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Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

Cell-Type-Specific Transcriptomic Signatures and Their Relevance to Transcriptional Reprogramming in HD Previous studies have investigated cell-type-specific transcrip- tomic signatures in the mouse striatum ( Gokce et al., 2016 ), providing insight into the signaling pathways that may distin- guish cell types. Here, we provide a novel resource for probing the molecular diversity and studying the biology of specific cell types in the mouse striatum, covering dSPNs, iSPNs, astrocytes, and microglia. Importantly, our data include transcriptomic sig- natures that are retained by our network analysis (i.e., spectral decomposition), in which data are analyzed against probabilistic functional networks (i.e., MouseNet) and the risk of false posi- tives and false negatives is reduced by virtue of the integration of orthogonal datasets. Spectral decomposition of gene expres- sion levels against probabilistic functional networks may retain genes not retained by traditional analysis of transcriptomic data, and vice versa. These network signatures are therefore strongly associated with the signaling systems that may underlie cellular identity in the mouse striatum. We detected no overlap with previously reported and larger signatures of human striatal neurons ( Kelley et al., 2018 ) and mouse D1 striatal neurons ( Gokce et al., 2016 ), as inferred from the data made available in this latter study. These comparisons suggest that network analysis is able to extract precise functional signatures not necessarily put forth by traditional analysis of transcriptomic data. These comparisons also suggest that mouse and human striatal neurons could differ in terms of molecular identity and the genes that are mostly enriched in these cell types. Addition- ally, network analysis notably showed that Eda2r, a member of the TNF receptor family associated with reactive astrogliosis, and the cell-to-cell or cell-to-matrix glycoprotein Thbs2 are two genes selectively and highly enriched in astrocytes. These genes are also strongly upregulated in the striatum of Hdh mice over time, highlighting their relevance to the reprogram- ming of transcription in the disease. Network analysis also
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Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability

Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability

( Muratore et al., 2014a ). That study was performed entirely in neurons differentiated to a forebrain fates of the cerebral cortex. However, iPSC-derived neurons can be efficiently patterned to different neuronal subtypes. Here, we directly compare control and APPV717I iPSCs differentiated to rostral, cortical fates with caudal neural fates of the hind- brain and spinal cord. We use this culture system to probe key questions regarding how neuronal cell type affects pro- cessing of APP by a-, b-, and g-secretases, as well as the responsiveness of different neuronal subtypes to Ab. We find that caudal neurons differ from rostral neurons in both their generation of and responsiveness to Ab species. APPV717I neurons directed to caudal neuronal fates generate Ab with a lower 42:40 ratio and higher 38:42 ratio than rostral telencephalic neurons. Further, we show that APPV717I neurons express higher levels of total and phos- pho-TAU proteins relative to control neurons when directed to a rostral neuronal fate, but not when directed to a caudal neuronal fate. Finally, we demonstrate that neu- rons of these different cell fates respond differentially to soluble extracts of clinically and neuropathologically typical ‘‘sporadic’’ late-onset AD (LOAD) brains. These AD brain extracts induce an elevation in the phosphorylation of TAU in forebrain neurons, and this is dependent upon the Ab present in these extracts. However, when exposed to the same AD extracts, TAU phosphorylation is not
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Cell type–specific genetic and optogenetic tools reveal hippocampal CA2 circuits

Cell type–specific genetic and optogenetic tools reveal hippocampal CA2 circuits

A preferential connection from CA2 to deep CA1 pyramidal cells establishes a novel trisynaptic circuit: DG-CA2-CA1 deep Previous studies using classical anatomical criteria showed that CA2 projects to CA1 8, 9, 21, 39 and forms functional synaptic connections with CA1 pyramidal cells 13 . However, how the newly defined CA2 cells project to the downstream CA1 region remains unclear. We infected the CA2- specific Cre knock-in mouse (MAP3K15 Cre) with a Cre-dependent virus, AAV9-EF1α-DIO- ChR2-YFP (Fig. 8a-c and Supplementary Fig. 16). All ChR2-YFP-positive cells expressed PCP4 confirming the high cell type specificity of the knock-in mouse (PCP4/YFP 97%±0.4, n=3 mice, Supplementary Fig. 16. Consistent with previous observations, CA2 axons traveled mainly in the stratum oriens (Supplementary Fig. 16) 8, 21, 39 . Optogenetic stimulation of ChR2- positive CA2 fibers during patch-clamp recordings from CA1 pyramidal cells revealed an excitatory response (average EPSC amplitude –120±20 pA, average EPSC onset 1.9±0.04 ms, n=28) that was sensitive to ionotropic glutamate receptor antagonists (Fig. 8d-g).
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Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP)

Cell type–specific mRNA purification by translating ribosome affinity purification (TRAP)

Each purification will require: 300 μl Streptavidin MyOne T1 Dynabeads, 120 μl Biotinylated Protein L (1 μg/μl in 1x PBS), and 50 μg each of GFP antibodies 19C8 and 19F7 (100 μg total antibody). For feasibility pilot experiments (see Experimental Design section), half and double the matrix component amounts, keeping ratios the same, can also be tried. Investigators should keep altering the amounts, keeping ratios the same, until an optimal amount of matrix is found that captures all tagged message. For example, if the amounts listed above are optimal for a new cell type, one would expect to see a halving of TRAP yield with half the amount of matrix used in TRAP purifications -- and no detectable purification of RNA from the non- TRAP control with either concentration. (We typically use a 1.5-fold excess of optimal concentration in our TRAP experiments, to account for matrix pipetting error.) Such a pilot experiment is recommended because the amount of affinity matrix needed will vary by cell type, as the abundance of the target cell type, its translational state, its size, and the local tissue background RNA binding levels are all characteristics that will vary between different investigators’ TRAP experiments. The amounts listed above were empirically determined to ensure complete binding of all epitope in a relatively abundant cell type (e.g. 0.7×10 6 spiny projection neurons in 30 mg striatal tissue), but rare cells may need a fraction of the amounts listed here. For such rare cell types, it is advisable to use the minimum amount of matrix needed to reduce background RNA binding. Upon receipt, record the two antibodies’ concentrations, as they will vary by batch. If the antibodies arrive unfrozen, mix each tube gently and aliquot each antibody into single experiment aliquots (to be used within a week), snap-freeze aliquots in liquid nitrogen, and store the aliquots at −80°C. If the antibodies arrive frozen, store immediately at −80°C; they should be thawed on ice and aliquoted before or at first use. On the day of use, thaw aliquot to be used that day on ice, spin tubes at maximum speed (>13,000 x g) in microcentrifuge for 10 minutes, 4 °C, and take supernatants (antibody) to new tubes. Add sodium azide as needed. Antibody can be kept at 4 °C for a few days. If interval between IPs is longer than this, aliquot, snap-freeze in liquid nitrogen, and store in single-use aliquots at −80 °C.
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Cell-Type-Specific Synchronization of Neural Activity in FEF with V4 during Attention

Cell-Type-Specific Synchronization of Neural Activity in FEF with V4 during Attention

The selective coupling of FEF visual neurons with V4 during sustained attention adds further evidence to the distinct contri- bution of FEF visual neurons to attentional mechanisms. Our finding that enhanced coupling occurs with attention only between FEF visual neurons and V4 suggests that V4 neurons have preferential connections with FEF visual neurons rather than any other FEF cell type. The pattern of anatomical connec- tions between FEF and V4 supports this conclusion. The majority of FEF projections to V4 arise from the supragranular layers ( Barone et al., 2000; Pouget et al., 2009 ), and neurons in the supragranular layers of the FEF subserve visual selection ( Thompson et al., 1996 ). With attention, an increase in gamma synchrony between FEF supragranular-layer visual cells and V4 with the appropriate phase relationships may increase effec- tive communication between the two areas to enhance process- ing of signals related to the attended location ( Fries, 2005; Gregoriou et al., 2009a; Gregoriou et al., 2009b ). Moreover, the absence of any effect of attention on synchrony between FEF movement cells and V4 further indicates that attentional mecha- nisms at the network level are largely independent and distinct from movement processing.
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Cell type-dependent control of NF-Y activity by TGF-β

Cell type-dependent control of NF-Y activity by TGF-β

Institut de Ge´ne´tique Mole´culaire de Montpellier, CNRS-UMR5535-IFR122, Montpellier, France Transforming growth factor b (TGF-b) is a pluripotent cytokine that regulates cell growth and differentiation in a cell type-dependent fashion. TGF-b exerts its effects through the activation of several signaling pathways. One involves membrane proximal events that lead to nuclear translocation of members of the Smad family of transcriptional regulators. TGF-b can also activate MAPK cascades. Here, we show that TGF-b induces nuclear translocation of the NF-YA subunit of the transcription factor NF-Y by a process that requires activation of the ERK cascade. This results in increased binding of endogenous NF-Y to chromatin and TGF-b- dependent transcriptional regulation of the NF-Y target gene cyclin A2. Interestingly, the kinetics of NF-YA relocalization differs between epithelial cells and fibro- blasts. NIH3T3 fibroblasts show an elevated basal level of phosphorylated p38 and delayed nuclear accumulation of NF-YA after TGF-b treatment. In contrast, MDCK cells show low basal p38 activation, higher basal ERK phosphorylation and more rapid localization of NF-YA after induction. Thus, NF-Y activation by TGF-b1 involves ERK1/2 and potentially an interplay between MAPK pathways, thereby opening the possibility for finely tuned transcriptional regulation.
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Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function

Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function

DISCUSSION We employed a BAC transgenic strategy to express ChR2-EYFP under the endogenous promoter elements that define the GABAergic, cholinergic, serotonergic, and Pvalb+ subtype of neurons throughout the CNS. While viral vectors have successfully been used by several laboratories to target ChR2 to defined neuronal populations within the intact mouse brain 3–18 , there are still important limitations and challenges to these strategies. Every experimental animal requires surgical stereotaxic delivery of virus encoding the ChR2 transgene. Inevitably, not every injected animal is suitable for experimentation due to experimenter error. Even those animals that are deemed suitable may have variable spread of the virus at the injection site, gradients of transgene expression levels in infected brain regions, or potential tissue damage. These potential confounding factors are eliminated by the development and use of cell-type specific ChR2-EYFP transgenic mouse lines. Our mouse lines have robust and functional transgene expression in defined populations of neurons that is stable in terms of both pattern and level of ChR2-EYFP expression across generations.
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CTCF-mediated transcriptional regulation through cell type-specific chromosome organization in the β-globin locus.

CTCF-mediated transcriptional regulation through cell type-specific chromosome organization in the β-globin locus.

Modeling cell type-specific interactions between CTCF sites To address the issue of whether differential cell type- specific CTCF contacts affect the conformation of the globin region, a polymer model of the 30 nm chromatin fiber was designed for each of the three cell types under investigation: K562, K562kd and 293T. Disregarding its internal structure, chromatin behaves as a flexible chain that self-avoids. We used the simplest continuous model, known as the WLC, which includes flexibility properties of a single chain (37–39) and considers the self-avoiding nature of a 30 nm thick fiber. Each WLC model represents a 1 Mbp region of human chromosome 11 containing the interacting CTCF sites and the b-globin locus. Interaction forces were determined by iterating the DNA-folding algorithm until the frequency of contacts between the sites corresponded to the experimentally measured CTCF-dependent chromatin contacts (Figure 1) (36). Importantly, CTCF interaction frequencies were the sole input to the polymer models and the inputs did not include the LCR or any gene promoter sites. This allowed us to Figure 1. CTCF-based chromatin loops differ between K562 cells and 293T cells. CTCF site interactions from Hou et al. (36) are depicted in a schematic diagram of the globin locus. Pairwise interaction frequencies detected by 3C are shown by arcs for K562 (red), K562 CTCF RNAi knockdown (green) and 293T (blue) cells. Black dots below the x-axis indicate CTCF-occupied sites confirmed by qPCR used in this analysis. The LCR is indicated by a gray vertical line. RNA seq data from the ENCODE Consortium (CalTech data set from Wold and Myers groups) for K562 cells (red track) and nine other non-erythroid cell types combined (multicolor track) are shown as normalized read density (reads per million, RPM) on a ln(x+1) scale. RefSeq gene models for the locus are shown, including globin genes (orange), odorant receptor genes (blue) and other genes (gray). All coordinates and data are for the hg19 assembly.
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Identification of a New Stromal Cell Type Involved in the Regulation of Inflamed B Cell Follicles

Identification of a New Stromal Cell Type Involved in the Regulation of Inflamed B Cell Follicles

expressed CD21 display the DTR on their surface, rendering them susceptible to deletion following DT injection. CD21cre-RFP mice were irradiated and reconstituted with CD21cre-DTR bone marrow cells in order to obtain animals in which FDCs and the new subset of stromal cells would express RFP in absence of the DTR, while all mature colorless B cells would express the DTR. Such chimeric mice were injected with CFA in the ears, and 3 wk later, mice were treated or not with DT for 3 consecutive days. At that time, inflamed dLNs were harvested and either analyzed by flow cytometry or sectioned, stained for B220 and CXCL13, and analyzed by confocal microscopy. Flow cytometry analysis revealed that DT injection induced the ablation of ,70%–90% of B cells in the LNs of treated mice (Figure 5A). In the LN sections of DT-treated chimeras, we determined that the remaining B cells were not evenly distributed in the areas formerly occupied by the enlarged B cell follicles but aggregated in small and compact B cell follicles centered around few CXCL13 + FDC-M2 + RFP + cells (Figure 5B and unpublished data). This rapid withdrawal of the B cell follicles provided a unique opportunity to investigate if the sudden release from B cell control affected the capacity of converted CD21 2 RFP + stromal cells to secrete CXCL13. To this aim, we extrapolated the shapes that each inflamed B cell follicle approximately occupied before DT treatment on LN sections and counted the number of FDC-M2 2 CXCL13 + and CXCL13 2 RFP + stromal cells present in these regions (see Materials and Methods, Figure 5, and Figure S6). Using this approach, we determined that only 12.9% (70 out of 543) of the RFP + cells located in a region previously occupied by inflamed B cell follicles remained positive for CXCL13 expression (Figure 5B). These results demonstrated that B cells were responsible for the conversion and the maintenance of this new stromal cell type into CXCL13 secreting cells.
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Compartment and cell-type specific hypoxia responses in the developing Drosophila brain

Compartment and cell-type specific hypoxia responses in the developing Drosophila brain

Fig. 6. The biosensor reveals cell-type specific hypoxia states in central brain and optic lobe. (A –E) Dorsal views (in relation to the neuraxis) of immunostained brains for cell-type specific markers (gray): (A) anti-Deadpan (neuroblasts, NB), (B) anti-Prospero (ganglion mother cells, GMC), (C) anti-Elav (neurons), (D) anti-Repo (glial cells). The cell-specific nuclear staining shown in (A –D) was utilized as segmentation signal to create a mask to obtain the corresponding ratiometric analysis of each cell type (A ′–D′). The anti-Discs large staining (E) was used to outline neuroepithelial cells. The corresponding ratiometric analysis (E ′) was based on manual segmentation of the IPC and OPC using TrakEM2. (F) Mean ratio −1 (oxygenation) for each cell type in central brain and optic lobe, represented as a function of mean distance to tracheoles. The dotted line shows the average trend, obtained by averaging the fits of all normoxic brains. (G) Decaying exponential fits for all cell types. (H) Boxplot comparing ratiometric values of all cell types both in central brain and optic lobe (n=4). (I) Exposure to hyperoxia has a stronger effect in neuroblasts than in neuroepithelial cells (n=6). (J) Cell-type specific hypoxia response in the central brain and optic lobe based on biosensor data. Scale bar is applicable to all panels and is 40 µm. Error bars in (F) show s.e.m. *P<0.05, **P<0.01, ***P<0.001 Student ’s t-test or Mann–Whitney Wilcoxon test.
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Cell-type specific determinants of NRAMP1 expression in professional phagocytes

Cell-type specific determinants of NRAMP1 expression in professional phagocytes

In summary, combined examinations of DNAse I footprinting and ChIP-Sequencing converge to reveal different types of protein-DNA interactions. Depending on cell type(s) in which they were found these DNA-protein associations may occur at different stages of, or be maintained through, the myelo-monocytic differentiation program. Data indicate that SLC11A1 locus spans 34.6 kb and it is activated early in myeloid development. Two candidate elements, both distal from the TSS and located at the ends of SLC11A1 locus, nested within CTCF boundaries, are predicted to bind ETS-related factors such as PU.1 and ELF-1 as early as the CMP stage. The whole locus appears extensively sensitive to DNAse I digestion along the myelo-monocytic pathway, with increasing intensity as maturation progresses. The data support prominent roles for C/EBPβ and PU.1 in the developmental control of SLC11A1 expression in mature phagocytes. Notably three C/EBPα/β sites, including the major TSS and two predicted upstream binding sites appear strongly mobilized to activate transcription in CD14 + MNs and MDMs, while another upstream site seems possibly involved earlier in promyelocytic development (NB4 cells). In contrast, most of the locus internal DNAse I hypersensitive areas disappear along the megakaryo-erythrocytic pathway implying suppression of SLC11A1 expression in this non-phagocytic myeloid lineage.
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Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

harbor mutations that associate with human brain disorders such as microcephaly and autism (Figure 2J–2M and below), suggesting cell type-specific mechanisms of human diseases. Alternatively Spliced Exons Alter Protein Domains and Subcellular Localization Exons that are differentially spliced between NPCs and neurons are remarkable in the extent to which they critically involve essential protein domains. Among 61 splicing changes in 49 genes regulating cytoskeleton (Table S3), 20 (33%) of them caused insertion or deletion of one or more entire protein domains (Figure 3A–3B, Figure S3A–S3C). Multiple genes showed AS of membrane-targeting domains (Figure 3B), which typically regulate subcellular localization. Another 13 (21%) alternative exons--including a microexon-- inserted extra amino acids (extraAA) into well-defined protein domains (Figure 3A, Figure S3A and S3C). Thus, more than half (54%) of differentially spliced regions directly regulate protein domains.
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Cell-type Specific Optogenetic Mice for Dissecting Neural Circuitry Function

Cell-type Specific Optogenetic Mice for Dissecting Neural Circuitry Function

commutator and then to the laser (Crystal Laser, 100 mW) and the laser power was controlled by an analog dial. The illuminated area was estimated as a 400 μm by 600 μm oval (0.24 mm 2 ) and this area was used to calculate the power density following direct measurement of the power output at the optic fiber tip using a power meter (Thor Labs). Pulsing of the laser was computer- controlled using custom “optogenetics” stimulation protocols designed in the pClamp 10.2 software. Light-induced inward currents were evoked with blue laser light delivered at 1–100 Hz frequencies (0.05–5 ms pulse duration), or by continuously applying blue laser light over one second or longer. Similar protocols were applied in the current clamp mode to monitor action potential firing of the recorded neurons in response to blue light stimulation from the resting membrane potential. For tonically active neurons (often seen for striatal cholinergic neurons, cerebellar Purkinje cells, and DRN 5-HT neurons) we assessed the ability to induce action potential firing either on top of basal firing rates or after silencing basal firing with the minimal necessary hyperpolarizing current injection. For experiments using VGAT-ChR2-EYFP bran slices to assess ChR2 mediated silencing in local circuits, a layer V cortical neuron was recorded in whole-cell current clamp mode and injected continuously with 100–200 pA of depolarizing current to induce tonic firing. Blue laser light was applied to nearby ChR2-expressing cortical interneurons to assess the extent of silencing or hyperpolarization in the layer V pyramidal neuron. Cell-attached recordings were performed in some experiments to monitor action potential firing without disruption of the cytosol. Cell-attached recordings were performed in voltage clamp mode with a zero holding current. Extracellular field recordings were obtained by placement of a glass recording electrode (1–2 MΩ tip) filled with aCSF into the slice and advanced to the depth that allowed discrimination of putative single units based on the response to blue laser light stimulation (in some cases multiple units were apparent). A Multiclamp 700B amplifier (Molecular Devices Corporation) and Digidata 1440A were used to acquire electrophysiological signals using Clampex 10.2 software (Molecular Devices). The signals were sampled at 20 kHz and low-pass filtered at 2 kHz. The series- resistance was ≤ 25 MΩ and was not compensated. Access to the recorded cells was continuously monitored, and only recordings with stable series resistance were included for analysis. All data analysis was performed in Clampfit 10.2 (Molecular Devices). Values are expressed as mean ± s.e.m. Data were tested for significance using a non-paired student t- test.
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Cell-type and endocannabinoid specific synapse connectivity in the adult nucleus accumbens core

Cell-type and endocannabinoid specific synapse connectivity in the adult nucleus accumbens core

The nucleus accumbens (NAc) is a mesocorticolimbic structure that integrates cognitive, emotional and motor functions. Although its role in psychiatric disorders is widely acknowledged, the understanding of its circuitry is not complete. Here, we combined optogenetic and whole-cell recordings to draw a functional portrait of excitatory disambiguated synapses onto D1 and D2 medium spiny neurons (MSNs) in the adult male mouse NAc core. Comparing synaptic properties of ventral hippocampus (vHipp), basolateral amygdala (BLA) and prefrontal cortex (PFC) inputs revealed a hierarchy of synaptic inputs that depends on the identity of the postsynaptic target MSN. Thus, the BLA is the dominant excitatory pathway onto D1 MSNs (BLA ⬎ PFC ⫽ vHipp) while PFC inputs dominate D2 MSNs (PFC ⬎ vHipp ⬎ BLA). We also tested the hypothesis that endocannabinoids endow excitatory circuits with pathway- and cell-specific plasticity. Thus, whereas CB1 receptors (CB1R) uniformly depress excitatory pathways regardless of MSNs identity, TRPV1 receptors (TRPV1R) bidirectionally control inputs onto the NAc core in a pathway-specific manner. Finally, we show that the interplay of TRPV1R/CB1R shapes plasticity at BLA-NAc synapses. Together these data shed new light on synapse and circuit specificity in the adult NAc core and illustrate how endocannabinoids contribute to pathway-specific synaptic plasticity.
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Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Tissue handling and staining. Mice used for all experiments were sacrificed by an iso flurane (Baxter KDG9623) overdose, mandibles were carefully dissected out, fixed in 4% paraformaldehyde pH 7.4 for 5–15 h, decalcified in 10% EDTA pH 7.4 for 7 days at +4 °C, cryopreserved in 30% sucrose overnight at +4 °C and embedded in OCT medium (Tissue-Tek, 4583) on dry ice. Samples were cut on cryostat (Leica CM1850UV) in sagittal orientation as 14- μm thick sections. Human teeth extracted for clinically relevant reasons were fixed in 4% paraformaldehyde pH 7.4 for overnight, decalcified in 10% EDTA pH 7.4 for 7 days at +4 °C and paraf fin embedded. Samples were cut on microtome (Leica SM2000R) as 2-μm thick sections. Before antibody staining antigen retrieval was performed (Dako S1699). Staining with primary antibodies was performed overnight at room tem- perature followed by Alexa-conjugated secondary antibodies staining at room temperature for 1 h (Invitrogen, 1:1000) or HRP-conjugated streptavidin-biotin antibody and immunoreactivity was visualized with ImmPACT DAB Peroxidase (Vecor Laboratories, SK4105).Used antibodies: ACTA2 (Protein Tech, 23081-1- AP; 1:500), AIF1 (Novus, NB100-1028; 1:500), CALB1 (Swant; CB-38a; 1:500); COL4 (AbD Serotec, 2150-1470; 1:500), CDH1 (Novus, Af748; 1:500), CSF1 (NSJ, R31901; 1:200), CLDN10 (Sigma–Aldrich, HPA042348; 1:200), DLX5 (LSbio, LS- C352119; 1:200), DPP4 (Novus, AF954; 1:200), EGR1 (Cell Signalling, 4154; 1:200), F4/80 (Abcam, ab6640; 1:200), LYVE1 (Novus, AF2125; 1:200), MKI67 (Zytomed, RBK027-05, 1:200), NOTUM (Sigma–Aldrich, HPA023041; 1:200), PIEZO2 (Sigma–Aldrich, HPA040616; 1:200), POSTN (Novus, NBP1-30042; 1:200), RYR2 (ThermoFisher, PA5-36121; 1:200), S100A13 (DAKO; IS504; 1:500), SALL1 (Abcam, ab31526; 1:200), SMOC2 (MyBioSource, MBS2527784; 1:200), SOX9 (Sigma–Aldrich, HPA001758; 1:200), SOX10 (Santa cruz, sc-365692; 1:200), THBD (RnD systems, MAB3894; 1:200). Cell nuclei counterstaining was performed with DAPI (Sigma–Aldrich, D9542) diluted 1:1000 in PBS + 0.1% Tween 20 (Sigma–Aldrich, P9416) and slides were mounted with 87% glycerol (Merck, 104094) or Fluoromount Aqueous Mounting Medium (Sigma –Aldrich, F4680). Imaging was performed using Zeiss LSM880 laser scanning confocal microscope and Lightsheet Z.1 microscope. ZEN2.1 (ZEISS) and Imaris (Bitplane) software was used for image processing. Conventional histological staining after Clodrosome or Encapsome treatments was performed after 4 weeks decalci fication of dissected mandibles in 19% EDTA. Mandibles were embedded in wax blocks and sectioned using 8μm thickness. Sections were stained using Masson’s Trichrome.
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Cell type-specific plasticity of striatal projection neurons in parkinsonism and L-DOPA-induced dyskinesia

Cell type-specific plasticity of striatal projection neurons in parkinsonism and L-DOPA-induced dyskinesia

Two-photon laser scanning microscopy and anatomy iSPNs and dSPNs in the dorsolateral striatum were identified by somatic enhanced green fluorescent protein (eGFP) and tdTomato, respectively, using two-photon laser scanning microscopy (2PLSM) with an Ultima Laser Scanning Microscope system (Prairie Technologies, Middleton, WI) or a Zeiss LSM710 NLO with MaiThai laser (Spectra Physics, Santa Clara, CA). A DODT contrast detector system was used to provide a bright- field transmission image in registration with the fluorescent images. The red tdTomato (554-581nm) and green eGFP (490-560 nm) were acquired with 810 nm excitation using a two-photon laser (Verdi/Mira laser) or 543 nm with a single photon laser (HeNe). SPNs were patched with video microscopy using either a Hitachi CCD camera and an Olympus X60/0.9 NA lens or an Axiocam CCD camera (Zeiss) and a W Plan-Apochormat 63×/1.0 NA objective (Zeiss). For anatomical assessments whole cell images were obtained with 0.389μm 2 pixels with 0.5μm z-steps and sholl's analysis performed on 3D reconstructions with NeuroLucida (Williston, VT) or Imaris (Bitplane, AG Zurich, Switzerland). High magnification images of dendritic segments (proximal: <80μm from soma; distal: >80μm from soma) were acquired with 0.15μm 2 pixels with 0.3μm z-steps. High magnification images were deconvolved in AutoQant (MediaCybernetics, Rockville, MD) and semi- automated spine counting performed using 3D reconstructions in NeuronStudio (CNIC, Mount Sinai School of Medicine, New York). Total spine estimates were calculated by averaging proximal and distal spine densities for each cell and multiplying by the following: median total dendritic length for treatment group – (30 * median number of primary dendrites for treatment group). Thirty multiplied by the number of primary dendrites was subtracted from the total dendritic length because the first 30μm of a primary dendrite are aspiny or have minimal spines.
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Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming

Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming

A. Gaignerie 1 , N. Lefort 2 , M. Rousselle 1 , V. Forest-Choquet 3 , L. Flippe 4,5,6 , V. Francois–Campion 4,5,6 , A. Girardeau 3 , A. Caillaud 3 , C. Chariau 1 , Q. Francheteau 1 , A. Derevier 1 , F. Chaubron 7 , S. Knöbel 8 , N. Gaborit 3 , K. Si-Tayeb 3 & L. David 1,4,5,6 Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.
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Cell-type specific contributions to Rett Syndrome : neuronal and astrocytic signaling and sensory processing

Cell-type specific contributions to Rett Syndrome : neuronal and astrocytic signaling and sensory processing

With these emerging findings we have provided additional evidence for the role of astrocytes in Rett pathophysiology by showing that MeCP2: (1) regulates astrocyte signaling p[r]

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Cell-type specific cholinergic modulation of the cortex

Cell-type specific cholinergic modulation of the cortex

Specifically, the cholinergic innervation of the neocortex by afferent fibers originating in the nucleus basalis (NB) of the basal forebrain (3) was found to be im[r]

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Role of ETS1 in the transcriptional network of diffuse large B cell lymphoma of the activated B cell-like type

Role of ETS1 in the transcriptional network of diffuse large B cell lymphoma of the activated B cell-like type

Based on these data, ETS1 appears to control the expression of genes mainly involved in the B- cell transcriptional program but also in RNA processing. Figure 1. Knockdown of ETS1 reduces the expression of genes with important functions in normal B cells and DLBCL cells. A qRT-PCR was used to determine the expression of the indicated genes following knockdown of ETS1 by siRNA in five ABC-DLBCL cell lines. The expression of each gene was normalized to GAPDH expression. Results shown are the average of three independent experiments; n = 3; error bars = standard deviation. Asterisk above bars indicates significant difference in expression. The ETS1 regulation of these transcripts was further validated after silencing ETS1 using shRNA in TMD8 and HBL1 cells. A downregulation in mRNA expression was confirmed for all of them (CD52, FCMR, RGS1, and HCST), as well for other genes (PTPN7, ARHGAP9, SASH3, and GPSM3) that had been identified only in the RNA-Seq analysis (Figure S6). However, as not all genes were significantly reduced in the TMD8 cell line, this suggests that cell-type dependency and other factors could be involved in regulating these genes.
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