Defining GRAMD2 expression in the alveolar epithelium using organoid and single cell approaches.

Alveolar epithelial regeneration is critical for normal lung function and disrupted in disease. Precise identification of alveolar epithelial cell populations, particularly type I (AT1) cells, remains a barrier to understanding alveolar homeostasis and repair. We previously identified GRAM domain-containing 2 (GRAMD2) as enriched in distal alveolar epithelium, with predominant expression in type I (AT1) cells. However, recent single-cell datasets reveal low level Gramd2 transcripts in other lung epithelial populations raising questions about specificity. To address this, we performed immunofluorescence of whole lung and cytospins of GFPhigh cells from Gramd2CreERT2;mTmG mice. Cre-induced lineage labeling predominantly marked cells expressing established AT1 markers, with minimal labeling of AT2 or club cells. GFPhigh cells were enriched for AT1 cells and formed organoids in culture that over time showed expression of AT2 and airway markers, suggesting plasticity. Despite apparent purity of the GFPhigh population by immunostaining, single-cell RNAseq identified AT2 cells and a small number of club cells in the labeled population, revealing discordance between GRAMD2 transcript and protein expression. Gramd2CreERT2;mTmG mice enable enrichment of AT1 cells but have limitations for definitive lineage tracing.