Neuroendocrine cell hyperplasia of infancy (NEHI) is a distinctive children lung disease with hyperplasia of bombesin-immunopositive neuroendocrine cells within distal bronchioles and alveolar ducts. NEHI results in significant morbidity in the newborn and young children and their pulmonary symptoms often last into adulthood. To date, NEHI remains significantly understudied with respect to molecular mechanisms of pathogenesis. The familial occurrence of NEHI suggests the possibility of a genetic etiology for this disorder. A heterozygous mutation in NKX2.1 (p.Arg191Leu) was identified in an extend family with NEHI. NKX2.1 is a transcription factor specifically expressed in the lung and regulates early lung development and cellular differentiation. In this proposal, we will deploy our human system as a novel way to investigate if this NKX2.1 mutation is a cellular intrinsic mechanism underlying the aberrant neuroendocrine cell prominence, a cardinal pathological phenotype in NEHI. By using our advanced stem cell technology and crispr-cas9 genome-editing tool, we will introduce multiple NKX2.1 missense point mutations (one NEHI-associated and three non-NEHI associated NKX2.1 mutations) into human airway stem cells. Then we will convert them into functional respiratory epithelium and study many NEHI pathogenic features. Furthermore, we will address our hypothesis that NEHI-associated NKX2.1 mutation causes NEHI pathogenic phenotypes by influencing neuroendocrine cell fate determination particularly through Wnt and Notch signaling pathways. We will also perform transcriptomic profiling and statistical analysis to identify transcriptional networks and regulatory programs unique to neuroendocrine cell. Finally, we will set up a screen platform to study the effect of signaling pathway modulators on neuroendocrine cell differentiation in human. We expect that our work will help to elucidate the role of NKX2.1 familiar mutation and key molecular signaling events for neuroendocrine cell differentiation and pathogenies of NEHI and other lung diseases exhibiting an increase in neuroendocrine cell number.