Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell–Specific Genes

Abstract

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Single-cell RNA sequencing identifies TGF-β as a key regenerative cue following LPS-induced lung injury

“…This suggests that the mechanisms underlying homeostatic turnover and regeneration are conserved and the rare AEC2s responsible for homeostatic turnover are likely not inherently different from the AEC2s mobilized to proliferate after injury but rather are specified by their location within the niche (1,9,15). Second, whereas the widely accepted AEC1 markers T1α and AQP5 were expressed by other lung epithelial cell types, as has been previously reported (47-49), we identified highly specific AEC1 markers that may be exploited to drive Cre expression or isolate AEC1s, invaluable techniques that remain somewhat elusive (2,34,40,48,(50)(51)(52). Finally, we identified immune pathways activated in the Injured AEC2-Derived cells, which could be investigated to elucidate the inflammatory and host defense functions of the alveolar epithelium.…”

“…This suggests the possibility that while TGF-β induces cell cycle arrest, TGF-β signaling must be inactivated to allow AEC1 differentiation. To test this, we again used cultured AEC2s, which begin to express AEC1 markers by day 3 of culture ( Figure 7, A-C), although they do not fully differentiate into AEC1s (15,(38)(39)(40). To determine whether TGF-β inactivation promotes transdifferentiation, cultured AEC2s were treated with the TGF-β inhibitor or neutralizing antibody.…”

Dlk1-Mediated Temporal Regulation of Notch Signaling Is Required for Differentiation of Alveolar Type II to Type I Cells during Repair

“…We expected that, in the mutant, the cell populations processed for RNA-seq included quiescent AT2 (Sp-C hi T1α neg ) that were not affected by the injury as well as the cuboidal AT2-like intermediate cells ( Figure 5F ) (Sp-C low T1α low ), whereas in the WT, only quiescent AT2 were included because, at this stage, the reparative AT2 should either have converted to AT1 or returned to the quiescent state ( Figures 5E and 5G ). The expression of ~40 each of the AT1 or AT2 markers ( Marconett et al, 2017 ; Wang et al, 2018 ) was compared between WT and mutant cells ( Figure 5G ; Table S2 ). We found that, in general, the mutant cells expressed higher levels of AT1 markers and lower levels of AT2 markers compared with the WT ( Figure 5G ; Table S2 ), consistent with the data in Figures 4 and 5E and 5F .…”

“…41 type I cell markers and 37 type II markers were selected for further analysis based on previous RNA sequencing or microarray studies ( Marconett et al, 2017 ; Wang et al, 2018 ). The mean value of these genes of 3 WT samples and 3 Mutant samples were listed in Table S2 .…”

A co-culture model of the bovine alveolus

“…Results section: In accordance with the comments of Pietro Mastroeni, a scientific discussion of the selected markers is missing. Inspiration for discussion could be taken from references 1 1 and 2 2 .Figure 6 A and B clearly show different magnificationsFigure 6 C and D show the formation of multilayered epithelia and are not further discussed.BPAEC in Figure 7 A looks as the same as in Figure 7 B. Is it the same?…”