Lab Projects
Holistic view of lung biology with cellular precision
As genome sequencing has cataloged DNA blueprints and cell atlas endeavor is cataloging constituent cell populations of each organ, our view of lung biology is increasingly genome-wide with a single-cell precision. This level of understanding is timely given the tremendous health and economic burden of lung diseases including lung cancers, pediatric and chronic respiratory diseases and infections, exacerbated by the ongoing pandemic. Our lab has been building up expertise in all resident lung cell types, namely its epithelial, endothelial, and mesenchymal lineages, as detailed below. This multi-lineage approach is necessary because the myriad of lung cell types are not compartmentalized, but intertwined for physical and chemical signaling in units of airways and alveoli that are repeated thousands to millions of times. Accordingly, normal development, homeostasis, and regeneration, as well as their derailment in diseases, are no longer an affair of a single gene or a single cell type, but a system-level resilience or deterioration. A holistic understanding of the entire genome and its use in all pertinent cell types, woven together with bioinformatics, is needed to advance lung biology and therapy.
Signaling roles of AT1 cells toward the vasculature and mesenchyme
Alveolar type 1 (AT1) cells, an ultrathin expansive epithelial cell type, cover 95% of the gas exchange surface. Recognizing their omnipresence, we have proposed and provided evidence that AT1 cells are not simply a structural component, but also have signaling roles toward surrounding vasculature and mesenchyme. We published that: (1) AT1 cells, instead of the surfactant-producing alveolar type 2 (AT2) cells, express the key angiogenic molecule Vegfa; (2) AT1 cell-derived Vegfa is required to specify a previously unknown capillary endothelial cell population marked by Car4, contrasting the conventional wisdom of the homogeneity of pulmonary capillaries; and (3) these Car4+ endothelial cells (also known as CAP2 or aCap cells) have unique morphology, localization, and function in alveologenesis – with implications in bronchopulmonary dysplasia (BPD). Our finding of the Vegfa signaling from AT1 cells prompted us to search for and identify additional signaling ligands, specifically Wnt3a and Wnt7a. We found evidence and are dissecting the hitherto unappreciated role of canonical Wnt signaling in the lung mesenchyme, building on our recent 3-axis classification of lung mesenchymal cells.
Transcriptional and epigenetic mechanisms of cell fate specification and activation
Combinatorial action of transcription factors and cell-type-specific deployment of epigenetic marks mold hundreds of cell types out of the same DNA blueprint. The lung alveolar epithelium provides a robust system to dissect this fundamental question of cell fate determination, as both AT1 and AT2 cells arise from Sox9+ progenitors and AT2 cells can self-renew and give rise to AT1 cells. We published that canonical Wnt signaling components including Ctnnb1 and the 4 Lef/Tcf transcription factors are required to promote the progenitor program, as well as suppress premature alveolar differentiation and the aberrant gastrointestinal fate in Sox9+ progenitors. We also published that, contrary to prior studies, the lung lineage transcription factor NKX2-1 is expressed by both AT1 and AT2 cells and is required for their specification and maintenance; and that cell-type-specific cofactors, YAP/TAZ/TEAD in AT1 cells, direct NKX2-1 to cell-type-specific binding sites. We are currently pursuing the corresponding cofactor CEBPA in AT2 cells. Our resulting experience in single-cell and bulk epigenomics is applied to AT2 cell activation during viral injury.
Never a dull cell – tools to study subcellular structures and apoptosis
Molecular mechanisms must be executed by cells whose toolbox includes proliferation, apoptosis, as well as subcellular structures such as cytoskeleton, junctions, adhesions, organelles, etc. How molecular events are converted into cellular changes driving tissue morphogenesis and homeostasis is poorly understood, especially in the lung made of cells with elaborate processes in 3D. This is partially due to lack of tools to visualize and manipulate the aforementioned cell biology. We have built ROSA knock-in alleles to conditionally express fluorescent fusion proteins to visualize microtubules, actin, adherens junctions, mitochondria, and lysosomes, all in distinct colors, as well as a BCL2 gene to block apoptosis. We have published the first of our tricolor cell biology reporter, dubbed Kaleidoscope. Applicable to any cell type in any organ, we are using these tools to study AT1/AT2 cells and endothelial cells and their changes upon injury, as well as the significance of developmental apoptosis of alveolar myofibroblasts.
Nature’s mutations – species-unique morphogenesis and cell fates
Mouse and human genetics has been instrumental to causally link gene mutations to disruptions in function. Evolution, in contrast, has engineered mutations to unlock species-unique morphology and physiology, representing an alternative, powerful path to identify gene function. Unlike traditional evolutionary biology that focuses on DNA changes and comparative anatomy, we are exploring single-cell evolutionary biology, leveraging the ability of single-cell genomics to not only depict tissue biology at a genomic scale with a single-cell precision, but also delineate the transcriptomic and epigenomic output of the DNA blueprint in the tissues of interest. Notably, the mammalian lungs use bidirectional air flow with alveoli forming at the end of the conducting airway tree, whereas the avian lungs use unidirectional air flow with alveoli forming on the side of parabronchi, like kernels on a corn cob. We are extending this reasoning and single-cell approach to the amphibian lung and, in the future and through collaboration, to any organ in any species with a sequenced genome.
Somatic functional genomics – harnessing the power of CRISPR and single-cell genomics
Classical mouse genetics engineers germline mutations that can be spatiotemporally activated in cell types and organs of interest. This process is time consuming mainly due to the necessity of generating and breeding germline alleles. However, the phenotypic analysis is often limited to targeted cells, raising the possibility of somatic functional genomics where genes are inactivated or activated in somatic tissues with the standard or variant CRISPR technology and the resulting tissues profiled via single-cell genomics. Furthermore, gRNAs can be bar-coded and decoded using the same single-cell platform to query multiple genes in a single experiment. We are setting up gene delivery system to target the lung. The same approach can be extended to human tissues where germline engineering is obviously not feasible.
