N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that  AR) [N-formyl peptide receptor (FPR) [Arrestins were originally described as cytosolic proteins recruited to ligand-activated and phosphorylated G protein-coupled receptors (GPCRs) –3. ThrouAR)  require AR) ,14. Howeor (FPR)  and the or (FPR) , do not More recently, arrestins have been described as scaffolds for G protein-independent GPCR-mediated signaling complexes –19. Sign1AR but not AT1AR stimulation [AR internalization and trafficking [Of the aforementioned signaling scaffolds, arrestin-Src kinase interactions are among the better described, with important functions in cell migration and degranulation ,28. In amulation . Finallyfficking ,29.N-formyl peptide receptor  derived from arrestin-2/3 double knockout mice, although most GPCRs no longer internalize , activator FPR, ,32) resu resuN-foor  [-/--3-/- FPR MEF) to assess the consequences of FPR activation in the presence or absence of transiently transfected wild type or mutant arrestins (as GFP fusion proteins) [-/--3-/- FPR MEFs expressing the EGFP vector (no arrestin present) stained with PI when stimulated with the agonist fMLP. Furthermore, when the FPR was stimulated in the presence of transfected wild-type arrestin-2-GFP, less than 5% of cells were again stained with PI. Finally, FPR activation in the presence of arr2-P91G/P121E-GFP resulted in extensive staining with PI, similar to the vector-only transfected cells. These results suggest that an interaction between arrestin-2 and Src kinase is necessary to ensure appropriate FPR signaling with respect to apoptosis.To determine whether the arrestin-2/Src kinase interaction plays a role in FPR signaling, we assessed the arr2-P91G/P121E mutant for its ability to regulate FPR-mediated apoptosis. We utilized mouse embryonic fibroblasts deficient in arrestin-2 and -3 (derived from arrestin-2ut mice)  stably eroteins) ,34. The roteins) ,34. On t-/-/-3-/- FPR MEFs expressing GFP-fused arrestins were assayed for PI staining in the presence of the pan-caspase inhibitor zVAD-FMK , Src kinase activity is still required for FPR-mediated apoptosis.Although previous publications have reported that overexpression of arr2-P91G/P121E inhibited ERK1/2 activation in response to β2-AR activation, this mutant did not affect ERK1/2 activation by the AT1AR . These r-/-/-3-/- FPR MEFs were cotransfected with GFP-fused arrestins and either wild type or kinase dead (K298M) Src kinase  and kinase dead Src kinase, less than 5% of cells stained with PI in response to FPR activation regardless of the absence or presence of arrestin (wild type or P91G/P121E), suggesting that Src kinase activity was specifically required to induce the apoptotic phenotype  of FPR internalization as well as the maximum extent of internalization of the FPR in the presence of the arr2-P91G/P121E mutant were similar to FPR internalization in the presence of EGFP alone or wild type arrestin-2  for 1 hour and ligand-FPR-arrestin complexes were visualized using confocal fluorescence microscopy  did not rescue FPR-mediated apoptosis. However, inhibition of Src kinase and ERK1/2 activity in the presence of this mutant prevented cell death, indicating that proper arrestin-Src kinase binding is necessary to control non-G protein-mediated FPR signaling events. Furthermore, these data suggest that without arrestin regulation, Src and ERK1/2 signal aberrantly resulting in cell stress leading to apoptosis. Where and how these signaling proteins are being aberrantly activated by FPR stimulation is currently unclear and could involve arrestin-independent G protein-mediated activation of these pathways . AlternaAR, without which the receptor remains trapped at the cell surface in clathrin-coated pits [Another interesting result from our study involves the role of arrestin-Src kinase interaction in FPR trafficking. The Src kinase binding-deficient arrestin mutant resulted in accumulation of FPR-arrestin complexes in the perinuclear region with Rab11, AP-2 and AP-1. This is perhaps not surprising given the fact that FPR-mediated apoptosis and FPR trafficking defects have been linked in previous work . Howeverted pits . Similarted pits . Our resFinally, our previous results have shown that FPR internalization from the plasma membrane is not dependent on clathrin , AP-2 3, or arreBased on our results, we propose a model involving two independent roles for Src kinase in FPR trafficking and signaling . Our modIn conclusion, this report elucidates two independent roles for Src kinase in FPR trafficking and signaling. To our knowledge, this is the first indication that Src kinase has multiple roles within the same GPCR signaling complex: one in control of its trafficking and one in mediating proper cellular signaling. Both of these roles are coordinated and regulated by binding to arrestin-2. Our results also clarify a role for arrestin-2 in the spatial and temporal control of non-G protein-mediated GPCR signaling and serve as a model for further study. Consistent with our results, recent reports also indicate a role for AP-2 in the apoptosis mediated by visual arrestin binding to a rhodopsin mutant found in certain humans diagnosed with autosomal dominant retinitis pigmentosa . Arresti5’-GCCCATATGGGCGACAAAGGGACGCGG-3’ and 5’-GCCCAGCTTCTTGATGAGGCGCTCCTGCAGCCGCGTCAGGGGCTTCTTGTCCTCACCGGCCGGCGGGAAAGACTGCACG-3’ and primer pairs 5’-GCAGGAGCGCCTCATCAAGAAGCTGGGCGAGCATGCCTACCCTTTCACCTTTGAGATCCCTGAGAACTCCCATGCTCTGTGACTTTGCAGCCG-3’ and 5’-GGATCCCGGGCCCATCTGTCGTTGAGCCGCGG-3’ were used to create N- and C-terminal fragments that contained the P91G and P121E mutations, respectively. Fragments were purified and amplified using the external primers to create an Arr2-P91G/P121E fragment containing HindIII/ApaI restriction sites. Standard subcloning methods were used to insert this fragment into the EGFP or mRFP1 vector. The presence of the correct sequence was verified by Sanger dideoxy sequencing of the entire coding sequence. Other constructs, including mRFP1, Arr2-WT-RFP, EGFP, Arr2-WT-GFP, Rab11-GFP, γ-subunit (AP-1)-GFP and α-subunit (AP-2)-GFP were previously described [All materials were from Sigma unless otherwise specified. Arr2-P91G/P121E-GFP was created by using a two-step PCR mutagenesis procedure. Briefly, primer pairs escribed . Murine -/-/-3-/- FPR cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin and 100units/mL streptomycin at 37°C and 5% CO2. Transient transfections of arr-2-/-/-3-/- FPR cells were performed with Lipofectamine 2000 according to manufacturer’s instructions.Arr-2-/-/-3-/- FPR cells were transiently transfected and plated on 12mm glass coverslips. The next day, cells were serum-starved for 30 min and incubated with serum-free medium (SFM) for 5 hours with 10 nM formyl-methionyl-leucyl-phenylalnine (fMLF) or vehicle at 37°C. Propidium iodide (PI) was then added to a final concentration of 100 pg/μL for 5–10 min at room temperature. Coverslips were washed and fixed with 2% paraformaldehyde and mounted using Vectashield. Random fields were viewed by fluorescence microscopy until 100–300 GFP expressing cells were assessed. GFP cells were scored for the presence of PI staining. Data are expressed as mean PI positive/GFP cell.Apoptosis assay was performed as previously described . Briefly-/-/-3-/- FPR cells were grown to confluence and harvested by trypsinization, which does not affect FPR binding of ligand . Cells were incubated with 1 μM fMLF at 37°C and aliquots were removed and placed in cold SFM at 0, 2, 5, 10, 20 and 30 min. Cells were washed extensively with cold SFM to remove excess fMLF. Cells were then resuspended in cold SFM containing 10 nM Alexa633-N-formyl-Leucyl-Leucyl-Phenylalanyl-Leucyl-Tyrosinyl-Lysine (633-6pep) and analyzed using a Becton Dickinson FACSCalibur at appropriate wavelengths. Live cells were gated using forward- and side-scatter parameters. Cells expressing the GFP-fused protein of interest were gated using FL-1 and mean channel fluorescence (MCF) was measured in FL-4 to determine amount of cell surface receptor remaining. Non-specific binding was determined by labeling arr-2-/-/-3-/- cells not expressing the FPR with 10 nM 633-6pep and was subtracted before further analysis. MCF from unstimulated cells represents 100% FPR cell surface expression with non-specific binding representing less than 3–5% of the signal in the presence of FPR expression. Cell surface expression from stimulated cells was calculated by dividing the MCF following treatment by the MCF from unstimulated cells. Internalization data were then plotted using GraphPad Prism to calculate maximum internalization using a one phase exponential decay.Internalization was performed as previously described . Briefly-/-/3-/- FPR cells transiently transfected with GFP-fused arrestins were harvested and resuspended in SFM. An aliquot was removed to measure total cell surface receptor. The remaining cells were stimulated with 1μM fMLF in SFM at 37°C for 1 hour and were then washed extensively to remove excess unlabelled ligand. Half the remaining cells were resuspended in pre-warmed SFM for 30 min at 37°C to allow the FPR to recycle. The other half was kept on ice to measure post-internalization cell surface receptor levels. All aliquots were then resuspended in SFM containing 10nM 633-6pep and assayed by flow cytometry using a Becton-Dickinson FACSCalibur. For analysis, cells were first gated for live cells using forward and side scatter parameters and then gated using FL-1 for GFP-fused arrestin mutant expression with the mean channel fluorescence measured in FL-4 to determine cell surface expression of the FPR. Non-specific binding was determined by labeling arr2-/-/3-/- MEF cells (not expressing the FPR) with 10nM 633-6pep and subtracting this value from FPR-expressing cell values. To account for differences in absolute recycling potentially due to differences in the initial extent of internalization, the fraction of recycled FPR  was divided by the fraction of internalized FPR. Data are expressed as a percentage of recycled/internalized receptor.FPR recycling assays were performed as previously described . Briefly-/-/-3-/- FPR cells were transiently transfected with the appropriate plasmids. Cells were plated on 25 mm coverslips, grown overnight and serum-starved for 30 min. Cells were then incubated in SFM containing 10 nM 633-6pep for the indicated times, washed with PBS, fixed with 2% paraformaldehyde and mounted using Vectashield. Fluorescence images were acquired using a Zeiss LSM 510 inverted laser scanning microscope equipped with He-Ne and Kr-AR lasers. To assess ligand/arrestin clusters outside the perinuclear region, cells were viewed and scored for the presence of ligand/arrestin outside the perinuclear Rab11-GFP-defined region in a blinded manner. Cells with less than 5 clusters outside of the Rab-11-GFP region were considered to exhibit accumulation without trafficking. To assess the extent of AP-2 or AP-1 colocalization, cells with arrestin clusters were viewed and scored for the presence of any corresponding AP-2 or AP-1 puncta, respectively. Data were expressed as the percentage of cells (mean ± SEM) with colocalized clusters from more than 25 cells/experiment.Microscopy was performed as previously described . BrieflyS1 Fig-/-/-3-/- FPR cells were transiently co-transfected with Rab11-GFP and either empty mRFP vector (Empty), wild type arrestin-2-RFP (WT) or arr2-P91G/P121E-RFP (P91G/P121E) along with the pUSE Src construct indicated below (D-F). Cells were treated and processed as described for ligand stimulation but in the absence of added ligand . A) No treatment, B) DMSO (PP2 vehicle), C) PP2, D) pUSE vector only (pUSE), E) wild type Src (Src WT) and F) kinase dead Src (Src KD). All images demonstrate a lack of arrestin colocalization with Rab11. Scale bars equal 10μm. Images are representative of three independent experiments.Arr-2(PDF)Click here for additional data file.S2 FigA) Arr-2-/-/-3-/- FPR cells were transiently transfected with RFP-fused arrestins (or vector only) and GFP-fused Rab11. Cells were incubated with DMSO (vehicle) for 30 min before and during stimulation as a control for PP2 treatment (see B) Arr-2-/-/-3-/- FPR cells were transiently transfected with RFP-fused arrestins and GFP-fused Rab11 and the pUSE empty vector as a control for wild type (see C below) and kinase dead Src (see C) Arr-2-/-/-3-/- FPR cells were transiently transfected with GFP-fused Rab11, RFP-fused arrestins and wild type Src kinase in pUSE as a control for kinase dead Src (see Rab11-GFP and either empty mRFP vector (Empty), wild type arrestin-2-RFP (WT) or arr2-P91G/P121E-RFP (P91G/P121E) along with the pUSE Src construct indicated below (B-C). Cells were stimulated with 10 nM 633-6pep for 60 min and viewed by confocal fluorescence microscopy. ment see . See Fig Src see . See Fig Src see . See Fig(PDF)Click here for additional data file.S3 Fig-/-/-3-/- FPR cells were transiently co-transfected with either Rab11-GFP (A), AP-2-GFP (B) or AP-1-GFP (C) and either empty mRFP vector (mRFP only), wild type arrestin-2-RFP (Arrestin-WT) or arr2-P91G/P121E-RFP (Arrestin-P91G/P121E). Cells were stimulated with 10 nM 633-6pep for 60 min and viewed by confocal fluorescence microscopy, followed by line intensity scanning in Zen software. Images are representative of three independent experiments.Arr-2(PDF)Click here for additional data file.


The Hedgehog (Hh) pathway is essential for embryonic development and tissue regeneration, and its dysregulation can lead to birth defects and tumorigenesis. Understanding how this signaling mechanism contributes to these processes would benefit from an ability to visualize Hedgehog pathway activity in live organisms, in real time, and with single-cell resolution. We report here the generation of transgenic zebrafish lines that express nuclear-localized mCherry fluorescent protein in a Gli transcription factor-dependent manner. As demonstrated by chemical and genetic perturbations, these lines faithfully report Hedgehog pathway state in individual cells and with high detection sensitivity. They will be valuable tools for studying dynamic Gli-dependent processes in vertebrates and for identifying new chemical and genetic regulators of the Hh pathway. Zebrafish have emerged as versatile models of vertebrate biology, due to their amenability to genetic and pharmacological manipulations, optical transparency during embryogenesis and larval development, and facile and economical husbandry In vivo reporters of developmental pathways are particularly instrumental, given the pervasive role of these signaling mechanisms in vertebrate biology. Several zebrafish lines that allow the real-time observation of cellular responses to Wnt, Hedgehog (Hh), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) family members have been described cis-regulatory elements specific to the signaling pathway of interest. While these transgenic animals can reveal tissue-specific differences in cell signaling, the biological properties of their reporters can limit their utility. All but one of these lines utilizes fluorescent reporters without subcellular targeting motifs, and the cytoplasmic distribution of these proteins decreases their detection sensitivity. As a result, the reporters typically cannot be observed by fluorescence microscopy until several hours after their transcription commences, especially if destabilized versions are utilized to improve reporter dynamics. Second, although cell-wide dispersion of the fluorescence signal can provide useful, collateral information on cell morphology, it can also obscure differences in pathway activity among neighboring cells.shha, shhb, and ihhb are expressed in distinct but overlapping domains, with transcripts initially detected within the dorsal mesoderm shortly after the onset of gastrulation . Hh ligand expression is restricted to the axial mesoderm as convergent extension proceeds, and these morphogens are produced by the developing notochord, medial floor plate, and ventral floor of the brain during somitogenesis. Zebrafish cells respond to Hh proteins through the Patched family of 12-transmembrane receptors (Ptch1 and Ptch2) The ability of zebrafish with Gli-dependent enhanced green fluorescent protein (EGFP) In vivo reporters of Wnt signaling have been enhanced in this manner Targeting fluorescent reporter signals to the nucleus can help overcome these limitations, providing transgenic lines with improved signal-to-noise properties and single-cell resolution. We have generated two Hh pathway reporter lines: one that expresses mCherry functionalized with a nuclear localization sequence (NLS) upon Gli activation and another that carries a destabilized form of the mCherry-NLS reporter. Gli-dependent mCherry fluorescence is clearly evident in these transgenic animals by the one-somite stage (11 hpf), and divergent responses to Hh signaling can be observed in these transgenic lines with single-cell resolution. Genetic and chemical perturbations further confirm that the fluorescent signals in these lines faithfully communicate Hh pathway state, and the reporter is functional in both embryos and adult fish. We anticipate that these transgenic lines will be valuable tools for studying Hh pathway-dependent development and physiology. They could also facilitate the discovery of new signaling components and small-molecule modulators, taking advantage of the zebrafish's amenability to genetic and chemical screens.All experiments involving zebrafish were approved by the Institutional Animal Care and Use Committee at Stanford University (Protocol ID: 10511).t4shhahi1640smotm79adzip1 mutants ts269gli1Tg(-2.4shha:GFP-ABC) zebrafish Wildtype AB, Transgenic fish were created through Tol2-based transgenesis as previously described SHGFPPAVAAQDDGT LPMSCAQESGMDRHPAACASARINV.8xGliBS-Luciferase SDPKKKRKVDPKK KRKVDPKKKRKVGYKKL, a triple tandem repeat of the SV40 large T antigen NLS, was assembled from the following four primers: 5′-GTACAAGTCCGATCCAAAAAAGAAGAGAAAG GTAGATCC-3′, 5′-AAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGG-3′, 5′-GTACCCTACCTTTCTCTTCTTTTTTGGATCTACCTTT-3′, and 5′-CTCTTCTTTTTTGGATCTAC CTTTCTCTTCTTTTTTGGATCGGACTT-3′. The primers were treated with T4 polynucleotide kinase to phosphorylate the 5′ ends, mixed, melted at 95°C for 1 minute, annealed at 65°C for 1 minute, cooled briefly to room temperature, and then ligated into an 8xGliBS-EGFP-Odc1 vector previously digested with BsrGI and dephosphorylated with Antarctic phosphatase (NEB) to yield 8xGliBS-EGFP-NLS-Odc1.cDNA encoding the polypeptide sequence 5′-TTATGCTAGCGACCGATCCTGAGAACTTCAGG-3′ and 5′-TTATAGATCTCTTTGCCAAAATGATGAGACAGC-3′. The amplicon was then digested with NheI/BglII and ligated into NheI/BglII-cut 8xGliBS-EGFP-NLS-Odc1 to yield 8xGliBS-IVS2-EGFP-NLS-Odc1The IVS2 intron was PCR-amplified from pEF1a-IVS2-EGFP-polyA-Tol2 5′-TTATAGATCTCCACCATGGTG AGCAAGGGCGAG-3′ and 5′-TTATTGTACACCTTGTACAGCTCGTCCATGCC-3′. The PCR product was digested with BglII/BsrGI and then ligated into BglII/BsrGI-digested 8xGliBS-IVS2-EGFP-NLS-Odc1 to yield 8xGliBS-IVS2-mCherry-NLS-Odc1.mCherry was PCR amplified from pRSET-mCherry 5′-TCGAGGCTAGCGTCGACGAATTCCTGCAGAAGCTTGATATCGATGGATCCT and 5′-GTACA GGATCCATCGATATCAAGCTTCTGCAGGAATTCGTCGACGCTAGCC-3′ which was then ligated into XhoI/BsrGI-digested pT2KXIG A multiple cloning sequence (MCS) was assembled with the primers: 5′-TAC TCGAGCGAGCTAACTTGTTTATTGCAGCT-3′ and 5′-ATGAATTCCTATCACTTCTTGTACCCT ACCTTTCTCTTCTT-3′. The amplicon was then digested with XhoI/EcoRI and ligated into XhoI/EcoRI-cut MCS-polyA-Tol2 to yield 8xGliBS-IVS2-mCherry-NLS-Odc1-polyA-Tol2.The 8xGliBS-IVS2-mCherry-NLS-Odc1 cassette was PCR-amplified from 8xGliBS-IVS2-mCherry-NLS-Odc1 using the primers: 5′-TACTCGAGCGAGCTAA CTTGTTTATTGCAGCT-3′ and 5′-ATGAATTCCTATCATACCTTTCTCTTCTTTTTTGGATCTAC -3′. The PCR product was then digested with XhoI/EcoRI and ligated into XhoI/EcoRI-cut MCS-polyA-Tol2 to yield 8xGliBS-IVS2-mCherry-NLS-polyA-Tol2.The 8xGliBS-IVS2-mCherry-NLS cassette was PCR-amplified from 8xGliBS-IVS2-mCherry-NLS using the primers: Tol2 transposase mRNA transcribed from pCS-TP Tg(8xGliBS:mCherry-NLS) and Tg(8xGliBS:mCherry-NLS-Odc1) lines in wildtype and mutant backgrounds were used in subsequent studies.The 8xGliBS-IVS2-mCherry-NLS-polyA-Tol2 and 8xGliBS-IVS2-mCherry-NLS-Odc1-polyA-Tol2 plasmids were maxiprepped, treated with proteinase K , subjected to a phenol/chloroform extraction , and then twice again with pure chloroform), and then precipitated with an equal volume of isopropanol after adjusting the solution pH to 4.5 with 0.3 M sodium acetate. Zebrafish zygotes were then co-injected with this RNAse-free plasmid DNA and Adult caudal fins were amputated by first anesthetizing the zebrafish with 0.2 mg/mL tricaine in fish-system water for approximately 5 minutes. The fish were next placed onto a clean paper towel, and their tails were snipped using spring scissors . The fish were then allowed to recover in system water.shha mRNA was transcribed from a pSP64TS-shha plasmid Zebrafish Brightfield and fluorescence images were acquired using either a Leica DM4500B compound microscope equipped with a Retiga SRV camera or a Leica MZFLIII stereomicroscope with a Leica DFC480 camera. GFP and mCherry fluorescence was visualized using GFP and Texas Red filtersets, respectively. For live-imaging studies, zebrafish embryos were manually dechorionated, anesthetized in E3 medium containing 0.05% (w/v) tricaine mesylate, and then placed in agarose wells. Adult fish were similarly anesthetized but imaged on a dark surface.in vivo Hh signaling with high detection sensitivity and single-cell resolution, we sought to generate transgenic zebrafish carrying Gli-dependent, nuclear-localized fluorescent reporters. Hh pathway-driven expression of exogenous genes in cultured cells or live organisms has been previously achieved using a minimal δ-crystallin promoter and eight tandem Gli binding sites derived from the murine Fox2A floor plate enhancer Tol2 mRNA.To visualize Tg(8xGliBS:mCherry-NLS) and Tg(8xGliBS:mCherry-NLS-Odc1) transgenes.Approximately 10–20% of adult zebrafish subjected to this transgenic procedure exhibited germline integration of the Gli-dependent reporter, and these F0 founders  typically gave rise to F1 carriers with a transmission efficiency of 5%. For each transgenic line, three founders were chosen for further analysis, and we identified those yielding progeny with robust, Hh pathway-dependent reporter expression that segregated as a single insertion allele in the F2 generation. Based on these criteria, we maintained one line each for the Tg(8xGliBS:mCherry-NLS-Odc1) larvae. These observations suggest that Hh morphogen-dependent patterning of these neural domains is largely complete by 84 hpf, where its actions within the cerebellum and telecephalon are ongoing. Differences between the two reporter lines could also be discerned at earlier stages upon addition of the Smo antagonist cyclopamine Tg(8xGliBS:mCherry-NLS) zebrafish previously exposed to cyclopamine for several hours, similarly treated Tg(8xGliBS:mCherry-NLS-Odc1) embryos lost mCherry signals in the ventroanterior brain, ventral spinal cord and posterior somites , several hours earlier than in previous Hh reporter lines Tg(8xGliBS:mCherry-NLS-Odc1) embryos (Tg(8xGliBS:mCherry-NLS-Odc1) line.Due to the enhanced temporal resolution afforded by the  embryos . In contTg(8xGliBS:mCherry-NLS-Odc1) reporter by assessing its responsiveness to chemically and genetically induced changes in Hh pathway state. We first treated Tg(8xGliBS:mCherry-NLS-Odc1) zygotes with cyclopamine. Consistent with the resulting Hh loss-of-function phenotypes, including ventral body curvature and U-shaped somites We next sought to confirm the fidelity of the Tg(8xGliBS:mCherry-NLS-Odc1) reporter output in mutant backgrounds with varying degrees of Hh pathway dysregulation, including the loss-of-function mutants t4shhahi1640smots269gli1tm79adzip1 mutants t4shha and ts269gli1 mutants lacked mCherry signals within the myotome but maintained a reduced reporter activity in anterior tissues, and tm79adzip1 mutants upregulated and downregulated Hh reporter expression within the somites and ventral brain, respectively. Taken together, our observations demonstrate that the Gli-dependent nuclear reporters are sensitive, specific indicators of Hh pathway state.As a third method to validate reporter fidelity, we analyzed shha is initially expressed around 30 hours post amputation (hpa) in a subset of basal epidermal cells at distal end of each ray ptch2-positive cells can subsequently be detected at 40 hpa in the epidermis, first overlapping with the distal shha-positive domain and then extending proximally We concluded our studies by investigating the functionality of our Hh pathway reporters in adult zebrafish. Caudal fin regeneration is a convenient model of Hh pathway-dependent physiology; previous studies have established clear domains of Hh ligand production and response during this process shha-producing cells and their ptch2-expressing responders, we crossed Tg(-2.4shha:GFP-ABC)Tg(8xGliBS:mCherry-NLS) zebrafish to obtain progeny carrying both fluorescent reporters. Shha-expressing cells in these transgenic organisms are labeled with GFP, and the corresponding cellular responses can be assessed by mCherry fluorescence. We cut the caudal fins of adult Tg zebrafish and monitored changes in GFP and mCherry reporter expression at 3 days post amputation. As expected, amputation caused a dramatic increase in GFP expression at the distal end of each fin ray  and Tg(8xGliBS:mCherry-NLS-Odc1) fish described here have certain advantages over previous Hh pathway reporter lines, including: (1) higher detection sensitivity that enables real-time observation of Hh signaling by the onset of somitogenesis; and (2) greater cellular resolution that allows pathway activity differences between neighboring cells to be discerned. We anticipate that these transgenic organisms will be valuable tools for studying the Hh pathway-dependent processes that contribute to embryonic development, tissue homeostasis, and tumorigenesis. They could also facilitate the identification of new genetic and chemical modulators of Hh signal transduction through high-content, image-based screens.Taken together, our results demonstrate the utility of nuclear-localized mCherry reporters of Hh pathway activity, particularly when used in conjunction with optically transparent model organisms such as the zebrafish. The Movie S1Real-time imaging of Hh signaling during early somitogenesis. Nuclear mCherry expression in a Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish between 11 and 15 hpf. The midline region between the future sixth and ninth somites was imaged at a rate of 1 frame/minute, and the movie is shown at rate of 30 frames/second. Embryo orientation: dorsal view and anterior up. Field of view: 180 µm×180 µm.(MOV)Click here for additional data file.Movie S2Real-time imaging of slow-twitch muscle precursor migration. Nuclear mCherry expression in a Tg(8xGliBS:mCherry-NLS-Odc1) zebrafish between 13 and 17 hpf. The midline region between the second and seventh somites was imaged at a rate of 1 frame/minute, and the movie is shown at rate of 30 frames/second. Embryo orientation: dorsal view and anterior up. Field of view: 260 µm×260 µm.(MOV)Click here for additional data file.


In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo. The signaling molecule Hedgehog promotes the production and release of the specific phospholipid PI(4)P, which directly interacts with Smoothened, activating it via phosphorylation and accumulation in the cilia. The Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis. A critical step in Hh signal transduction is the inhibition of Smoothened (Smo), an atypical G protein-coupled receptor (GPCR), by the Hh receptor Patched (Ptc). It is a longstanding question how Ptc inhibits Smo and how Hh promotes Smo phosphorylation and activation. It is unlikely that Ptc inhibits Smo by direct interaction. Here, we uncover that phosphatidylinositol 4-phosphate (PI(4)P), a specific phospholipid, directly interacts with Smo through an arginine motif in the Smo C-terminal tail and promotes Smo phosphorylation, activation, and ciliary localization. Ptc also interacts with PI(4)P, which is inhibited by Hh stimulation, indicating that Hh triggers the release of PI(4)P from Ptc. We further uncover that Hh stimulation induces the overall production of PI(4)P, likely by regulating PI(4)P kinase and phosphatase. Finally, in addition to the direct role in regulating Smo phosphorylation, G protein-coupled receptor kinase 2 (Gprk2) facilitates PI(4)P interaction with Smo. This study suggest that PI(4)P acts as a special small molecule shuttling between Ptc and Smo to modulate Hh responses. Drosophila, the Hh signal is transduced through a receptor system at the plasma membrane, which includes the receptor complex Patched-Interference Hh (Ptc-Ihog) and the signal transducer Smo . ,15. 9,15f PI(4)P . We furtectively . To accu,4,5)P3) , we opti,4,5)P3)  induced ,4,5)P3)  stimulatptc-luciferase (ptc-luc) reporter assay to monitor the activity of Hh signaling . Su. Sudpp-leraction , suggesteraction . To furtd PI(4)P  and the d PI(4)P . In addid PI(4)P . SmoRA4 uc assay . Using tto SmoWT . RA4 mutpression , the explls Figs  and S4G.th SmoWT , and thaSmoSD123  and was nt cells . These ding disc  and highmulation . Taken tThe binding position of PI(4)P in Smo is very critical, because fusion of the PH domain from OSBP to either the third intracellular loop (SmoL3PH) or the C-tail (SmoPH) retained PI(4)P with Smo  but compC765-SmoPKA12 (en expression in gprk2 mutant cells (ΔC) abolished its ability to rescue en expression (OSBP) restored this ability P transport protein (PITP) facilitates the interaction between PI(4)P and Smo, since Smo itself does not contain a PH domain. We used RNAi lines from the VDRC to screen a total of 15 typical PH domain-containing PITPs in the fly genome for their ability to modulate Hh phenotypes; inactivation of these proteins by RNAi did not affect Smo accumulation in wing discs, although RNAi of some candidate PITPs modified the wing phenotype of SmoPKA12 . Interesnt cells . Howeverpression , whereas ability . This isin vitro , indicatΔC and found that the expression of Gprk2 elevated the levels of both Smo and PI(4)P (ΔC had no effect (OSBP (Gprk2 resulted in increased PI(4)P and Smo accumulation P , whereaso effect . Similarct  interacted directly with PI(4)P; mutation of arginine (PHGprk2RA) or phenylalanine (PHGprk2FA) abolished this interaction (OSBP interaction with Smo (Gprk2 interaction with Myc-SmoWT was increased by Hh and PI(4)P treatments in cultured S2 cells P-induced Smo phosphorylation detected by the anti-SmoP antibody . RNAi ta did not , suggest did not . Moreoveeraction . Finallywith Smo , the PHGS2 cells . Taken tS2 cells  and in cS2 cells , our finWT), HA-tagged Ptc lacking its SSD domain (HA-PtcΔSSD), and HA-tagged SSD domain (HA-SSD). We transfected S2 cells with these constructs and evaluated the ability of each to interact with PI(4)P. When expressed in S2 cells, all proteins were expressed at low levels, detected only after immunoprecipitation (WT and HA-SSD strongly bound PI(4)P, whereas HA-PtcΔSSD did not bind (The finding that expression of the PH domain from OSBP accumulated PI(4)P  prompted2 or PIP3 phospholipids (ptc or knockdown of Ptc by RNAi increased PI(4)P levels in the wing disc P P in wing discs.To further determine whether the SSD domain from Ptc directly interacts with PI(4)P, we used the solid phase lipid-binding assay similar to that used for detecting Smo binding. We found that the SSD fragment protein purified from bacteria strongly associated with PI(4)P, but not with PIPholipids , suggestholipids  suggestsholipids ; these ming disc , similarf PI(4)P , which iIn addition to promoting the production of PI(4)P, Hh may also regulate the pools of PI(4)P between Smo and Ptc. To test this hypothesis, we purified Smo and Ptc proteins from S2 cells treated with Hh-conditioned medium or control medium and accessed the protein interaction with PI(4)P. As shown in It would be interesting to understand how Hh regulates PI(4)P. However, we found that Hh treatment did not significantly change the mRNA levels of Stt4 and Sac1 . In addiGli-luc reporter , which mimics mSmo phosphorylation by GRK2 and CK1 FRT42D/hs-GFP FRT42D. Inp54p  and IpgD  were subcloned into HA-UAST backbones. A phosphatase-dead version of Inp54p with a D281A mutation (Inp54pD281A) was generated by site-directed mutagenesis. GST-SmoWT containing aa 656–755 has been described [RA12, SmoRA34, and SmoRA4 were generated by PCR on the backbone of GST-SmoWT. GST-PHGprk2, GST-PHGprk2RA, and GST-PHGprk2FA were constructed by the same approach. HA-PtcWT, HA-PtcΔSSD, and HA-SSD were generated by PCR with Ptc or its fragments inserted into HA-UAST backbones, and transgenic lines were generated using the VK5 attP locus. His-SSD was generated by in-frame fusion of Ptc SSD domain (aa421-589) to pET30a backbone. Myc-tagged mSmoWT, mSmoRA mutants, and mSmoSD (previous mSmoSD0-5) have been described [WT and mSmoRA34 were generated by subcloning each cDNA into pEGFP-N1 backbone. Flag-tagged Stt4 (Flag-Stt4) was generated by combining seven RT-PCR fragments with unique cloning sites into the SK+ backbone and, finally, subcloning into Flag-UAST backbone. HA-tagged Sac1 (HA-Sac1) was constructed by inserting the RT-PCR fragment into the HA-UAST backbone. pGE-mPtch1-Myc and pGE-mPtch2-Myc were generated by subclone of the open reading frame from pEGFP-mPtch1 and pEGFP-mPtch2 (Gift from Dr. Chi-Chung Hui), respectively. MS1096-Gal4, ap-Gal4, C765-Gal4, and Gprk2 deletion mutants have been described [prd-Gal4 (#1947) were obtained from VDRC or Bloomington Stock Center (BSC). Generation of Gprk2 RNAi lines have been described [Generation of the Myc-Smoescribed . Myc-Smoescribed ,20. Myc-promoter  was insebackbone . RFP-PHOescribed . Flag-taescribed ,18. Stt47 S2 cells (or 4 × 106 NIH3T3 cells) from a 100 mm dish were harvested and washed once with ice-cold PBS and suspended in 340 μL H2O and 1500 μL quench mix with 25 ng of 17:0–20:4 PI(4)P  in 10 μL methanol as the internal standard. Lipids were extracted with 1450 μL CHCl3 and 340 μL 2 M HCl and subsequently derivatized with trimethylsilyl diazomethane (Sigma), as previously described [PtdIns lipid extraction and quantification by mass spectrometry were carried out as previously described . Brieflyescribed . After wescribed . PIP con2 monoclonal antibodies (Echelon Biosciences) were added for 1 h, followed by goat anti-Mouse IgG-HRP (Jackson ImmunoResearch) for 30 min, with 3 PBS washes after each inculation. Finally, chemiluminescence substrate  was added to the microplate, and luminescence intensity was determined by a luminometer.For PI(4)P quantification by ELISA assay, similar PtdIns lipid extraction procedures were used without adding the PI(4)P internal control. After washing and drying with nitrogen stream, lipid extracts were dissolved in ethanol and loaded into a microplate, dried under a vacuum, and incubated with 2% BSA in PBS at room temperature for 30 min. Mouse anti-PI(4)P or anti-PIPGST-Smo fusion proteins expressed in bacteria were pooled by GST beads , then eluted with elution buffer (10mM Glutathione pH 8.0 in 50 mM Tris) at 4°C overnight. Myc-tagged Smo proteins expressed in S2 cells were immunoprecipitated with anti-Myc antibody combined with beads of protein A ultralink resin, followed by two sequential elutions with Myc peptide . The eluted purified GST-fusion proteins and Myc-tagged Smo proteins were concentrated by the Centrifugal filter units (Millipore) and incubated with lipid-dotted strips according to manufacturer’s instruction (Echelon Biosciences), followed by western blot with the anti-GST (Santa Cruz), anti-Myc (Santa Cruz), or anti-His (Millipore) antibodies.Gprk2, GST-PHGprk2RA, and GST-PHGprk2FA proteins were expressed in bacteria and purified using the protocol employed for GST-Smo purification. The purified and concentrated GST-fusion proteins or epitope-tagged proteins were incubated with PI(4)P beads (Echelon Biosciences) with wash/binding buffer , and subjected to western blot to detect PI(4)P bound proteins.For the PI(4)P beads pull-down experiments, HA-tagged Gprk2 proteins were expressed in S2 cells, immunoprecipitated with mouse anti-HA antibody , eluted with HA peptide , and concentrated by the Centrifugal filter units (Millipore). GST-PH4Cl for 15 min, followed by incubation with the anti-PI(4)P antibody at 4°C overnight. For Drosophila embryo primary antibody staining, stage 11 fly embryos with specific genotypes were dechorionated, fixed with Heptane solution, and immunostained with similar procedures. To examine mSmo ciliary localization, NIH3T3 cells were transfected with mSmo-GFP variants, treated with Shh or PI(4)P, and immunostained for mSmo localization in the cilium. Primary antibodies in this study were: mouse anti-Myc , anti-Flag , anti-SmoN (DSHB), anti-En (DSHB), and anti-PI(4)P ; rabbit anti-β-Gal (Cappel), anti-GFP (Clontech), anti-Acetylated tubulin (Sigma), anti-PS1 [Wing discs from third instar larvae were dissected in PBS and then fixed with 4% formaldehyde in PBS for 20 min. After permeabilization with 1% PBST, discs were incubated with primary antibodies for 3 h and appropriate secondary antibodies for 1 h, and washed three times with PBST after each incubation. Affinity-purified secondary antibodies (Jackson ImmunoResearch) for multiple labeling were used. It was a challenge for disc staining with the mouse anti-PI(4)P antibody. We have adopted/modified a critical method for PI(4)P immunostaining from previous publications ,65. Discanti-PS1 , and ratS1 Data(XLSX)Click here for additional data file.S1 FigC765-Gal4 do not exhibit phenotypes. (D) A wing from flies expressing SmoPKA12 by C765-Gal4. Arrow indicates a reproducible wing phenotype with partial fusion between Vein 3 and Vein 4, a phenotype indicating the partial loss of Hh signaling activity. (E) A wing from flies coexpressing SmoPKA12 with Stt4RNAi by C765-Gal4. Arrowheads indicate enhanced fusion between Vein 3 and Vein 4. (F) A wing from flies coexpressing SmoPKA12 with Sac1RNAi by C765-Gal4. Arrow indicates the weakened fusion between Vein 3 and Vein 4. (G) Cuticle prep with a WT larva shows the well-organized abdominal cuticles. Arrows indicate the cuticle lines. (H) Larva with prd-Gal4 driven Sac1 RNAi shows cuticle lines similar to WT. Arrows indicate the cuticle lines. (I) Larva with prd-Gal4 driven Hh RNAi shows the loss of cuticles. Arrow indicates where the cuticles are supposed to be located. (J) Larva with prd-Gal4 driven Hh RNAi combined with Sac1 RNAi shows partially recovered cuticles. Arrows indicate the cuticle lines rescued by Sac RNAi.(A) A WT adult wing showing interveins 1–5. (B–C) Wings from flies expressing Stt4RNAi (B) or Sac1RNAi (C) by (TIF)Click here for additional data file.S2 Fig-3P, a constitutively active form of Ci, was immunostained for PI(4)P and HA. Arrow indicates the elevated accumulation of PI(4)P in the wing disc. Shown on the right is an enlarged image indicating the accumulation of PI(4)P. (E) A wing disc from flies expressing Myc-SmoSD123, a constitutively active form of Smo, was immunostained for PI(4)P and Myc. Arrow indicates the elevated accumulation of PI(4)P in the wing disc.(A–C) WT stage 11 embryos were stained for En, Smo, PI(4)P, and Ci, respectively. En staining indicates the Hh expression domain. The domain of Smo accumulation correlates the domain of PI(4)P accumulation. Arrows in C indicate PI(4)P accumulation. (D) A wing disc from flies expressing HA-Ci(TIF)Click here for additional data file.S3 Figptc-luc activity. PI(4)P treatment for 6 h induces the peak Hh signaling activity. Right panel, to monitor phospholipids delivery, ELISA assay with the anti-PI(4)P or anti-PIP2 antibody was used. *P < 0.01. † no statistical difference detected. (B) S2 cells in 6-well plates were cotransfected with Myc-SmoWT and different amounts of Hh cDNA and assayed for ptc-luc activity. Empty UAST vector was used as a transfection control, and carrier 3 was used as a treatment control. PI(4)P consistently increased the low and high Hh signaling activity. (C) In left panel, the efficiency of RNAi knockdown was monitored by Real-Time PCR. * p < 0.05 versus control (GFP dsRNA). In middle and right panels, RNAi efficiency was also confirmed by knocking down the expression of the transfected constructs in S2 cells. (D) The efficiency of GFP RNAi. * p < 0.05 versus control. GFP RNAi efficiency was monitored by either Real-Time PCR (left panel) or western blot with the anti-GFP antibody (right panel). The underlying data of panels A–D can be found in (A) Left panel, S2 cells were treated with PI(4)P at the indicated time points and assayed for (TIF)Click here for additional data file.S4 FigWT was immunostained for Ci and Myc. An enlarged view in B” shows the localization of Myc-Smo. Arrow indicates the elevated Ci staining induced by the expression of Myc-SmoWT. (C–C”) A wing disc expressing Myc-SmoPH was immunostained for Ci and Myc. An enlarged view in C” shows the localization of Myc-SmoPH, which had no obvious difference compared to Myc staining in B”. Arrow indicates the less elevated Ci staining compared to B. (D–D”) A wing disc from flies expressing Myc-SmoL3PH was immunostained for Ci and Myc. An enlarged view in D” shows the localization of Myc-Smo. Arrow indicates Smo accumulation in punctate dots, compared to B” and C”. Of note, Myc-SmoL3PH does not induce any Ci elevation in D. (E) S2 cells were transfected with the indicated constructs, treated with either carrier 3 or carrier 3 plus PI(4)P, and assayed for the ptc-luc reporter activity. Empty UAST vector served as transfection control. SmoPH and SmoL3PH have little to no ptc-luc reporter activity. PI(4)P enhances the activity of SmoWT and SmoSD123 but not the activity of SmoRA4 and SmnoSD123RA4. *p < 0.05 versus SmoWT treated with carrier. † p < 0.05 versus SmoWT treated with PI(4)P. ** p < 0.01 versus SmoSD123 treated with carrier. †† p < 0.01 versus SmoSD123 treated with PI(4)P. *** p < 0.01 versus SmoWT treated with carrier. ††† p < 0.01 versus SmoWT treated with PI(4)P. **** p < 0.01 versus SmoWT treated with carrier. †††† P < 0.01 versus SmoWT treated with PI(4)P. (F) FRET efficiency from the indicated WT or mutant Smo with CFP or YFP tagged to their C-terminus. S2 cells were transfected with the indicated constructs, treated with Hh-conditioned medium or control medium, carrier 3 or carrier 3 plus PI(4)P, and assayed for FRET. SmoRA4 have significantly reduced FRET value compared to SmoWT (* p < 0.01 versus SmoWT), and have much less responsiveness to Hh and PI(4)P stimulation P). (G) Image enlarged from (A) Illustrations of SmoPH and SmoL3PH are shown in the left panel. S2 cells were transfected with Myc-SmoPH or Myc-SmoL3PH and immunoprecipitated with the anti-Myc antibody to enrich Smo protein. Purified Smo proteins were incubated with PI(4)P beads to examine the interaction between PI(4)P and Myc-Smo (shown in the right panel). The high mobility shift of SmoL3PH, compared to SmoPH, was due to the additional PH domain inserted. (B–B”) A wing disc from flies expressing Myc-Smo(TIF)Click here for additional data file.S5 Figap-Gal4 was stained for PI(4)P and Ci. GFP indicates RNAi domain of the dorsal compartment. Arrow indicates the decreased accumulation of PI(4)P caused by knockdown of Gprk2. (B) A wing disc expressing RFP-PHGprk2 by MS1096-Gal4 was stained for PI(4)P and Ci. Overexpression of the PH domain of Gprk2 increases the level of PI(4)P (arrow). (C) A wing disc expressing RFP-PHGprk2 by MS1096-Gal4 was stained for Smo. Arrow indicates the increased accumulation of Smo by the overexpression of the PH domain of Gprk2.(A) A wing disc expressing Gprk2RNAi by (TIF)Click here for additional data file.S6 FigMS1096-Gal4 were stained for Flag and GFP. A/P boundary was defined by Ci staining. There were no Flag and GFP staining differences between A and P compartments, indicating Hh does not regulate the stability of the protein. The underlying data of panel A can be found in (A) The levels of Stt4 and Sac1 mRNA were monitored by Real-Time PCR when S2 cells were treated with 60% Hh-conditioned medium or 60% conditioned medium plus Hh cDNA transfection (to achieve the highest levels of Hh activity). No statistical differences detected. (B–C) Wing discs expressing Flag-Stt4 or GFP-Sac1 by (TIF)Click here for additional data file.S7 Figp < 0.001 versus control (first column); ** no statistical difference detected versus control (first column); † no statistical differences detected versus cyclopamine treatment alone (fourth column). Right panel, Ptc1 RNAi efficiency in NIH3T3 cells was shown by western blot with the anti-Myc-antibody to detect the transfected Myc-Ptc1, and shRNA1 was used in this study. (B) NIH3T3 cells transfected with YFP-Ptc1 or GFP-Ptc2 were immunostained to show the expression of Acetylated (Ac)-tubulin , YFP , and GFP . Images in the inserts are enlarged views with shifted overlays to show the ciliary localization of Ptc1 or Ptc2. About 100 ciliated cells were counted for each set. Quantification of ciliary localization of Ptc1 or Ptc2 is shown in (A) Left panel, NIH3T3 cells were treated with the indicated combinations of Shh, Ptc1 dsRNA, and cyclopamine, a potent Smo antagonist. PI(4)P was detected by ELISA. * (TIF)Click here for additional data file.S1 MethodsWe provide additional information for the generation of various constructs, transgenic lines, and mutants. We also describe the procedures for GST fusion protein purification and in vitro kinase assay, luciferase assay, and cuticle preparation.(DOCX)Click here for additional data file.S1 TableThe results and phenotypes from knockdown of the indicated PITP by RNAi using different Gal4 lines are shown in the table. Cell culture assays for Smo stability and phosphorylation are also shown with the primers used for the synthesis of individual dsRNA.(DOCX)Click here for additional data file.


Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis . Rec8-co  •Rec8-mediated chromosome cohesion is established in fetal oocytes•Bivalent cohesion is maintained without detectable turnover in arrested oocytes•Irreversible cohesin loss underlies age-related chromosome missegregation How chromosome cohesion is maintained in female germ cells arrested for months or decades is poorly understood. Burkhardt et al. show that cohesion is built in fetal oocytes and after birth is maintained without detectable renewal for months. This implies that the oocyte’s inability to renew cohesion contributes to maternal age-related trisomies. The frequency of clinically recognized trisomic pregnancies increases with maternal age . Most anRec8TEV/TEV oocytes converts 100% of bivalents to chromatids. To test whether new cohesion is built, Rec8-Myc that is not cleavable by TEV protease is induced in addition to endogenous Rec8TEV that establishes cohesion during DNA replication Stop/Rec8-Myc. Cre recombinase deletes the Stop cassette and activates Rec8-Myc transgene expression. Deletion of the Stop cassette using (Tg)Sox2-Cre in the early embryo, before primordial germ cell specification, rescues bivalent cohesion in mature oocytes [The entrapment of sister DNA molecules by cohesin complexes can be measured indirectly and directly using biochemical and cell biological approaches , 23, 24.lication B. If Rec oocytes . TherefoRec8TEV/TEV oocytes expressing wild-type but not mutant TEV protease (TEVmut), demonstrating that bivalent cohesion is intact without TEV cleavage  stage oocytes isolated from sexually mature females are cultured in 3-isobutyl-1-methylxanthine (IBMX)-containing medium to inhibit germinal vesicle breakdown (GVBD). GV oocytes are microinjected with mRNA encoding H2B-mCherry to visualize chromosomes, TEV protease and another marker such as CenpB-EGFP that localizes to kinetochores. After expression of the mRNA constructs, oocytes are released into IBMX-free medium to resume meiosis and followed by confocal time-lapse microscopy. Bivalents are converted to chromatids within 3–4 hr in cleavage C. TherefRec8-Myc transgene in arrested oocytes shortly after birth. To achieve this, we selected the widely used deleter strain (Tg)Gdf9-iCre in which Cre expression is controlled by the promoter of growth differentiation factor 9 (Gdf9) [(Tg)Gdf9-iCre is thought to delete conditional alleles in oocytes shortly after birth, i.e., days after DNA replication and meiotic recombination  , 33, 34.bination A.Rec8-Myc transgene activation occurs after meiotic DNA replication, we carried out due diligence to confirm that Gdf9-iCre does not delete during DNA replication. Using a conditional LacZ reporter strain (Rosa26-LacZ) [Rosa26-LacZ (Tg)Gdf9-iCre ovaries on embryonic day E13.5 when oocytes enter meiosis. Unexpectedly, seven out of ten fetal ovaries contained X-gal positive cells ((Tg)Stop/Rec8-Myc (Tg)Gdf9-iCre females Stop/Rec8-Myc (Tg)Gdf9-iCre females resulted in variable rescue efficiencies (Gdf9-iCre for cell cycle phase-specific deletion studies.Since it is important that 26-LacZ) , we analve cells B, sugges females C and 2D. embryos D. Overalficiency , but theciencies A and S2BRec8-Myc transgene after meiotic DNA replication might rely on Cre recombinase controlled by a promoter driving expression of a protein required for recombination. The topoisomerase-like enzyme Spo11 generates DNA double-strand breaks that initiate recombination [Spo11-Cre. If Spo11-Cre deletes after DNA replication and before or during recombination, then this system will test whether cohesion is built at all after DNA replication. It would not be possible to distinguish whether cohesion is generated during recombination or the dictyate-stage arrest.A second approach to activate the bination . TherefoSpo11-Cre, we used Rosa26-LacZ and (Tg)Stop/Rec8-Myc mice Stop/Rec8-Myc Spo11-Cre females Gdf9-iCre that are thought to delete in arrested oocytes before growth, we chose to directly control the timing of Cre-mediated deletion by injection of 4-hydroxytamoxifen (4-OHT). Specifically, the germ cell-specific Dppa3 promoter drives expression of Cre fused to mouse estrogen receptors (MERCreMER) and a PEST degradation motif in (Tg)Dppa3-MCM-P mice [To demonstrate whether arrested oocytes maintain cohesion with or without turnover, it is important to activate M-P mice  (Table SM-P mice .Rosa26-LacZ (Tg)Dppa3-MCM-P females resulted in no X-gal positive oocytes in ovary sections, consistent with the negligible background reported by others (Rosa26-LacZ (Tg)Dppa3-MCM-P females resulted in ∼25% X-gal positive oocytes, indicating that Cre-mediated deletion had occurred (Stop cassette in Rec8-Myc will be identified by single-cell PCR genotyping after the cohesion rescue assay ((Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P females are fertile for up to 12 litters, and 28% of offspring inherited (Tg)Rec8-Myc . Therefore, 4-OHT and Cre-mediated deletion do not grossly affect oocyte maturation and female fertility.The challenges with this approach are a risk of deletion without 4-OHT, inefficient deletion with 4-OHT, and effects of 4-OHT on fertility. Since background deletion could result in a false positive cohesion rescue, we first tested whether there is any deletion without 4-OHT. Reassuringly, vehicle injection into y others . On the occurred . Since tue assay . Lastly,Rec8TEV/TEV(Tg)Stop/Rec8-Myc females mated to Rec8TEV/TEV(Tg)Dppa3-MCM-P males were injected with 4-OHT on embryonic day E10.5 to activate Rec8-Myc in replicating oocytes of female embryos (F1 generation) (Rec8TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P F1 females were sacrificed at 2 months for oocyte isolation (Rec8TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P F1 female (Rec8TEV/TEV(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P F1 females with a deleted Stop cassette  A. Rec8TEsolation B. If Rec1 female C and 3D.cassette E and 3F.(Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P adult females with vehicle or 4-OHT and performed RT-PCR on whole ovaries. (Tg)Rec8-Myc ovaries served as a positive control to identify Rec8-Myc mRNA; the presence of oocytes was confirmed using the germ cell-specific transcripts Nobox and Smc1β. Rec8-Myc was expressed specifically in 4-OHT- and not in vehicle-injected (Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P ovaries Stop/Rec8-Myc oocytes, little but detectable Rec8-Myc localized to chromosomes from oocytes isolated from (Tg)Stop/Rec8-Myc (Tg)Dppa3-MCM-P females injected with 4-OHT Stop/Rec8-Myc (Tg)Dppa3-MCM-P adult females were injected with 4-OHT to activate Rec8-Myc in oocytes, and the cohesion rescue assay was performed 2 or 4 months after activation ((Tg)Dppa3-MCM-P activation, most likely during DNA replication. Since genetic tools like Spo11-Cre cannot distinguish temporally between meiotic DNA replication and homologous recombination (In the key experiment, we asked whether cohesion is maintained with or without turnover in oocytes arrested for months at the dictyate stage of prophase I. tivation C. Followtivation . Importativation  and S2. Overall our results show that sister chromatid cohesion mediated by Rec8-containing cohesin complexes is established in fetal oocytes and maintained without detectable turnover after birth, both during the prolonged dictyate-stage arrest and the weeks of oocyte growth . To fullRec8TEV/+ oocytes [BAC, and we have investigated the mechanism of cohesion maintenance relevant to the wild-type.The advantage of the TEV cleavage assay of cohesin combined with an inducible transgenic rescue construct is that cohesion of sister chromatids is revealed in live cells. At the same time, it is challenging to empirically determine the sensitivity of the assay toward newly built cohesion. Certainly 50% turnover is robustly detected as bivalents remain intact following TEV protease expression in  oocytes . Given t oocytes , it is lThe discovery that bivalent cohesion is established predominantly, if not exclusively, in fetal oocytes has important implications for aging oocytes with increasing chromosome segregation errors , 20, 21.For material and methods, see Conceptualization, K.T.-K., S.B., and M.B.; Methodology, K.T.-K., S.B., M.B., and S.A.W.; Formal Analysis, S.B. and M.B.; Investigation, K.T.-K., S.B., M.B., and A.S.; Resources, J.G., P.E.C., M.S., and T.H.; Writing – Original Draft, K.T.-K.; Writing – Review & Editing, K.T.-K., S.B., and M.B.; Visualization, S.B.; Supervision, K.T.-K.; Project Administration, K.T.-K.; Funding Acquisition, K.T.-K.


These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment  The mucosa-associated invariant T (MAIT) cells recognize antigens that are byproducts of the riboflavin biosynthesis pathway shared by many microbes. These antigens are presented by the MHC class I-like MR1 molecules and trigger rapid activation of MAIT cells in an innate-like fashion with deployment of effector mechanisms including cytokine production and cytolysis. Here, we investigated the MAIT cell response to bacteria in humans infected with HIV-1, and possible means to restore functionality to these cells. MAIT cell dysfunction in HIV-infected patients included an inability to express components of the cytolytic effector machinery. Impairment of the MAIT cell population involved the loss of expression of the transcription factors T-bet and Eomes. Interestingly, IL-7 had strong effects on MAIT cells, including the antigen-independent arming of cytolytic function and enhanced sensitivity for low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively associated with the size of the MAIT cell population, and IL-7 could rescue their function. These findings indicate that MAIT cell impairment in HIV-1 infection is broad-based, includes loss of critical transcription factors, and loss of cytolytic function. Furthermore, the data support the notion that IL-7 is a strong candidate for immunotherapy in diseases associated with MAIT cell loss. In. In11]. PBMCs were isolated from peripheral blood by Ficoll-Hypaque density gradient centrifugation (Pfizer-Pharmacia or Axis-Shield), and either allowed to rest overnight in complete medium, or cryopreserved in liquid nitrogen until required.2 in RPMI medium supplemented with 10% fetal calf serum and 50 μg/ml gentamicin (Gibco) (RF10 medium) prior to functional assay. MAIT cell functions were determined in vitro using a paraformaldehyde (PFA)-fixed E. coli stimulation  in the presence of 1.25 μg/ml anti-CD28 mAb (BD Biosciences) [E. coli in the presence of anti-CD28 for six days as described [PBMCs were cultured in 10 ng/ml recombinant human IL-7 (R&D Systems), a combination of IL-12 and IL-18  for 24–48 h, as indicated or left untreated at 37°C and 5% COciences) . PBMCs w+ T cells were purified from freshly isolated PBMCs using Vα7.2-APC antibody, followed by anti-APC microbeads (Miltenyi Biotec) and positive selection using MACS Cell Separation (Miltenyi Biotec). The purity of enriched Vα7.2+ T cells was typically >95%, with minimum MAIT cell purity of 90%. Vα7.2+ T cells were cultured in RF10 medium supplemented with 25 ng/ml recombinant human IL-7 or in RF10 medium alone for 72 h. Human 293T cells stably transfected with human MR1  [E. coli at an MOI of 10 for three hours, followed by the addition of anti-MR1 mAb  or IgG2a isotype control  for 60 min prior to the addition of Vα7.2+ T effector cells at the indicated effector to target (MAIT:293T-hMR1) ratio. The MAIT:293T-hMR1 ratio was adjusted to take into accounts the overall purity of MAIT cells within the Vα7.2+ T cell population as the contaminating Vα7.2+CD161- T cells did not mediate cytotoxicity. Target cell apoptosis was detected through the fluorescent inhibitor of caspases (FLICA) flow cytometry-based methodology. Briefly, the FLICA reagent  was added at a final concentration of 0.2% (v/v) to the MAIT-293T-hMR1 cell culture at the beginning of the assay, and anti-CD107a PECy7 at a total concentration of 0.4 μg/ml was also added to detect MAIT cell degranulation. After 24 h of culture, cells were harvested and stained to simultaneously detect MAIT cell cytotoxicity and 293T-hMR1 cell death.Vα7.2 Hansen)  were incAnti-CD3 Alexa Fluor (AF)700 (clone UCHT1), anti-CD4 APC-H7 (clone SK3), anti-CD38 PECy7 (clone HIT2), anti-CD161 PECy5 (clone DX12), anti-GrzB AF700 (clone GB11), anti-HLA-DR APC-H7 (clone L243), anti-RORγt PE (clone Q21-559), anti-TNF PECy7 (clone MAb11) were from BD Biosciences; anti-CD3 Brilliant Violet (BV)510 and BV785 (clone OKT3), anti-CD4 BV711 (clone OKT4), anti-CD8 BV570 (clone RPA-T8), anti-CD57 Pacific Blue, anti-CD107a PECy7 (clone H4A3), anti-CD127 BV650 (clone A019D5), anti-CD161 BV605 (clone HP-3G10), anti-granulysin PE (clone DH2), anti-granzyme (Grz)A AF700 (clone CB9), anti-GrzB FITC (clone GB11), anti-IFNγ BV785 (clone 4S.B3), anti-IL-17A BV421 and BV711 (clone BL168), anti-Ki67 BV421 (clone Ki-67), anti-perforin BV421 (clone B-D48), anti-T-bet BV605 and BV711 (clone 4B10), and anti-Vα7.2 APC, PE, and PECy7 (clone 3C10) were from Biolegend; anti-Eomes FITC (clone WD1928), and anti-Helios eFluor 450 (clone 22F6) were from Ebioscience; anti-CD8 Q-dot 655 (clone 3B5), and live/dead aqua and near infrared fixable cell stain were from Life Technologies; anti-PLZF APC (clone 6318100), and anti-TIM-3 AF488 (clone 344823) were from R&D systems.Cell surface staining was performed using directly conjugated antibodies and fixed in Cytofix/Cytoperm or in Transcription Factor Fixation/Permeabilization buffer (both from BD Biosciences) as appropriate. Intracellular staining was performed using the relevant mAbs in Perm/Wash or Transcription Factor Perm/Wash buffer as appropriate (both from BD Biosciences). Samples were acquired on an LSRFortessa 18-colour flow cytometer (BD Biosciences) equipped with 405, 488, 561 and 639 nm lasers. Single-stained polystyrene beads (BD Biosciences) were used for compensation purposes. Software-based compensation was performed using the compensation platform in FlowJo software version 9.6 (Tree Star).Circulating IL-7 was detected in plasma (diluted 1:4) using the Quantikine HS Human IL-7 Assay , according to the manufacturer’s instructions. Each sample was assayed in duplicate.Significant differences in independent samples were assessed using Fisher’s exact test for categorical variables. Continuous variables were first assessed for normality, and differences in independent samples were assessed using t-test or Mann-Whitney test for continuous variables as appropriate. The Wilcoxon signed rank test or paired t-test as appropriate was used to determine significance between paired samples. The Friedman test followed by Dunn’s post-hoc test was used to detect differences across multiple, paired samples. Correlations were evaluated using Spearman’s rank correlation. Statistical analyses were performed on raw data using Prism version 6.0f (GraphPad), and two-sided p-values < 0.05 were considered significant.S1 Fig+ T cell population sufficiently identifies the MAIT cell population. Freshly isolated PBMCs from 10 healthy individuals were stained for the transcription factors PLZF and RORγt. The co-expression patterns for these transcription factors, PLZF+ RORγt+ (gate no. 1), PLZF- RORγt+ (gate no. 2), PLZF+ RORγt- (gate no. 3) were determined in total T cells, CD8+ T cells, and CD4- CD8- double-negative (DN) T cells. MAIT cells were identified from these sub-populations by their Vα7.2 TCR and high levels of CD161 co-expression. Box and whisker plot shows median, IQR and the 10th to the 90th percentile. Significance across multiple paired samples was determined by the Friedman test followed by Dunn’s post-hoc test. Representative FACS plots from a single individual are shown.(A) Most of MAIT cell cytolytic proteins expressed at steady state are co-expressed with perforin (Prf). FACS plots representative of data from 20 healthy individuals are shown. (B) PLZF and RORγt co-expression in the CD8(PDF)Click here for additional data file.S2 FigE. coli (MOI 10) (D), before staining for cytolytic proteins and IFNγ. (B) To determine the MR1-dependency of IL-7 arming of MAIT cell cytotoxicity, freshly isolated PBMCs were incubated with 10 ng/ml IL-7 in the presence of anti-MR1 or IgG2a isotype control for 24 h. Cells were then harvested and stained for cytolytic proteins and IFNγ. Representative FACS plots from two independent donors are shown. Error bars represent mean and standard deviation.Freshly isolated PBMCs from three healthy donors were incubated with a range of IL-7 concentrations as indicated or left untreated for 24 h. Cells were then cultured for another 24 h in the absence , or presence of PFA-fixed (PDF)Click here for additional data file.S3 FigE. coli stimulation (MOI 10)  before staining for MAIT cell cytolytic proteins and IFNγ. (C) MAIT cell frequency following E. coli stimulation in the presence or absence of IL-7, or in a combination of IL-12 and IL-18, was determined in three healthy individuals (left panel) or in eight healthy individuals (right panel). The Friedman test followed by Dunn’s post-hoc test was used to determine significance across multiple, paired samples. Error bars represent median and IQR, and box and whisker plot shows median, IQR and the 10th to the 90th percentile. (D) Spearman rank correlation between the capacity of healthy donor (n = 18) MAIT cells to upregulate GrzB and degranulate following a 24 h co-culture with E. coli-pulsed 293T-hMR1 cells, and the 293T-hMR1 cell death.(A) Freshly isolated PBMCs from three healthy donors were incubated with 10 ng/ml of IL-7, a combination of IL-12 and IL-18 , or left untreated for 48 h. Cells were further cultured for the next 24 h in the absence or presence of PFA-fixed (PDF)Click here for additional data file.S4 Figdim Eomeshi and T-betneg Eomesneg MAIT cells from nine HIV-1 infected ART-untreated patients. (B) PBMCs from seven healthy controls were incubated with 10 ng/ml of IL-7 for 48 h and then stained for transcription factor expression as described in (A) The levels of CD127 expression in T-bet(PDF)Click here for additional data file.S5 Fig+ CD161- T cells from 20 healthy controls and 31 untreated HIV-infected patients. (B) The expression of CD38, HLA-DR, CD57, and TIM-3 was then determined on the MAIT cell population from these individuals. Significance was determined using the Mann-Whitney test. Box and whisker plots show median, IQR and the 10th to the 90th percentile. (C) Spearman’s rank correlation between CD38hi-expressing MAIT cells and the frequency of MAIT cells in the peripheral blood of 31 untreated HIV-infected patients.(A) The frequency of MAIT cells and Vα7.2(PDF)Click here for additional data file.S6 FigRelationships between plasma IL-7 levels and CD4 counts (A), plasma viral loads (B), as well as with the MAIT cell activation markers CD38 (C), HLA-DR (D), CD57 (E), and TIM-3 (F) was assessed using Spearman’s rank correlation in 31 ART-untreated HIV-infected patients.(PDF)Click here for additional data file.


C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth.Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in  C. elegans to investigate how new membrane and dendrite proteins are trafficked. The PVD neuron is easy to visualize and has remarkably long and widely branched dendrites positioned along the skin of the worm, which transmits information about harsh touch and cold temperature to the nervous system. We have discovered that a key organizer of vesicle trafficking, the RAB-10 protein, localizes to membrane vesicles and is required to traffic these vesicles that contain plasma membrane and dendrite proteins to the growing PVD dendrite. Further, our work revealed that a complex of proteins, termed the exocyst, that helps fuse membrane vesicles at the plasma membrane, localizes with RAB-10 and is required for dendrite branching. Together, our work has revealed a novel mechanism for how neurons build dendrites that could be used to help repair damaged neurons in human diseases and during aging.Dendrites are cellular extensions from neurons that gather information from other neurons or cues from the external environment to convey to the nervous system of an organism. Dendrites are often extensively branched, raising the question of how neurons supply plasma membrane and dendrite specific proteins from the source of synthesis inside the cell to developing dendrites. We have examined membrane trafficking in the PVD neuron in the nematode worm  Drosophila, loss-of-function mutations in genes encoding the small GTPases Rab1 and Sar1, which are key regulators of ER-to-Golgi vesicular transport . In . In rab-cycling) –33, had ehbp-1, however, had normal PVD dendritic arbors  fully rescued the dendritic arborization defect in exoc-8 (ok2523) mutant worms, indicating that the exocyst complex functions in the PVD to promote dendritic arborization )—a strain where both copies of the endogenous sec-5 genes are tagged with zf1  during PVD dendrite morphogenesis.Homozygous mutants of two other exocyst components, e defect . To testwith zf1  37]. Co. Cosec-5with zf1 . In someendrites . Controlrphology . We concrab-10, exoc-8 and sec-8 mutants might in part be due to the failure of docking of post-Golgi vesicles. To test this, we built fluorescent reporters for the dendritic membrane proteins, DMA-1 and HPO-30, transmembrane proteins expressed in PVD neurons that function to mediate dendritic branching and stabilization  v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02058}attP2 (BL 26289), y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF03139}attP2 (BL 28712) and from the Vienna Drosophila Resource Center: w[1118]; P{GD16778}v46791/CyO; (v46791), w[1118]; P{GD11816}v30112/TM3; (v30112). UAS- RNAi lines were crossed to ppktdGFP; ppkgal4, UAS-dcr2 virgins. Larvae resulting from this cross were imaged at third larval instar. The dendritic arbor was quantified as described .Hippocampal neurons cultured from E19 Long-Evans rats (Charles River Lab) were plated at a density of 200,000 neurons per 18 mm acid treated glass coverslips (Fisher) coated with 0.06 mg/ml poly-D-lysine (Sigma) and 2.5 ug/ml Laminin (Sigma). Neurons were plated using plating media , 0.45% dextrose, 0.11 mg/ml sodium pyruvate, 2mM glutamine, Reagents from UCSF cell culture facility). Cultures were transferred to maintenance media  4 hours post plating. Half of the media was replaced with fresh media every 4 days.TTCAAGAGA as the loop sequence. Sequences used for constructing the shRNAs are: 1) Rab10 (NM_017359.2 Start position: 2373) “TTGACTCTATCATTGTTTA” 2) Exoc84 (NM_139043.1 Start position: 310) “CGCAGAACCTGAAGCGCAA” 3) Sec8 (NM_053875.1 Start position: 887) “CCGTTAAAGCCATTAAAGA”. ShRNA transfections were performed using Lipofectamine-2000 (Invitrogen) following manufacturer’s guidelines. Transfections were done at DIV7 and DIV14 and fixed 48 hours post transfection at DIV9 and DIV16, respectively using 4% PFA+4% sucrose at room temperature for 15 min. We did not observe toxicity or neuronal death in any of the shRNA treatment conditions. Neurons were blocked in blocking buffer  for an hour followed by overnight incubation with primary antibodies . Coverslips were washed thrice in PBS and incubated for 2 hours with secondary antibodies  followed by three washes in PBS before mounting onto slides for analysis using Fluromont mounting media from EMS).All shRNA constructs were made in the pLentilox3.7 vector backbone using Imaging of rat hippocampal neurons was performed on Leica Sp5 scanning confocal microscope with Laser lines 488 and 561 using a 40X NA 1.25 objective with zoom 0 for dendrite length and zoom 3.0 for dendritic spine imaging. Confocal image stacks were acquired at 1024X1024 pixels with 0.4micron z spacing between two frames such that the entire depth of the neuron/dendrite was imaged. Analysis was performed on maximum projected image stacks using ImageJ. ShRNA transfected cells were identified by GFP expression. Neurite length was calculated using the ‘Measure’ function of ImageJ and dendrite density was calculated by manually counting the number of spines per 100 microns of dendrite length.One-way ANOVA followed by post-hoc comparisons using the Dunnett’s test was used for all the figures, except S1 Figrab-10(ok1494), rab-10(dx2), exoc-8(ok2523) and sec-8(ok2187) worms from maximum intensity projections of z-stacks using Image J. Axons were visualized using the wdIs51 strain. At least 15 animals were quantified for each genotype. A one-way ANOVA was used to compare wild-type and mutant animals. Error bars report ±SEM. ns: not significant.Length of axons was quantified for wild-type, (TIF)Click here for additional data file.S2 Figrab-10(ok1494) mutant animals  are shown. All images are maximum z projections. L4 or young adult stage animals were examined. Scale bar, 20 μm.Morphology of  FLP neuron,  OLL neuron and  AWB and AWC neurons in wild-type  and (TIF)Click here for additional data file.S3 FigIs[ser2prom3>gfp::rab-10 DN] and  transgenic animals carrying Ex[ser2prom3>rab-8 DN]. PVD morphology was visualized using the PVD>gfp marker strain wdIs51. All images are maximum z projections. L4 stage animals were examined. Scale bar, 50 μm. (E) Quantification of animals with wild-type or defective PVD dendrite morphology. Two independent lines were quantified for dominant negative RAB-10 and RAB-8, respectively. At least 30 worms were examined. D+P defect means that dendrite arborization in both distal and proximal regions were reduced (shown in A and C). Proximal defect means that only dendrites in the proximal region were affected (shown in B and D). No defect means the morphology of PVD dendrite arbors were indistinguishable from that of wild-type worms.(A-D) Images showing the morphology of PVD neuron in  transgenic animals carrying (TIF)Click here for additional data file.S4 Fig(A-B) Fluorescence images show the morphology of PVD neuron using the PVD>myr-gfp marker strain wyIs592 in (A) wild-type and (B) GFP::RAB-10 Q68L over-expression. Both images are maximum intensity projections of z-stacks. L4 stage animals were examined. Scale bar, 50 μm. (C-E) Quantification of number of (C) secondary dendrites, (D) tertiary dendrites and (E) quaternary dendrites per PVD neuron in the above three genotypes (n = 12 for each strain). A one-way ANOVA was used to compare wild-type and RAB-10 Q68L over-expressing lines. Error bars report ±SEM. ns: not significant.(TIF)Click here for additional data file.S5 FigwdIs51 in (A) rab-10(ok1494), (B) rab-10(ok1494); Ti[ser2prom3>gfp::rab-10], (C) dma-1(tm5159), (D) dma-1(tm5159); Is[ser2prom3>dma-1::gfp], (E) hpo-30(ok2047), and (F) hpo-30(ok2047); Is[ser2prom3>hpo-30::gfp]. All images are maximum intensity projections of z-stacks. L4 or young adult stage animals were examined. Scale bars, 50 μm. (G) Quantification of cell-autonomous rescue of dendritic morphogenesis defect by cell-specific expression of gfp::rab-10, dma-1::gfp and hpo-30::gfp transgenes. At least 30 animals were quantified for each genotype.(A-F) Fluorescence images show the morphology of PVD neuron using the PVD>GFP marker strain (TIF)Click here for additional data file.S6 FigwdIs51. Fluorescence images show the morphology of PVD neuron in (A) wild type, (B and C) ser2prom3>rab-1 DN transgenic animals. See (A-C) PVD morphology was visualized using the strain (TIF)Click here for additional data file.S7 Figrab-10 (ok1494) and (G-I) exoc-8 (ok2523) in the distal, middle and tail regions, respectively. All images are maximum intensity projections of z-stacks. L4 stage animals were examined. Insets are enlarged 2.5 fold. Scale bar, 10 μm. (J) Quantification of number of HPO-30::GFP vesicles/vesicle clusters per area (76.8μm x 76.8μm). 10 animals at the L4 stage were quantified for each genotype. (K) Quantification of the intensity of HPO-30::GFP at the surface of the primary dendrites in wild-type, rab-10 (ok1494) and exoc-8 (ok2523) mutant worms in the distal, middle, and tail regions, respectively. 10 animals at the L4 stage were quantified for each genotype. A one-way ANOVA followed by post-hoc comparisons using the Dunnett’s test was used to compare wild-type and mutant animals. **: P<0.01. Error bars report ±SEM.(A-I) Fluorescence images show subcellular localization of HPO-30::GFP in (A-C) wild-type, (D-F) (TIF)Click here for additional data file.S8 FigNumber of vesicles or vesicle clusters labeled by DMA-1::GFP was quantified. At least 60 secondary dendrites were quantified for each genotype. A one-way ANOVA followed by post-hoc comparisons using the Dunnett’s test was used to compare wild-type and mutant animals. **: P<0.01. Error bars report ±SEM.(TIF)Click here for additional data file.S9 Figgfp marker strain wdIs51 in rab-10(ok1494); rrf-3(pk1426) fed with (A) control RNAi strain (harboring L4440 empty vector) and (B) dma-1 RNAi strain. Both images are maximum intensity projections of z-stacks. L4 or young adult stage animals were examined. Scale bar, 50 μm.(A-B) Fluorescence images show the morphology of PVD neuron using the PVD>(TIF)Click here for additional data file.S10 Figrab-10 (ok1494) mutant. Both images are maximum intensity projections of z-stacks. L4 or young adult stage animals were examined. Scale bars, 10 μm. The inset images are enlarged views (2.5 fold) of the regions indicated by the boxes. Arrows indicate vesicles labeled by EXOC-8::GFP reporter.(A-B) Fluorescence images showing EXOC-8::GFP in (A) wild-type and (B) (TIF)Click here for additional data file.S11 Figppk-tdgfp for the Drosophila class IV dendritic arborization neurons in (A and B) wild type, (C and D) rab10 RNAi and (E and F) exo84 RNAi treated animals. The images shown in  are magnified 2.5 fold from the boxed regions shown directly above. Scale bars, 100 μm. (G-H) Quantification of the number of (G) end points and (H) total dendritic arbor in wild type, rab10 RNAi (2 independent lines) and exo84 RNAi (2 independent lines) in Drosophila class IV dendritic arborization neurons. At least 7 neurons were quantified for each genotype. A one-way ANOVA followed by post-hoc comparisons using the Holm-Sidak test was used to compare wild-type and mutant animals. *: P<0.05; ns: not significant. Error bars report ±SD.(A-F) Fluorescence images showing expression of a reporter (TIF)Click here for additional data file.S12 FigRab10 (E and F) shRNA against Sec8 and (G and H) shRNA against Exo84. Immunolocalization was conducted using antibodies against GFP  and MAP2 . Scale bars, 20 μm. (I-J) Quantification of (I) total dendrite length at DIV9 and (J) dendritic spine density at DIV16 in control shRNA, Rab10 shRNA, Sec8 shRNAi and Exo84 shRNA transfected rat hippocampal neurons. 10 neurons were quantified for each genotype. A one-way ANOVA followed by post-hoc comparisons using the Dunnett’s test was used to compare wild-type and mutant animals. ns: not significant; **: P<0.01. Error bars report ±SEM.(A-H) Confocal fluorescence maximum projected images of cultured rat hippocampal neurons transfected with (A and B) control shRNA, (C and D) shRNA against (TIF)Click here for additional data file.S1 MovieThe time-lapse shows GFP::RAB-10 labeled vesicles moving bi-directionally in the PVD dendrites. Frames were collected every 0.8 sec. for a total of 96 sec using a spinning disk confocal . The movie was generated using 7 frames per second. Scale bar: 10μm.(AVI)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Cine cardiac magnetic resonance (cine-CMR) provides excellent endocardial delineation, enabling accurate quantification of left atrial (LA) chamber size. Relative utility of cine-CMR linear dimensions and area-based indices for prediction of LA-associated arrhythmias is unknown.The study comprised patients with coronary artery disease (CAD) included in a multimodality registry. Cine-CMR (1.5T) was performed using a standard 2-dimensional steady state free precession (SSFP) pulse sequence . Cine-CMR evidenced mitral regurgitation (MR) severity was graded in accordance with established conventions based on size of MR associated inter-voxel dephasing of the regurgitant jet. LA size was measured on cine-CMR at atrial end-diastole using two established methods: [1] linear diameter (measured in 3-chamber long axis orientation), [2] area (planimetered in 4-chamber long axis orientation), with both indices indexed to body surface area. Clinical follow-up was performed via medical record review, with atrial fibrillation (AF) or flutter (AFl) verified based on physician documentation.336 patients with CAD were studied ; LA diameter (mean 2 ± 0.4 cm/m2) and area (12 ± 3 cm2/m2) yielded similar prevalence of chamber dilation  assigned using established cine-CMR population-based cutoffs. LA indices correlated highly .


To evaluate the new robot-assisted navigation system for CT-guided lung tumour proceduresImaging-guided lung procedures are usually challenging due to patient breathing. This is an ongoing prospective study with 50 patients targeted in a university-based hospital. This was an initial assessment of efficacy involving 10 patients with lung tumours who underwent CT-guided lung interventions utilizing the robot-assisted Navigation system . The targeted needle pathway was planned on Maxio Robotic system based on pre-procedural CT-scans. The primary endpoint was satisfactory instrument position for intended intervention. Lesion size and depth from skin were noted. Performance level was documented on a five-point scale (5-1: excellent-poor). Total radiation doses were recorded and compared against 20 patients with conventional CT-guidance and CT-fluoroscopy lung procedures (ratio 1:1).There were 7 male and 3 female patients in the robotic group. Average age was 72.1 years (range 67-78). 8 patients underwent lung biopsy while the rest had thermal ablation or fiducial marker insertion. Average lesion size was 2.8cm (range 1.9-4.1cm). Average lesion depth was 6.2cm (range 3.7-8.6cm). All interventions met the primary endpoint of satisfactory instrument positioning. Average performance levels were 4.5. Average radiation dose (Dose Linear Product) was 480.4 (range 196.5-959.8) whereas conventional CT-guidance was 645.4 (range 285.1-1043.5) and CT-fluoroscopy was 460.1 (range 214.2-1157.0).Our initial experience demonstrated effectiveness of the robot-assisted navigation system for CT-guided lung tumour interventions with lower radiation dose compared with conventional CT-guided procedures. Radiation doses were similar to CT-fluoroscopy without radiation exposure to interventional radiologists. Targeting success rate for satisfactory intervention was 100%.


Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor cryptochrome (CRY). Naïve male flies exhibited significantly increased courtship activities when they were exposed to a ≥ 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. Our results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila.The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. Here, we report that courtship activity of male  All organisms on Earth are exposed to the planet’s natural magnetic field, which is approximately 0.3–0.5 Gauss (G). Some animals use this geomagnetic field for navigation and orientation –3. ThreeDrosophila melanogaster) .–16.white mutants .cry mutants under enhanced magnetic field conditions. The cryb mutation affects a highly conserved protein domain that is likely involved in FAD binding, which is necessary for both CRY and photolyase functions [crym mutation truncates the C-terminal domain of CRY, leaving the photolyase domain intact [cryb and crym mutants did not increase over that in the natural environment (0 G), suggesting that this behavioral phenotype results from magnetoreception through a CRY-dependent pathway . The effectiveness of each UAS-cryRNAi was validated using quantitative PCR (elav-GAL4/UAS-cryRNAi) showed significant knockdown in cry mRNA compared with the control group (elav-GAL4/+). We further tested whether targeted knockdown of cry expression in cry-GAL4-positive neurons would diminish increased courtship activity in a magnetic field. In the 20-G magnetic field, knockdown of CRYs using the cry-GAL4 driver in UAS-cryRNAi flies diminished the increases in male courtship activity caused by the magnetic field , l-LNvs, and s-LNvs, as well as neurons in small subsets of the ellipsoid body  but also in the ellipsoid body of the deep brain  flies were obtained from the Vienna Drosophila Resource Center (VDRC).Flies were raised on standard cornmeal media at 25°C and 60% relative humidity under a 12-h light:12-h dark cycle. The white-eyed Canton-S , elav-GATwo Helmholtz coils were used to build the platform. Each coil was wound with 332 copper wires and was 20 cm in diameter. The distance between the two coil rings was 10 cm . The pla2, and both test males and target females  were transferred to the behavior chamber, where the magnetic field was established. The courtship index is defined as the percentage of time that the tested male spent courting the target female during a 10-min recording period . For the TrpA1 studies, all of the flies were raised at 23°C before the experiments, and were placed in a 23°C or 30°C environment 10 min before and during the courtship behavioral assays.To evaluate courtship behavior, one male and one virgin female fly were placed in a chamber on the magnetic apparatus, and their courtship behaviors were recorded with a video camera. The courtship behavioral assay followed procedures established by a previous study . Naïve mDrosophila brains were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature. After fixation, the brain samples were incubated in PBS containing 1% Triton X-100 and 10% normal goat serum (PBS-T) and degassed in a vacuum chamber to expel tracheal air with six cycles of depressurizing to 270 mm Hg followed by holding for 10 min). Next, the brain samples were blocked and penetrated in PBS-T at 25°C for 2 h and then incubated in PBS-T containing mouse 4F3 anti-discs large (DLG) monoclonal antibody  at 25°C for one day. After the samples were washed in PBS-T three times, the samples were incubated in a biotinylated goat anti-mouse antibody  at 25°C for one day. Next, the brain samples were washed and incubated in Alexa Fluor 635-conjugated streptavidin  at 25°C for one day. After extensive washing, the brain samples were cleared and mounted in FocusClear (CelExplorer) for confocal imaging.Fly brain samples were imaged using a Zeiss LSM 700 confocal microscope with a 40X C-Apochromat water-immersion objective lens. To overcome the limited field of view, the brain samples were imaged twice, one for each hemisphere, with an overlap in between. We then combined the two parallel image stacks into a single dataset with ZEN image-processing software, using the overlapping region to align the two stacks.t-tests. All data are presented as the mean + standard error of the mean (SEM).All data were analyzed parametrically with Prism 5 statistical software (GraphPad). Data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests or evaluated by paired S1 Figcryb or crym mutants, white-eyed Canton-S did not show a significant difference in courtship activity in a 0-Gauss environment (left panel), but these flies significantly increased their courtship activity in a 20-Gauss magnetic field (right panel). Each value represents the mean + SEM . Genotypes: (1) w/Y; +/+; +/+, (2) w/Y; +/+; cryb/cryb, (3) w/Y; +/+; crym/crym. (B) A restricted wavelength of light (> 500 nm) did not affect normal courtship activity in white-eyed Canton-S male flies .(A) Compared with (TIF)Click here for additional data file.S2 FigUAS-cryRNAi line used in this study. Quantitative polymerase chain reaction (PCR) analysis showed that there was less targeted mRNA in the elav-GAL4/UAS-cryRNAi(v7238) and elav-GAL4/UAS-cryRNAi(v7239) flies than in the control elav-GAL4/+ flies. The results were normalized to the relative amount of 60S ribosomal protein L32 (RpL32). Each value represents the mean + SEM. (n ≥ 3). The forward and reverse primers used were 5′-AGGGTATAGCCCTAATTCCCG-3′ and 5′-GCATCCGATTGTAACCCACATT-3′, respectively. Genotypes: (1) w/elav-GAL4; +/+; +/+, (2) w/elav-GAL4; +/+; +/UAS-cryRNAi(v7238), (3) w/elav-GAL4; +/+; +/UAS-cryRNAi(v7239). (B) RNAi-mediated knockdown of cry in cry-GAL4-expressing neurons inhibited the increase in courtship indices in the 20-G magnetic field, compared with the 0-G control. Each value represents the mean + SEM . Genotypes: (1) w/Y; cry-GAL4/+; +/+, (2) w/Y; +/+; +/UAS-cryRNAi(v7238), (3) w/Y; +/+; +/UAS-cryRNAi(v7239), (4) w/Y; cry-GAL4/+; +/UAS-cryRNAi(v7238), (5) w/Y; cry-GAL4/+; +/UAS-cryRNAi(v7239).(A) Effectiveness of knockdown in the (TIF)Click here for additional data file.S3 Figcry-GAL4 > UAS-mCD8::GFP; UAS-mCD8::GFP male individual. Genotype: w/Y; cry-GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP. (B) GFP expression pattern (green) in the brain of a cry-GAL4; cry-GAL80 > UAS-mCD8::GFP; UAS-mCD8::GFP male individual. Genotype: w/Y; cry-GAL4/UAS-mCD8::GFP; cry-GAL80/UAS-mCD8::GFP. (C) GFP expression pattern (green) in the brain of a pdf-GAL4 > UAS-mCD8::GFP; UAS-mCD8::GFP male individual. Genotype: w/Y; pdf-GAL4/UAS-mCD8::GFP; +/UAS-mCD8::GFP. The brains were immunostained with anti-DLG antibody (magenta). The scale bars represent 50 μm.(A) GFP expression pattern (green) in the brain of a (TIF)Click here for additional data file.S4 FigVT4244-GAL4 > UAS-mCD8::GFP; UAS-mCD8::GFP male individual. Genotype: w/Y; +/UAS-mCD8::GFP; VT4244-GAL4/UAS-mCD8::GFP. The brain was immunostained with anti-DLG antibody (magenta). The scale bar represents 50 μm. (B) Activating VT4244-GAL4-expressing neurons did not increase the courtship index. Each value represents the mean + SEM . Genotypes: w/Y; +/+; VT4244-GAL4/UAS-TrpA1.(A) Green fluorescent protein (GFP) expression pattern (green) in the brain of a (TIF)Click here for additional data file.


To evaluate whether pretreatment magnetic resonance imaging (MRI) determined imaging parameters are predictive for outcome in hepatocellular carcinoma (HCC) treated with radio-frequency ablation (RFA).Thirty-seven patients with HCC treated by RFA were evaluated. Lesion number, size and segmental location, T2-weighted (w), arterial, portal-venous and venous contrast-phase, b600 diffusion-w imaging (DWI) and delayed phase contrast-enhanced imaging pattern were assessed at MRI. The separate imaging patterns as well as pretreatment clinical variables were correlated with outcome  using a chi-square test with multiple variables and Mann-Whitney U test respectively. Pretreatment clinical variables and imaging parameters were correlated with Keratin 19 and microvascular invasion status at the biopsy during RFA.None of the pretreatment patient- or tumour-related parameters correlated to disease free survival (p>0.5).The portal-venous, venous phase and b600 DWI imaging pattern showed strongest correlation with disease free survival . Also correlation was found for T2w imaging pattern (p=0.007), and hepatobiliary phase imaging pattern (p=0.017). Patients with tumour recurrence within 1 year (n=14) showed persistent venous rim- or nodular enhancement in 13 patients and b600 DWI rim-like hyperintensity in 9 patients correlating with microvascular invasion at biopsy (p=0.04). Patients disease free for at least 1 year (n=23) showed venous wash-out in 22 of 23 patients and whole-lesion hyperintensity b600 DWI in 18 patients.Pretreatment venous rim-enhancement and rim-like intensity at b600 DWI were strongest predictors of treatment failure within the first year after RFA of HCC.