclaved before use, ambient temperature maintained at 23 °C, and 5% Clidox-S was used as a disinfectant. Experimental breeding cages were randomly housed on three different racks in the vivarium, and all cages were kept on automatic 12-h light/dark cycles. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. A group of the resistant Ccm2ECKO colony was exported to the Centenary Institute, Sydney, Australia, where the mice were permanently maintained as an inbred colony in a quarantine facility. After several generations, this colony uniformly converted to lesion susceptibility. Cages were changed on a weekly basis; ventilated cages, bedding, food and acidified water (pH 2.5–3.0) were autoclaved before use. Ambient temperature was maintained between 22–26 °C, and 80% ethanol and F10SC (1:125 dilution of the concentrate, a quaternary ammonium compound) were used as disinfectants. Experimental breeding cages were randomly distributed throughout the vivarium, and all cages were kept on 12-h light/dark cycles. The Sydney Local Health District Animal Welfare Committee approved all animal ethics and protocols. All experiments were conducted under the guidelines/regulations of Centenary Institute and the University of Sydney. Germ-free Swiss Webster mice were purchased from Taconic and directly transferred into sterile isolators (Class Biologically Clean Ltd) under the care of the Penn Gnotobiotic Mouse Facility. Food, bedding and water (non-acidified) were autoclaved before transfer into the sterile isolators. Ventilated cages were changed weekly, and all cages in the vivarium were kept under 12-h light/dark cycles. Microbiology testing (aerobic and anaerobic culture, 16S qPCR) was performed every ten days and faecal samples were sent to Charles Rivers Laboratories for pathology testing on a quarterly basis. Further details regarding the sterile C-section fostering can be found below. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols. For all neonatal CCM mouse model experiments, at one day post-birth (P1), pups were intragastrically injected by 30-gauge needle with 40 μg of 4-hydroxytamoxifen (4OHT, Sigma Aldrich, H7904) dissolved in a 9% ethanol/corn oil (volume/volume) vehicle (50 μl total volume per injection). This solution was freshly prepared from pre-measured, 4OHT powder for every injection. Before injection, the pup skin was sanitized using ethanol wipes. The P1 time point was defined by checking experimental breeding pairs every evening for new litters. The following morning (P1), pups were injected with 4OHT. All experimental pups were subjected to this induction regimen. For the Tlr4 rescue experiment (Fig. 2), and all lineage-tracing experiments, an additional dose of 40 μg 4OHT was intragastrically delivered at P2 (P1+2, two total doses). Pups were then harvested as previously described9 at the specified time points. Tissue samples were fixed in 4% formaldehyde overnight, dehydrated in 100% ethanol, and embedded in paraffin. 5-μm-thick sections were used for haematoxylin and eosin (H&E) and immunohistochemistry staining. The following antibodies were used for immunostaining: rat anti-PECAM (1:20, Histo Bio Tech DIA-310), rabbit anti-pMLC2 (1:200, Cell Signaling 3674S), goat anti-KLF4 (1:100, R&D AF3158), and rabbit anti-RFP (1:50, Rockland 600-401-379). Littermate control and experimental animal sections were placed on the same slide and immunostained at the same time under identical conditions. Images were taken at the same time using the same exposure times and colour channels, and were subsequently overlaid using ImageJ. Intra-abdominal abscesses were dissected and triturated in 500 μl of SOC medium. Drops of the mixture were placed on a microscope slide, briefly exposed to heat, and Gram staining was performed using a kit from Sigma Aldrich (77730) following the manufacturer’s protocol. Eyes from euthanized P17 mice were removed and fixed overnight in cold 4% PFA/PBS solution. The following day, retinas were dissected, cut into petals, and stained with isolectin-B4 conjugated to Alexa488 fluorophore (Thermo Fisher I21411) as previously described46. The retinas were then whole-mounted on microscopy slides in a flat, four-petal shape for fluorescence imaging. B. fragilis was purchased directly from the ATCC (strain 25285) and grown in chopped meat glucose (CMG) broth (Anaerobe Systems AS-813) under anaerobic conditions at 37 °C. Autoclaved, degassed caecal contents (ACC) were generated by collecting caecal contents from the colons of euthanized adult mice between 2–8 months of age. Caecal contents were then autoclaved and pulverized in an equal volume of CMG broth. This slurry was filtered through a 70-μm nylon strainer and degassed overnight in the anaerobic chamber. 1 ml of CMG broth was inoculated with B. fragilis and grown overnight to an optical density of between 0.8 and 1.0. An equal volume of ACC was mixed with the overnight bacterial culture. 100 μl of this B. fragilis–ACC mixture was injected intraperitoneally into five-day-old pups with a 31-gauge needle. Control littermates were simultaneously injected intraperitoneally with 100 μl of ACC alone. Pups were harvested at P17. Spleen weight was measured immediately after dissection, and all tissue was subsequently processed as described above. LPS from E. coli O127:B8 was purchased from Sigma (L3129) and administered to the low-lesion-penetrance, resistant Ccm2ECKO neonatal CCM disease model. At P5, a 3 μg dose of LPS dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, a 5 μg dose of LPS was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Peptidoglycan from Bacillus subtilis (a Gram-positive gut commensal) was purchased from Invivogen (tlrl-pgnb3) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Poly(I:C) was purchased from Invivogen (tlrl-picw) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the LPS experiments. Mouse IL-1β was purchased from Genscript (Z02988) and administered to the resistant Ccm2ECKO neonatal CCM disease model. At P5, a 5 ng dose of IL-1β dissolved in sterile PBS was administered retro-orbitally in a total 30 μl volume by 31-gauge needle. At P10, an 8-ng dose of IL-1β was administered retro-orbitally in a total 50 μl volume by 31-gauge needle. Control animals were identically injected with PBS alone. Pups were euthanized and brains dissected at specified time points. Mouse TNFα was purchased from Genscript (Z02918) and administered to the resistant Ccm2ECKO neonatal CCM disease model under identical conditions as the IL-1β experiments. For all experiments using microCT quantification of CCM lesion volume, brains were harvested and immediately placed in 4% PFA/PBS fixative. Brains remained in fixative until staining with non-destructive, iodine contrast and subsequent microCT imaging performed as previously described47. All tissue processing, imaging and volume quantification were performed in a blinded manner by investigators at the University of Chicago without any knowledge of experimental details. We blinded samples at three distinct points in the analysis. First, neonatal CCM model pups were injected with 4OHT without knowledge of genotypes. Second, hindbrains from genotyped animals were given randomized, de-identified labels to provide for blinded microCT scanning by an independent operator. Third, randomized microCT image stacks were analysed in a blinded manner by individuals not involved in any prior experimental steps. Mice were anaesthetized with Avertin and underwent intra-cardic perfusion with 10 ml of cold PBS. The brain was separated from the brainstem, and the cerebellum was separated from the remaining brain and processed in parallel. The tissue was minced with scissors, placed in digestion buffer (RPMI, 20 mM HEPES, 10% FCS, 1 mM CaCl , 1 mM MgCl , 0.05 mg ml−1 Liberase (Sigma), 0.02 mg ml−1 DNase I (Sigma)), and incubated for 40 min at 37 °C with shaking at 200 r.p.m. The mixture was passed through a 100-μm strainer and washed with FACS buffer (PBS, 1% FBS). Cells were resuspended in 4 ml of 40% Percoll (GE Healthcare) and overlaid on 4 ml of 67% Percoll. Gradients were centrifuged at 400g for 20 min at 4 °C and cells at the interface were collected, washed with 10 ml of FACS buffer, and stained for flow cytometric analysis. Neonatal P10 mice were anaesthetized with Avertin and underwent intracardiac puncture/blood draw using a 27-gauge needle/syringe coated with 0.5 M EDTA, pH 8.0 immediately before use. Cells were pelleted by centrifugation at 300g for 5 min at 4 °C. Serum was removed and red blood cells were lysed using ACK lysis buffer. Spleens were dissected in parallel, hand-homogenized using a mini-pestle and red blood cells were lysed using ACK lysis buffer. Cells from both sets of tissues were passed through a 70-μm cell-strainer, pelleted and resuspended in FACS buffer (PBS, 2% FBS, 0.1% NaN ) for immunostaining and subsequent FACS analysis. Cells were isolated from the indicated tissues. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Multi-laser, flow cytometry analysis procedures were performed at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analysers running FACSDiva software (BD Biosciences). Two-laser, flow cytometry analyses were performed at the University of Pennsylvania iPS Cell Core using BD Accuri C6 instruments. FlowJo software (v.10 TreeStar) was used for data analysis and graphics rendering. All fluorochrome-conjugated antibodies used are listed as follows (Clone, Company, Catalog Number): CD11b (M1/70, Biolegend, 101255); CD11c (N418, Biolegend, 117318); CD16/32 (93, Biolegend, 101319); CD16/32 (93, eBiosciences, 56D0161D80); CD19 (6D5, Biolegend, 115510); CD3ε (145D2C11, Biolegend, 100304); CD4 (GK1.5, Biolegend, 100406); CD45 (30-F11, Biolegend, 103121 or 103151), CD8a (53D6.7, Biolegend, 100725); Foxp3 (FJK-16 s, eBiosciences, 50-5773-82); Ly-6G (1A8, Biolegend, 127624); Live/Dead (N/A, Thermofisher, LD34966); NK1.1 (PK136, Biolegend, 108745); RORγt (B2D, eBiosciences, 12-6981-82); Siglec-F (E50D2440, BD, 562757); TCRγδ (UC7-13D5, Biolegend, 107504) At the specified time points, cerebellar endothelial cells were isolated through enzymatic digestion followed by separation using magnetic-activated cell sorting by anti-CD31-conjugated magnetic beads (MACS MS system, Miltenyl Biotec), as previously described9. Lung endothelial cells were isolated through enzymatic digestion, as previously described, followed by separation using anti-CD31-conjugated magnetic beads and the MACS MS system48. Isolated endothelial cells were pelleted and total RNA was extracted using the RNeasy Micro kit (Qiagen 74004). For qPCR analysis, cDNA was synthesized from 300 ng to 500 ng total RNA using the SuperScript VILO cDNA Synthesis Kit and Master Mix (Thermo Fisher 11755050). Real-time PCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher 4368577) using the primers listed (all mouse): Gapdh forward: 5′-AAATGGTGAAGGTCGGTGTGAACG-3′; Gapdh reverse: 5′-ATCTCCACTTTGCCACTGC-3′; Klf2 forward: 5′-CGCCTCGGGTTCATTTC-3′; Klf2 reverse: 5′-AGCCTATCTTGCCGTCCTTT-3′; Klf4 forward: 5′-GTGCCCCGACTAACCGTTG-3′; Klf4 reverse: 5′-GTCGTTGAACTCCTCGGTCT-3′; Krit1 forward: 5′-CCGACCTTCTCCCCTTGAAC-3′; Krit1 reverse: 5′-TCTTCCACAACGCTGCTCAT-3′; Il1b forward: 5′-GCAACTGTTCCTGAACTCAACT-3′; Il1b reverse: 5′-ATCTTTTGGGGTCCGTCAACT-3′; Sele forward: 5′-ATGCCTCGCGCTTTCTCTC-3′; Sele reverse: 5′-GTAGTCCCGCTGACAGTATGC-3′; Tlr4 forward: 5′-ACTGGGGACAATTCACTAGAGC-3′; Tlr4 reverse: 5′-GAGGCCAATTTTGTCTCCACA-3′. As part of the Brain Vascular Malformation Consortium (BVMC) CCM study (Project 1), a large cohort of familial CCM individuals with identical KRIT1(Q455X) mutations were enrolled between 2009–2014 at the University of New Mexico. All study protocols were approved by the Institutional Review Boards at the University of New Mexico and University of California San Francisco (UCSF) and all procedures were performed in accordance with these protocols. Prior to participation in the study, written informed consent was obtained from every patient and properly documented by UNM investigators. At study enrollment, participants received a neurological examination and 3T MRI imaging using a volume T1 acquisition (MPRAGE, 1-mm slice reconstruction) and axial TSE T2, T2 gradient recall, susceptibility-weighted, and FLAIR sequences. Lesion counting by the neuroradiologist was based on concurrent evaluation of axial susceptibility-weighted imaging with 1.5-mm reconstructed images and axial T2 gradient echo 3-mm images. Participants also provided blood or saliva samples for genetic studies. Genomic DNA was extracted using standard protocols. De-identified samples were normalized, plated on 96-well plates, and genotyped at the UCSF Genomics Core Facility using the Affymetrix Axiom Genome-wide LAT1 Human Array. Affymetrix Genotyping Console (GTC) 4.1 Software package was used to generate quality control metrics and genotype calls. All samples had genotyping call rates of ≥ 97% and were further checked for sample mix-ups (sex check, Mendelian errors and cryptic relatedness), resulting in 188 samples for genetic analysis. 21 candidate genes were further examined in the TLR4 and MEKK3–KLF2/4 signalling pathways (TLR4, CD14, MD-2, LBP, MYD88, TICAM1, TIRAP, TRAF1-6, MAP3K3, MEK5, ERK5, MEF2C, KLF2, KLF4, ADAMTS4, ADAMTS5) including 467 SNPs within 20 kb upstream or downstream of each gene locus using UCSC Genome Browser coordinates (GRCh37/hg19). Because total lesion counts are highly right-skewed, raw counts were log-transformed and analysis was performed on residuals (adjusted for age at enrollment and sex). To identify genotypes associated with log-transformed residual counts, linear regression analysis was implemented using QFAM family-based association tests for quantitative traits (PLINK v1.07 software), with stringent multiple testing correction (Bonferroni correction for the number of SNPs tested within each gene) given that some SNPs on the Affymetrix array were in linkage disequilibrium with each other, that is, statistically correlated with R2 > 0.8. The Fehrmann dataset used for eQTL lookups consisted of peripheral blood samples from the UK and the Netherlands49, 50. Samples were genotyped with Illumina HumanHap300, HumanHap370 or 610 Quad platforms. Genotypes were input by Impute v2 (ref. 51) using the GIANT 1000G p1v3 integrated call set for all ancestries as a reference52. Gene expression levels were measured by Illumina HT12v3 arrays. Gene expression pre-processing involved quantile normalization, log transformation, probe centring and scaling, population stratification correction (first four genetic multi-dimensional scaling components were removed from gene expression data) and correction for unknown confounders (first 20 gene expression principal components not associated with genetic variants were removed from gene expression data). Identification of potential sample mix-ups was conducted by MixupMapper21 and finally 1,227 samples remained. All pre-processing steps were performed with the QTL mapping pipeline v1.2.4D (https://github.com/molgenis/systemsgenetics/tree/master/eqtl-mapping-pipeline - downloading-the-software). These results are corroborated by an independently conducted GTEX Consortium study (http://www.gtexportal.org/home/snp/rs10759930 and http://www.gtexportal.org/home/snp/rs778587). TAK-242 was purchased from EMD Millipore (614316) and administered to the neonatal CCM disease model. Five, seven and nine days after birth, a 60-μg dose of TAK-242 was dissolved in DMSO/sterile intralipid (Sigma, I141) vehicle and administered retro-orbitally in a total volume of 30 μl. Control animals were identically injected with sterile DMSO/intralipid vehicle alone. Pups were euthanized and brains dissected 10 days after birth. LPS-RS ultrapure was purchased from Invivogen (tlrl-prslps) and administered to the neonatal CCM disease model. Starting at P5, a 20 μg dose dissolved in sterile PBS was administered retro-orbitally in a total volume of 30 μl every 24 h. Control animals were identically injected with sterile PBS alone. Pups were euthanized and brains dissected 10 days after birth. Experimental breeding pairs of mice, yielding susceptible neonatal CCM pups, were identified by induction of a neonatal CCM litter and evaluation of lesion burden. These breeding pairs then underwent timed matings and at E14.5, both male and female adult mice received antibiotic-laced drinking water mixed with 40 g l−1 of sucralose and red food colouring. Antibiotic water was replaced daily. The following antibiotics were mixed with 0.22-μm-filtered water: penicillin (500 mg l−1), neomycin (500 mg l−1), streptomycin (500 mg l−1), metronidazole (1 g l–1) and vancomycin (1 g l−1). Antibiotics were purchased from the Hospital of the University of Pennsylvania pharmacy. The neonatal CCM model was induced as described above. At P10, pups were euthanized and antibiotic water switched to normal drinking water. Experimental breeding pairs were then mated to obtain third generation, post-antibiotic pups. Co-housed, susceptible Krit1fl/fl females underwent evening–morning timed matings with a single susceptible Krit1ECKO male. Upon detection of a plug in the morning, the females were subsequently separated into singly-housed cages. At E14.5, female mice were received either vancomycin (1 g l−1)-laced or untreated (vehicle) sterile-filtered drinking water, changed daily. The drinking water was further mixed with 40 g l−1 sucralose and red food colouring. Pups were harvested at P11. The entire neonatal gut was dissected, snap-frozen on dry ice, and stored at −80 °C. The QIAamp DNA Stool Mini Kit (Qiagen 51504 or 51604) was used to extract bacterial DNA from the neonatal gut. Before commencing the standard Qiagen protocol, the frozen gut was mixed in the included stool lysis buffer and homogenized with a 5 mm stainless steel bead in a TissueLyser LT (Qiagen 69980) at 50 Hz for 10 min at 4 °C. Concentration of the extracted DNA was equalized and 16 ng of DNA was used per qPCR reaction with universal bacterial 16S rRNA gene primers53, two different sets of previously characterized Bacteroidetes s24-7 primers54, 55, and Firmicutes primers56. Universal 16S rRNA forward: 5′-ACTGAGAYACGGYCCA-3′; universal 16S rRNA reverse: 5′-TTACCGCGGCTGCTGGC-3′; Bacteroidetes s24-7 rRNA set 1 forward: 5′-GGAGAGTACCCGGAGAAAAAGC-3′; Bacteroidetes s24-7 rRNA set 1 reverse: 5′-TTCCGCATACTTCTCGCCCA-3′; Bacteroidetes s24-7 rRNA set 2 forward: 5′-CCAGCAGCCGCGGTAATA-3′; Bacteroidetes s24-7 rRNA set 2 reverse: 5′-CGCATTCCGCATACTTCTC-3′; Firmicutes rRNA forward: 5′-TGAAACTYAAAGGAATTGACG-3′; Firmicutes rRNA reverse: 5′-ACCATGCACCACCTGTC-3′. Evening–morning timed matings to generate donor susceptible or resistant females yielding Krit1ECKO or Ccm2ECKO pups were performed and timed pregnant Swiss Webster females (Charles River 024) served as foster mothers. To prevent delivery of the pups, at E16.5, donor females were injected subcutaneously with 100 μl of a 15 μg ml−1 solution of medroxyprogesterone (Sigma Aldrich, M1629) dissolved in DMSO. The morning of E19.5, the donor mother was euthanized by cervical dislocation and submerged in a warm sterile solution of 1% VirkonS/PBS (weight/volume) for one minute. The uterus was then dissected in a sterile laminar flow hood, submerged in a warm sterile solution of 1% VirkonS/PBS for one minute and quickly rinsed with warm sterile PBS. Pups were then removed from the uterus and fostered to the Swiss Webster recipient female. The following morning, induction of the neonatal CCM model was performed as described above. Timed matings were performed using germ-free Swiss Webster mice housed in sterile isolators under care of the University of Pennsylvania Gnotobiotic Mouse Facility. Simultaneous evening–morning timed matings were also performed using co-housed, susceptible Krit1fl/fl females and Krit1ECKO males previously characterized to yield CCM-susceptible pups. Medroxyprogesterone was administered to donor females and the sterile C-section was performed at E19.5 as described in the previous section. The intact uterus was passed through a J-tube filled with warm 1% VirkonS/PBS that was hermetically sealed to the sterile isolator. Pups were dissected from the uterus inside the sterile isolator and fostered to the recipient germ-free Swiss Webster mother. Approximately one week later, faecal samples were collected for microbiology testing. Germ-free status was further confirmed by 16S qPCR of bacterial DNA isolated from maternal faeces and neonatal guts. Fresh faecal pellets were collected from experimental females yielding susceptible or resistant pups one day after harvesting the pups to determine phenotypic severity. Collection was performed between 16:00 and 18:00, pellets were immediately snap-frozen on dry ice, and stored at −80 °C. DNA was extracted from stool samples using the Power Soil htp kit (Mo Bio Laboratories) following the manufacturer’s protocol. Library preparation was performed by using previously described barcoded primers targeting the V1/V2 region of the 16S rRNA gene57. PCR reactions were performed in quadruplicate using AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen). Each PCR reaction consisted of 0.4 μM primers, 1× AccuPrime Buffer II, 1 U Taq, and 25 ng DNA. PCRs were run using the following parameters: 95 °C for 5 min; 20 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s; and 72 °C for 8 min. Quadruplicate PCR reactions were pooled and products were purified using AMPureXP beads (Beckman-Coulter). Equimolar amounts from each sample were pooled to produce the final library. Positive and negative controls were carried through the amplification, purification and pooling procedures. Negative controls were used to assess reagent contamination and consisted of extraction blanks and DNA-free water. Positive controls were used to assess amplification and sequencing quality and consisted of gBlock DNA (Integrated DNA Technologies) containing non-bacterial 16S rRNA gene sequences flanked by bacterial V1 and V2 primer binding sites. Paired-end 2 × 250 bp sequence reads were obtained from the MiSeq (Illumina) using the 500 cycle v2 kit (Illumina). Sequence data were processed using QIIME version 1.9.1 (ref. 58). Read pairs were joined to form a complete V1/V2 amplicon sequence. Resulting sequences were quality filtered and demultiplexed. Operational taxonomic units (OTUs) were selected by clustering reads at 97% sequence similarity59. Taxonomy was assigned to each OTU with a 90% sequence similarity threshold using the Greengenes reference database60. A phylogenetic tree was inferred from the OTU data using FastTree61. The phylogenetic tree was then used to calculate weighted and unweighted UniFrac distances between each pair of samples in the study62, 63. Microbiome compositional differences were visualized using principle coordinates analysis (PCoA). Community-level differences between mice genetic background as well as disease susceptibility groups were assessed using a PERMANOVA test64 of weighted and unweighted UniFrac distances. To assess significance in the PERMANOVA test, each cage was randomly re-assigned to groups 9,999 times. Differential abundance was assessed for taxa present in at least 80% of the samples, using generalized linear mixed-effects models. For tests of taxon abundance, the cage was modelled as a random effect, as previous research has established that the faecal microbiota of mice are correlated within cages65. The P values were corrected for multiple testing using Benjamini–Hochberg method. Sample sizes were estimated on the basis of our previous experience with the neonatal CCM model and lesion volume quantification by microCT9. Using 40 historically collected, susceptible Krit1ECKO and Ccm2ECKO P10 brains, we calculated a sample standard deviation of 0.250 mm3. Between Krit1ECKO and Ccm2ECKO genotypes, an F-test to compare variances confirmed no significant difference (P = 0.340). Thus, for a two-group comparison of lesion volumes, each sample group requires seven animals for a desired statistical power of 95% (β = 0.05), and a conventional significance threshold of 5% (α = 0.05) assuming an effect size of 50% (0.5) and equal standard deviations between sample groups. These predictive calculations were corroborated by our recent publication in which larger effect sizes (>90%) were found to be statistically significant with four to five samples per group9. All experimental and control animals were littermates and none were excluded from analysis at the time of harvest. Experimental animals were lost or excluded at two pre-defined points: (i) failure to properly inject 4OHT and observation of significant leakage; (ii) death before P10 because of injection or chaos. Given the early time points, no attempt was made to distinguish or segregate results based on neonatal genders. P values were calculated as indicated in figure legends using an unpaired, two-tailed Student’s t-test; one-way ANOVA with multiple comparison corrections (Holm–Sidak or Bonferroni); PERMANOVA; or linear mixed effects modelling. As indicated in the figure legends, the standard error of the mean (s.e.m.), 95% confidence interval, or boxplot is shown. All relevant data are available from the authors upon reasonable request.

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