The biomechanical basis of biased epithelial tube elongation in lung and kidney development

ABSTRACT During lung development, epithelial branches expand preferentially in a longitudinal direction. This bias in outgrowth has been linked to a bias in cell shape and in the cell division plane. How this bias arises is unknown. Here, we show that biased epithelial outgrowth occurs independent of the surrounding mesenchyme, of preferential turnover of the extracellular matrix at the bud tips and of FGF signalling. There is also no evidence for actin-rich filopodia at the bud tips. Rather, we find epithelial tubes to be collapsed during early lung and kidney development, and we observe fluid flow in the narrow tubes. By simulating the measured fluid flow inside segmented narrow epithelial tubes, we show that the shear stress levels on the apical surface are sufficient to explain the reported bias in cell shape and outgrowth. We use a cell-based vertex model to confirm that apical shear forces, unlike constricting forces, can give rise to both the observed bias in cell shapes and tube elongation. We conclude that shear stress may be a more general driver of biased tube elongation beyond its established role in angiogenesis. This article has an associated ‘The people behind the papers’ interview.


SUPPLEMENTARY FIGURES
: Measuring epithelial morphology in growing epithelial tubes (A) 3D morphometric measurements of branch length and average circumference for a developmental timeline of a mouse lung. Specimens were serially isolated between E10.5 and E14.5 and carried the Shh GC/+ ; ROSA mT/mG reporter, which enabled the visualization of the embryonic epithelium. Branch length was measured from below the carina to the most distal tip, while average branch circumference was calculated for tubular cross-sections. Scale bars 50 µm. (B) E12.5 mouse lung with iso-surface overlays denoting tubular sections used for morphometric quantifications (blue). Scale bar 200 µm. (C) 2D morphometric measurements of length and diameter for an E11.5 lung cultured on a filter for 48h. Width scale in µm. Scale bars 200 µm.  Figure S4. Narrow luminal spaces are not the result of dissection /clearing conditions 3D rendering of mouse lung explants (top) and cross-sectional slices (bottom) of specimens (A) cleared using a short CUBIC regimen (2 days in reagent-1, 2 days in reagent-2) and a long CUBIC regimen (4 days in reagent-1, 4 days in reagent-2) displaying narrow luminal spaces. Similarly, (B) both explants dissected on ice and at room temperature (RT), and cleared using a short CUBIC regimen, also showed collapsed lumens. Furthermore, the same luminal morphology was observed in (C) CUBIC cleared whole-embryos imaged through thoracic cavity. Scale bars 100 µm. Development: doi:10.1242/dev.194209: Supplementary information  control cut below larynx cut above carina E11.5 E11.5 + 24h E11.5 + 48h Development: doi:10.1242/dev.194209: Supplementary information
Movie 4. Mesenchyme-free ureteric bud culture Time-lapse movie of a mesenchyme-free ureteric bud dissected at E11.5 and cultured for 60h. HoxB7/myr-Venus expression is shown in green, brightfield in grey. (AVI 10.8 MB).

Movie 5. Lung explant cultures with FGFR inhibitor SU5402
Culture of embryonic lungs (E11.5) under control conditions and with the treatment of the FGFR inhibitor SU5402 at different concentrations for 48h. (AVI 8.16 MB).

Movie 6. Lung explant cultures with MMP inhibitor GM60001
Culture of embryonic lungs (E11.5) under control conditions and with the treatment of the MMP inhibitor GM6001 at different concentrations for 60h. (AVI 5.3 MB).

Movie 7. High-resolution light-sheet microscopy time-lapse imaging of lung bud elongation
Time-lapse movie showing the development of an E11.5 left lung rudiment carrying the Shh GC/+; ROSA mT/mG construct. The specimen was mounted in a hollow cylinder made from low-melting-point agarose and filled with matrigel to replicate the native microenvironment and promote near-physiological growth. Imaging was done using the Zeiss Z.1 Lightsheet system for 34h. (AVI 2.38 MB). Development: doi:10.1242/dev.194209: Supplementary information Movie 8. Tracking of beads in the lung lumen Time-lapse movie of a 2 h spinning disk confocal acquisition (1 min time steps between frames) of fluorescent beads injected into the lung lumen. Bead tracks were generated in Imaris, green dots mark the detected bead at any given time point. (AVI 9.2 MB).

Movie 9. Outflow of fluid from the trachea opening
Spinning disk confocal imaging of fluorescent beads (488nm excitation channel, shown in green) overlayed with a brightfield image of the trachea of an E11.5 lung after 1h in low-volume culture. (AVI 2.5MB)