Poster U17 - Diffusion of inhibitory morphogens determines spacing between branches in the embryonic lung

Introduction: Tissue shape is closely related to tissue function. For example, the airways of the vertebrate lung exhibit a branched structure, which maximizes surface area within a confined volume and enables efficient delivery of air to the gas-exchange surface. Building this epithelial tree requires that branches avoid contact with each other. Several studies have suggested that such self-avoidance is intrinsic to the epithelial branches of the developing lung, but the underlying mechanism remains elusive. Here, we used the embryonic chicken lung as a model system to study how epithelial branches sense and avoid colliding with each other. Our data suggest that the embryonic lung epithelium uses inhibitory morphogens to control the spacing between developing branches.

Materials and

Methods: Fertilized chicken (Gallus gallus) eggs were incubated at 38°C in a humidified atmosphere. Lungs of embryos at embryonic day 5 (E5) to day 8 (E8) were microdissected and cultured ex-vivo at the air-liquid interface on a porous membrane floating on DMEM/F12 medium (without HEPES) supplemented with 5% fetal bovine serum and antibiotics. To examine patterns of cell proliferation, lung explants were pulsed with 5-ethynyl-2'-deoxyuridine (EdU) for 30 minutes. To examine the effects of branch proximity, either the first (b1) or the second (b2) secondary bronchus was physically removed using tungsten needles prior to culture. In inhibition studies, E6 lung explants were cultured in either RepSox or Dorsomorphin-containing culture media for 24 hours and then pulsed with EdU. Branches were visualized by fixing lungs in paraformaldehyde and carrying out immunofluorescence analysis for E-cadherin; samples were counterstained with DAPI to label nuclei.

Results, Conclusions, and Discussions: In the embryonic chicken lung, several secondary bronchi emerge from the primary bronchus between E5 and E8. While the number of secondary bronchi quadruples during this time, the average spacing between the branches remains nearly constant, with the stalks of neighboring secondary bronchi oriented parallel to each other. EdU analysis revealed that proliferative epithelial cells are evenly distributed throughout each branch at E6. However, we found a local decrease in proliferation of the epithelium adjacent to neighboring branches at E7, suggesting a correlation between branch orientation and patterns of growth.
To test whether this local decrease in proliferation results from interactions with the adjacent branch, we surgically removed one of the secondary bronchi from E6 lungs prior to culture for 24 hours. In contrast to the control, we found that epithelial cells remained proliferative when their neighboring branch was removed.
Previously, it has been shown that blocking either the transforming growth factor-beta (TGFβ) or bone morphogenetic protein (BMP) signaling pathway disrupts the spacing between branches in the developing kidney. To test whether epithelial branches of the embryonic lung also communicate with each other via TGFβ and/or BMP, we inhibited the receptors for these ligands using the small molecules RepSox and Dorsomorphin, respectively. Surprisingly, we found that adding Dorsomorphin to the culture media had no effect on the pattern of proliferation in the epithelial branches. In contrast, we found that the epithelium of lungs cultured in RepSox-containing media remains proliferative and eventually collides with its neighboring branch.
Using the chicken lung, which exhibits uniform spacing between its branches, allowed us to elucidate how epithelial branches sense each other during embryonic development. We showed that close proximity to a neighboring branch decreases epithelial cell proliferation, an effect that is lost when the neighboring branch is surgically removed. Our experimental results suggest that signaling from TGFβ, but not BMP, is responsible for tuning this pattern of epithelial proliferation and setting the spacing between branches. Collectively, these data show that diffusion of inhibitory morphogens regulates the spacing between branches and prevents them from colliding with each other in the developing chicken lung.

Acknowledgements (Optional): This work was supported in part by the National Science Foundation.