Title: Airway Basal Cells in Development, Injury-Repair, and Homeostasis
Description: Basal cells (BCs) are multipotent tissue-specific stem cells of a variety of organs including the skin, digestive and respiratory tract. BCs are broadly identified by expression of Krt5, Krt14 and the transcription factor p63. In the adult airways, BCs are not only important for normal maintenance but also crucial for epithelial repair after injury. However, the embryonic origin of these adult stem cells remains elusive. Previous reports showed that p63+ cells appear early during airway development, but these do not express markers of adult BCs, raising the question whether these cells represent BC precursors. Moreover, little was known whether embryonic BCs have an impact in the adult pool of progenitors that mediate responses of the lung to injury or pulmonary diseases. The goal of this thesis is to address these gaps of knowledge using a variety of technologies, including functional and lineage tracing analysis in vivo in mouse genetic models, injury modeling, high-throughput profiling and gene regulation approaches.

This thesis is to comprehensively characterize airway BCs in development, injury-repair, and homeostasis. These studies revealed a previously unrecognized broader role of embryonic p63+ cells in the establishment of the stem cell pools of the lung pre and postnatally. Surprisingly, lineage analysis showed that early in development these cells were able to generate all epithelial cell types of the airways and alveolar compartment. However, as development proceeds, they underwent two sequential lineage segregation events to finally generate two regionally distinct adult stem cell pools. One of these became the well-known BCs that populate extrapulmonary airways through an undescribed maturation process from the perinatal stage to adulthood, and the other was identified as a rare stem cell pool in the pseudostratified epithelium of intrapulmonary airways which maintained immature and quiescent throughout lifetime. Moreover, the latter responded uniquely to lung injury induced by H1N1 viral infection.

Recent studies have demonstrated that BC-like p63+ Krt5+ cell clusters (“Krt5+ pods”) are ectopically present in the areas of severe alveolar injury by H1N1 viral infection. The presence of these pods has been associated with pathological scars in several human pulmonary diseases including idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS). However, their cellular origin has been intensely debated. This thesis showed that this rare progenitor pool is established during embryonic development when airways are still branching. Further characterization demonstrated a p63 gene dosage dependency in the specification/maintenance of this rare progenitor pool. By utilizing multiple lineage-tracing lines, an underappreciated diversity of this pool was revealed by showing a novel subpopulation carrying secretory lineage marker spatially restricted to intrapulmonary airways. Further molecular characterization and genetic manipulation of this rare progenitor pool may provide valuable cues to understand the pathogenesis about pulmonary disorders and to develop effective therapies.

Moreover, the molecular signatures of tracheal embryonic E18.5 preBCs and adult TrBCs were generated through high-throughput profiling, which provided hints about the genetic regulation of airway BC maturation process and generated potential molecular landmarks for the in vitro ES/iPS cell differentiation towards airway BCs. In addition, single cell RNA-sequencing analyses revealed heterogeneity of adult BCs in the tracheal and esophageal epithelia. Lastly, candidate master regulators of their differentiation programs in homeostatic and metaplastic states were identified through unbiased systems biology algorithms, which will be further validated in functional assays in the near future. Taken together, the studies in this thesis comprehensively characterized airway BCs in development, injury-repair and homeostasis. This thesis work showed the newly identified p63+ airway progenitors before E10.5 are multipotent for all lung epithelial lineages and this multipotency gets restricted to proximal fate at E10.5.

In the adult injury-repair, this thesis work for the first time revealed that the H1N1-induced Krt5+ pods are generated by bronchial p63+ Krt5- progenitors, which originate from a subpopulation of E13.5 intrapulmonary p63+ progenitors. At homeostasis, this thesis work uncovered a previously underappreciated heterogeneity of BCs in both airways and esophagus, and provided molecular foundations for further explorations into the mechanistic perspectives of BC cellular identity maintenance.