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Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C4 maize (Zea mays L.) plants. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of abBS cells and support a modification to the canonical phloem loading pathway in maize.

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis., Methods: TGR5-expression and-localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4(-/-) , Abcb4(-/-)/Tgr5(Tg) and ursodeoxycholic acid (UDCA)-or 24-norursodeoxycholic acid (norUDCA)-fed Abcb(4-/-) mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models., Results: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4(-/-) livers, in Abcb4(-/-) extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4(-/-) mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfb2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4(-/-) BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4(-/-) livers., Conclusions: Reduced TGR5 levels in BECs from patients with PSC and Abcb4(-/-) mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA., Lay summary: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.

The histone demethylase UTX (gene: KDM6A) directs cell and tissue differentiation during development. Deleterious mutations in KDM6A occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood, plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation, namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14high precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14low cells. UTX knockdown induced apoptosis and enriched KRT14high cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of KDM6A mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.

Currently, the clinical diagnosis of schizophrenia relies solely on self-reporting and clinical interview, and likely comprises heterogeneous biological subsets. Such subsets may be defined by an underlying biology leading to solid biomarkers. A transgenic rat model modestly overexpressing the full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1) protein (tgDISC1 rat) was generated that defines such a subset, inspired by our previous identification of insoluble DISC1 protein in post mortem brains from patients with chronic mental illness. Besides specific phenotypes such as DISC1 protein pathology, abnormal dopamine homeostasis, and changes in neuroanatomy and behavior, this animal model also shows subtle disturbances in overarching signaling pathways relevant for schizophrenia. In a reverse-translational approach, assuming that both the animal model and a patient subset share common disturbed signaling pathways, we identified differentially expressed transcripts from peripheral blood mononuclear cells of tgDISC1 rats that revealed an interconnected set of dysregulated genes, led by decreased expression of regulator of G-protein signaling 1 (RGS1), chemokine (C–C) ligand 4 (CCL4), and other immune-related transcripts enriched in T-cell and macrophage signaling and converging in one module after weighted gene correlation network analysis. Testing expression of this gene network in two independent cohorts of patients with schizophrenia versus healthy controls (n = 16/50 and n = 54/45) demonstrated similar expression changes. The two top markers RGS1 and CCL4 defined a subset of 27% of patients with 97% specificity. Thus, analogous aberrant signaling pathways can be identified by a blood test in an animal model and a corresponding schizophrenia patient subset, suggesting that in this animal model tailored pharmacotherapies for this patient subset could be achieved.

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) – an autoimmune disease of the CNS – increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction., Here the authors provide a single-cell characterization of cerebrospinal fluid and blood of newly diagnosed multiple sclerosis (MS) patients, revealing altered composition of lymphocyte and monocyte subsets, validated by other methods including the interrogation of the TFH subset in mouse models of MS.