INTEGRATION OF MECHANICAL AND MOLECULAR CUES DURING DISTAL LUNG DEVELOPMENT

Project Summary
The developing alveolus is lined by two epithelial cell types: thin alveolar type 1 (AT1) cells that provide the gas-
exchange surface, and cuboidal AT2 cells that secrete pulmonary surfactant. During embryonic development
both AT1 and AT2 cells derive from a distal progenitor pool. Alveolar epithelial differentiation is influenced by
multiple molecular signals –– especially FGF–– and recent work suggests that another key driver in late gestation
is mechanical stretch. Questions remain as to how these signals are regulated to precisely time alveolar
differentiation (as both are present at and critical for earlier stages of lung development), and to what extent are
they reactivated in adulthood during regeneration. Recent work in embryonic stem cells (ESCs) points to a
specific cell-intrinsic mechanical property (known to resist cell stretch) that blocks differentiation – cellular
membrane tension (CMT). In ESCs, high CMT represses differentiation by blocking the endocytosis of a FGF
receptor, which modulates its downstream signaling. While upstream FGF signaling was always active in ESCs,
it was the shunting of its downstream signaling that was CMT-dependent and that was critical in timing
differentiation. Could CMT also be involved in the embryonic lung to block alveolar differentiation? Does CMT
re-emerge in the adult lung during alveolar epithelial regeneration? If so, how does it affect activation of
facultative AT2 progenitors? To address these questions, we propose to investigate CMT in both the developing
and adult mouse lung. We hypothesize that alveolar epithelial differentiation is blocked by high CMT, which
regulates downstream signaling via inhibiting endocytosis and resisting stretch. During alveolar regeneration, we
hypothesize that facultative AT2 progenitors are activated by increased CMT that subsequently reduces to direct
AT1 and AT2 differentiation. To answer these questions, we will use a combination of culture and transgenic
mouse experiments wherein measurements will be taken primarily by confocal microscopy. Our preliminary data
indicate that CMT reduces immediately prior to (and is required for) differentiation during development.
Concomitantly, we observe an increase of endocytosis prior to differentiation and that its inhibition also blocks
differentiation in culture. Finally, we developed two approaches to measure mechanical properties of individual
cells within living lung tissue. In doing so, we observed higher CMT in progenitors versus adult AT2 cells, as well
as increased stiffness of AT2 cells relative to neighboring cells in the adult lung. Our approach is innovative, as
neither CMT nor endocytic regulation of FGF has been studied before in the context of lung development and
regeneration. Moreover, we have established two novel approaches to measure mechanics in living lung tissue
with cellular resolution. If successful, we will have established a novel mechanism of cell-intrinsic
mechanoregulation for the lung field which will be informative in understanding pulmonary diseases with aberrant
epithelial differentiation. Further, our findings will guide development of novel diagnostics or therapeutics, either
for direct use in patients or in culture to control stem cell differentiation.

Grant Number: 5R01HL171056-02
NIH Institute/Center: NIH
Principal Investigator: Douglas Brownfield