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The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.
Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.
Copyright © 2018 Elsevier Inc. All rights reserved.
The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.
Copyright © 2017 Elsevier Inc. All rights reserved.
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
RHOA, a founding member of the Rho GTPase family, is critical for actomyosin dynamics, polarity, and morphogenesis in response to developmental cues, mechanical stress, and inflammation. In murine small intestinal epithelium, inducible RHOA deletion causes a loss of epithelial polarity, with disrupted villi and crypt organization. In the intestinal crypts, RHOA deficiency results in reduced cell proliferation, increased apoptosis, and a loss of intestinal stem cells (ISCs) that mimic effects of radiation damage. Mechanistically, RHOA loss reduces YAP signaling of the Hippo pathway and affects YAP effector epiregulin (EREG) expression in the crypts. Expression of an active YAP (S112A) mutant rescues ISC marker expression, ISC regeneration, and ISC-associated Wnt signaling, but not defective epithelial polarity, in RhoA knockout mice, implicating YAP in RHOA-regulated ISC function. EREG treatment or active β-catenin Catnb mutant expression rescues the RhoA KO ISC phenotypes. Thus, RHOA controls YAP-EREG signaling to regulate intestinal homeostasis and ISC regeneration.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Flow of fluids through the gut, such as milk from a neonatal diet, generates a shear stress on the unilaminar epithelium lining the lumen. We report that exposure to physiological levels of fluid shear stress leads to the formation of large vacuoles, containing extracellular contents within polarizing intestinal epithelial cell monolayers. These observations lead to two questions: how can cells lacking primary cilia transduce shear stress, and what molecular pathways support the formation of vacuoles that can exceed 80% of the cell volume? We find that shear forces are sensed by actin-rich microvilli that eventually generate the apical brush border, providing evidence that these structures possess mechanosensing ability. Importantly, we identified the molecular pathway that regulates large vacuole formation downstream from mechanostimulation to involve central components of the autophagy pathway, including ATG5 and LC3, but not Beclin. Together our results establish a novel link between the actin-rich microvilli, the macroscopic transport of fluids across cells, and the noncanonical autophagy pathway in organized epithelial monolayers.
© 2017 Kim et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
The TGF- and Wnt/-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF- has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF- signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF- type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF- receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF- receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF- receptor impaired -catenin activity and Genetically restoring -catenin activity in proximal tubules lacking the TGF- receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF- receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of -catenin activity or an indirect effect of -catenin interacting with other growth factors. In conclusion, blocking TGF- and -catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Copyright © 2017 by the American Society of Nephrology.
Purpose - Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE).
Methods - Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins.
Results - Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function.
Conclusions - Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.
ALDH3A1 is a corneal crystallin that protects ocular tissues from ultraviolet radiation through catalytic and non-catalytic functions. In addition, ALDH3A1 plays a functional role in corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation. We have previously shown that Aldh3a1 knockout mice in a C57B6/129sV mixed genetic background develop lens cataracts. In the current study, we evaluated the corneal phenotype of Aldh3a1 knockout mice bred into a C57B/6J congenic background (KO). In vivo confocal microscopy examination of KO and wild-type (WT) corneas revealed KO mice to exhibit corneal haze, manifesting marked light scattering from corneal stroma. This corneal phenotype was further characterized by Imaging Mass Spectrometry (IMS) with spatial resolution that revealed a trilayer structure based on differential lipid localization. In these preliminary studies, no differences were observed in lipid profiles from KO relative to WT mice; however, changes in protein profiles of acyl-CoA binding protein (m/z 9966) and histone H4.4 (m/z 11308) were found to be increased in the corneal epithelial layer of KO mice. This is the first study to use IMS to characterize endogenous proteins and lipids in corneal tissue and to molecularly explore the corneal haze phenotype. Taken together, the current study presents the first genetic animal model of cellular-induced corneal haze due to the loss of a corneal crystallin, and strongly supports the notion that ALDH3A1 is critical to cellular transparency. Finally, IMS represents a valuable new approach to reveal molecular changes underlying corneal disease.
Copyright © 2016. Published by Elsevier B.V.