Title: Proximal-distal patterning of the lung
Description: The mammalian lung is an exquisitely designed organ with two structurally distinct compartments, one that comprises multiple generations of branched tubules to conduct and clean the air (airways) and another that consists of a vast network of thin-walled alveolar structures to allow gas exchange (alveoli). In the embryo these compartments arise from highly dynamic patterning events during branching morphogenesis that define two major domains, a proximal (Sox2+) and a distal (Sox9+), which ultimately form the airways and alveoli, respectively. Although the signaling pathways controlling branching morphogenesis are increasingly understood, the mechanisms that regulate the transition zone (TZ) between the proximal and distal domains are still elusive. The goals of this thesis are to identify markers and molecular regulators of the TZ, to examine the role of Hippo-Yap signaling in the establishment of the TZ and to investigate the evolutionary conservation of this process in the lung of the snake Pantherophis guttata, which lacks a branched airway tree.

Using a combination of mouse genetics, single cell RNAseq, computational approaches and immunofluorescence-confocal analyses I show that Yap transcriptional activity and nucleocytoplasmic shuttling are essential for patterning of the lung by being pivotal for initiation of the events that give rise to the TZ, as well as for subsequent lineage differentiation of compartment-specific progenitors. I show that cytoplasmic sequestration of Yap in Sox2+ epithelial progenitors is a crucial mechanism to prevent the deleterious effects of maintaining nuclear Yap once airway progenitors are specified. Moreover, PISCES-inferred protein activity profiling identified Hspa8, Krt19, Btg2, Anxa2, Cldn10 and Icam1 in the TZ. Notably, analyses of Yap loss and gain function in mice revealed Icam1 as a key marker of the TZ and a downstream target of Yap. Lastly, I show that Sox2 and Sox9 are conserved markers of proximal (bronchiolar) and distal (respiratory) cell fate in the respiratory tract. However, in the snake Pantherophis guttata, the early proximal-distal event that specifies the Sox9+ compartment in the mouse appears delayed.

I speculate that proximal-distal patterning in murine lung development actually represents a precocious specification event of respiratory identity, as well as that this ultimately enabled the incorporation of a program of branching morphogenesis in the ancestral program of lung development. Considering that in humans the primordial lungs are double Sox2+ Sox9+, this suggests an unsuspected heterogeneity in the early lung developmental events of human, mice, and reptiles. Altogether, the findings revealed by this work open new avenues of research to further understand the molecular mechanisms that drive lung development.