Integration of scRNA-seq data from millions of cells revealed a high diversity of cell types in the healthy and diseased human lung. In a large and complex organ, constantly exposed to external agents, it is crucial to understand the influence of lung tissue topography or external factors on gene expression variability within cell types. Here, we apply three spatial transcriptomics approaches, to: (i) localize the majority of lung cell types, including rare epithelial cells within the tissue topography, (ii) describe consistent anatomical and regional gene expression variability within and across cell types, and (iii) reveal distinct cellular neighborhoods in specific anatomical regions and examine gene expression variations in them. We thus provide a spatially resolved tissue reference atlas in three representative regions of the healthy human lung. We further demonstrate its utility by defining previously unknown imbalances of epithelial cell type compositions in chronic obstructive pulmonary disease lungs. Our topographic atlas enables a precise description of characteristic regional cellular responses upon experimental perturbations or during disease progression.

Background  Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear.

Methods  We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)β1.

Measurements and main results  We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1⁺ cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1⁺ transitional fibroblasts and Cthrc1⁺ myofibroblasts. TGFβ1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1⁺ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFβ1-induced fibroblast invasion and RHOA pathway activity.

Conclusions  Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFβ1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.

Several airway epithelial cell types and subpopulations have been defined, using high throughput single-cell mRNA profiling. However, the spectrum and topology of cell populations within the secretory cell lineage has not been explored. The subject of this thesis is the investigation of diverse secretory cell states, the identification of their progenitors and the exploration of genetic mechanisms establishing distinct secretory cell states in the airways.

Hedgehog (HH) signaling is important for airway development. The paracrine function of epithelial-derived HH expression is to pattern the adjacent developing mesoderm and give rise to the future mesenchyme. In turn, patterning of the mesenchyme facilitates proper airway epithelial differentiation. However, a possible autocrine role of HH signaling on the developing epithelium and whether or not it participates in airway epithelial differentiation has remained unexplored. We utilized knockout mouse models and an in vitro culture setup of human bronchiolar epithelial cells (HBECs), to investigate the autocrine function of HH signaling, in mice and humans. Epithelial specific inactivation of the Smoothened (Smo) effector in the developing trachea, rendered epithelial cells unresponsive to HH signaling. Tracheal epithelial cells, deficient for Smo, showed reduced proliferation of epithelial cells and their differentiation towards ciliated and secretory cell types. The observed phenotype was milder than the one caused by epithelial inactivation of the ligand sonic hedgehog (Shh), presumably due to changes in the mesenchyme that signals in a paracrine fashion to regulate epithelial differentiation. We found that autocrine function of HH signaling in tracheal epithelial cells promotes secretory and ciliated cell differentiation from epithelial progenitor cells. Pharmacological in vitro inactivation of Smo activity in HBECs shows a conserved function of HH signaling in airway development in mammals. So, our data conclude that Smo activity in tracheal epithelial cell controls the proliferation of epithelial progenitors and their differentiation in cell-autonomous manner.

Secretory cells are the bulk cell type of the airway epithelium. To further investigate the potential heterogeneity within the secretory cell lineage and the mechanisms that control the balance between the secretory subpopulations, we combined single cell transcriptomic profiling with a multiplex hybridization approach. We found opposing gradients of differentiation, along the proximodistal axis of the adult lung epithelium. Within these gradient programs, we defined two distinct secretory cell states S1 and S2, each expressing a unique set of mature markers. A third, secretory state is defined by the low levels of expression of both S1 and S2 markers, suggesting that it represents an intermediate default state. The three secretory states show distinct localization along the proximal-distal airway axis. Using transgenic mice, we inactivated fibroblast growth factor receptor (FGFR) signaling shortly after birth, specifically in all secretory cells. We found that FGFR deficient cells reduced the levels of distally expressed markers, including epithelial type 2 (AEC2) -related genes and upregulated AEC1-related genes. This suggests FGFR activity promotes proper distalization of the airway epithelium and is maybe required for the function of distal bronchiolar secretory cells in homeostasis and upon injury.  

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.