Advanced Functional Materials
Zamani, M;Cheng, Y;Charbonier, F;Gupta, V;Mayer, A;Trevino, A;Quertermous, T;Chaudhuri, O;Cahan, P;Huang, N;
| DOI: 10.1002/adfm.202203069
Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.
Xu, J;Farsad, H;Hou, Y;Barclay, K;Lopez, B;Yamada, S;Saliu, I;Shi, Y;Knight, W;Bateman, R;Benzinger, T;Yi, J;Li, Q;Wang, T;Perlmutter, J;Morris, J;Zhao, G;
| DOI: 10.1038/s43587-023-00363-8
A, Upset plot showing the overlap between putamen conserved marker genes of Ast-0, Ast-1 and Ast-2 astrocyte with marker genes of mouse DAA and Gfap-high astrocytes from Habib et al., 2020. B, Violin plots showing the expression level distributions of orthologous genes of murine DAA and Gfap-high astrocyte marker genes in the putamen astrocytes. C, PCA plot using murine DAA and Gfap-high astrocyte marker gene logFC of gene expression (comparing murine DAA and Gfap-high astrocyte with Gfap-low astrocytes, downloaded from Habib et al., 2020) and the logFC of the human orthologous genes (comparing putamen Ast-1 and Ast-2 with Ast-0 astrocytes). D,E, Violin plots showing the expression level distributions of reactive astrocyte marker genes in astrocytes from the (D) putamen and (E) prefrontal cortex. F, Violin plots showing the expression level distributions of A1-, A2-specific activated astrocyte markers and JAK-STAT3 pathway genes. G, Top 10 GO terms in the Biological Process category enriched in the astrocyte subpopulation signature genes (hypergeometric test, FDR-adjusted P value < 0.05, ≥ 5 query genes). Conserved marker genes plotted in panel (B), (D) and (E) were determined by FindConservedMarkers using Wilcoxon Rank Sum test and _metap_ R package with meta-analysis combined P value < 0.05 comparing gene expression in the given cluster with the other cell clusters for AD (n = 4), PD (n = 4) and the controls (n = 4). Genes plotted in (F) were not statistically significantly higher in any of the astrocyte subpopulations.
Smith, RJ;Liang, M;Loe, AKH;Yung, T;Kim, JE;Hudson, M;Wilson, MD;Kim, TH;
PMID: 36717563 | DOI: 10.1038/s41467-023-36228-2
Epithelial-mesenchymal signaling in the gastrointestinal system is vital in establishing regional identity during organogenesis and maintaining adult stem cell homeostasis. Although recent work has demonstrated that Wnt ligands expressed by mesenchymal cells are required during gastrointestinal development and stem cell homeostasis, epigenetic mechanisms driving spatiotemporal control of crosstalk remain unknown. Here, we demonstrate that gastrointestinal mesenchymal cells control epithelial fate and function through Polycomb Repressive Complex 2-mediated chromatin bivalency. We find that while key lineage-determining genes possess tissue-specific chromatin accessibility, Polycomb Repressive Complex 2 controls Wnt expression in mesenchymal cells without altering accessibility. We show that reduction of mesenchymal Wnt secretion rescues gastrointestinal fate and proliferation defects caused by Polycomb Repressive Complex 2 loss. We demonstrate that mesenchymal Polycomb Repressive Complex 2 also regulates niche signals to maintain stem cell function in the adult intestine. Our results highlight a broadly permissive chromatin architecture underlying regionalization in mesenchymal cells, then demonstrate further how chromatin architecture in niches can influence the fate and function of neighboring cells.
The circadian clock gene, Bmal1, regulates intestinal stem cell signaling and represses tumor initiation
Cellular and molecular gastroenterology and hepatology
Stokes, K;Nunes, M;Trombley, C;Flôres, DEFL;Wu, G;Taleb, Z;Alkhateeb, A;Banskota, S;Harris, C;Love, OP;Khan, WI;Rueda, L;Hogenesch, JB;Karpowicz, P;
PMID: 34534703 | DOI: 10.1016/j.jcmgh.2021.08.001
Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.
He, S;Bhatt, R;Brown, C;Brown, EA;Buhr, DL;Chantranuvatana, K;Danaher, P;Dunaway, D;Garrison, RG;Geiss, G;Gregory, MT;Hoang, ML;Khafizov, R;Killingbeck, EE;Kim, D;Kim, TK;Kim, Y;Klock, A;Korukonda, M;Kutchma, A;Lewis, ZR;Liang, Y;Nelson, JS;Ong, GT;Perillo, EP;Phan, JC;Phan-Everson, T;Piazza, E;Rane, T;Reitz, Z;Rhodes, M;Rosenbloom, A;Ross, D;Sato, H;Wardhani, AW;Williams-Wietzikoski, CA;Wu, L;Beechem, JM;
PMID: 36203011 | DOI: 10.1038/s41587-022-01483-z
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Investigative Ophthalmology & Visual Science
Zhu, X;Xu, M;Grachtchouk, M;
RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells. Transcriptional profiling of GLI2ΔN-expressing and control MGs revealed that forced GLI2ΔN expression caused greatly increased expression of _Lrig1_ and _Lgr6_ and suppressed expression of meibocyte differentiation genes.
Martin, M;Vermeiren, S;Bostaille, N;Eubelen, M;Spitzer, D;Vermeersch, M;Profaci, CP;Pozuelo, E;Toussay, X;Raman-Nair, J;Tebabi, P;America, M;De Groote, A;Sanderson, LE;Cabochette, P;Germano, RFV;Torres, D;Boutry, S;de Kerchove d'Exaerde, A;Bellefroid, EJ;Phoenix, TN;Devraj, K;Lacoste, B;Daneman, R;Liebner, S;Vanhollebeke, B;
PMID: 35175798 | DOI: 10.1126/science.abm4459
The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a "hit-and-run" adeno-associated virus-assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.
