The mammalian lung is a highly branched network, in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types remain obscure, in part due to the limited number of lineage markers1-3 and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations1–5. We used microfluidic single cell RNA sequencing (RNA-seq) on 198 individual cells at 4 different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell type diversity in the distal lung and led to the discovery of many novel cell type markers and transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full lifecycle of the alveolar type 2 cell lineage. This single cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.

Summary Individual mammalian cells exhibit large variability in cellular volume even with the same absolute DNA content and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, mostly likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S-phase. Our results provide a framework for quantitatively understanding the relationships between DNA content, cell size and gene expression variability in single cells.

Constitutive Hedgehog (HH) signaling underlies several human tumors, including basal cell carcinoma (BCC). Recently, Bijlsma and colleagues reported a new biologic function for vitamin D3 in suppressing HH signaling in an in vitro model system. On the basis of that work, we have assessed effects of vitamin D3 on HH signaling and proliferation of murine BCCs in vitro and in vivo. We find that indeed in BCC cells, vitamin D3 blocks both proliferation and HH signaling as assessed by mRNA expression of the HH target gene Gli1. These effects of vitamin D3 on Gli1 expression and on BCC cell proliferation are comparable to the effects of cyclopamine, a known inhibitor of the HH pathway. These results are specific for vitamin D3, because the precursor 7-dehydrocholesterol and the downstream products 25-hydroxy vitamin D3 [25 (OH)D] and 1,25-dihydroxy vitamin D3 [1,25(OH) 2 D] are considerably less effective in reducing either Gli1 mRNA or cellular proliferation. Moreover, these effects seem to be independent of the vitamin D receptor (VDR) because short hairpin RNA knockdown of VDR does not abrogate the anti-HH effects of D3 despite reducing expression of the VDR target gene 24-hydroxylase. Finally, topical vitamin D3 treatment of existing murine BCC tumors significantly decreases Gli1 and Ki67 staining. Thus, topical vitamin D3 acting via its HH inhibiting effect may hold promise as an effective anti-BCC agent.