Investigating the Role of NKX2-1 in Early Human Lung Development Using an Induced Pluripotent Stem Cell (iPSC) Model

点击翻译按钮获取中文摘要

NK2 Homeobox 1 (NKX2-1) is a critically important transcription factor in lung development, with all lung epithelia being derived from an NKX2-1+ progenitor pool. Nkx2-1 knockout mice have hypoplastic lungs, and humans with NKX2-1 mutations suffer from respiratory insufficiency, hypothyroidism, and neurological problems, a disease known broadly as brain-lung-thyroid syndrome. It is known that NKX2-1’s importance is derived from its ability to influence the expression of downstream target genes, which include genes like surfactant proteins and secretoglobins, which contribute significantly to lung function. Despite its known importance, NKX2-1’s role in early human development is not fully defined. Studies into early human development have been significantly hindered by difficulties in access to human fetal tissue and issues with genetically manipulating fetal derived cells. A recently developed technology that allows researchers to investigate early human development is the induced pluripotent stem cell (iPSC) system, which allows for generation and subsequent differentiation of stem cells to tissue types of choice. Our lab and others have developed protocols to generate airway and alveolar cells from iPSCs, actively recapitulating development along the way. Our group has also gained expertise in technologies to characterize and manipulate our cells, including single cell transcriptomics and CRISPR interference (CRISPRi). In this project, we seek to synergistically combine the components of this scientific toolbox to study how NKX2-1 influences early human development. Using our iPSC system, we seek to test the hypothesis that NKX2-1 plays a central role in lung specification and patterning, and that the downstream targets of NKX2-1 are context dependent and vary based on cell-type and developmental time point. We seek to do this through genomic binding assays to identify NKX2-1 binding loci at several points in development. In addition, we will perform single cell mRNA sequencing with an NKX2-1 mutant cell line in comparison with an isogenic cell line with this mutation corrected. Using these two approaches we hope to identify NKX2-1’s binding loci across development, and to observe the molecular consequences that come with an NKX2-1 mutations. We also seek to modulate expression of downstream targets of NKX2-1, including SOX2, TP63, and NKX2-1 itself to further deduce the gene regulatory network by which NKX2-1 acts. Our iPSC-based model allows for the unprecedented coupling of genetic manipulation and high-resolution transcriptomics to further understand how a critical transcription factor influences development.