Fgf10 patterning at E13.5.

<p>Ventral and dorsal views of mouse lung at embryonic day E13.5. Whole mount in situ hybridization shows that <i>Fgf10</i> expression is strongly restricted to the distal part of the mesenchyme.</p

FGF10 diffusion accounts for Spry2 patterning.

<p>(<b>A</b>) <i>Spry2</i> whole mount in situ hybridizations before and after a branching event, courtesy of S. Bellusci <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036925#pone.0036925-Mailleux1" target="_blank">[11]</a>. Before the branching event, <i>Spry2</i> expression spreads on the entire bud’s width. After branching has occurred, <i>Spry2</i> expression splits and focuses on each new bud, while it weakens at the branching point. As <i>Spry2</i> expression is induced by FGF10, <i>Spry2</i> reports FGF10 reception. (<b>B</b>) Calculation of FGF10 flux predicted by the model in the same geometry. No adjustable parameters are used. Before the branching event, FGF10 diffusive flux spreads on the entire bud’s width. After branching has occurred, FGF10 flux spontaneously focuses on distal tips, just as <i>Spry2</i> expression. Although it only considers FGF10 diffusion from distal mesenchyme, the model accounts for the patterning of <i>Spry2</i>.</p

FGF10 diffusion accounts for branching morphogenesis.

<p>(<b>A</b>) Time lapse sequence of a growth simulation. The simulation relies on the laplacian model and couples the motion of the epithelial and mesothelial surfaces. Left image is a plot of the sigmoid growth response to FGF10 corresponding to the simulation. Colors in the mesenchyme stand for FGF10 flux, which focuses on distal tips. Branching occurs spontaneously and branches never meet each other, as observed in vivo. The epithelium to mesothelium distance is conserved, as branches never reach the mesothelium. (<b>B</b>) Results of three simulations with their respective growth responses. B1 features an other sigmoid growth response with a different spread. B2 and B3 feature linear growth responses with different values of <i>g</i>. The initial condition is always the initial tube displayed in A. The arbitrary scale is chosen the same for all (A) and (B) simulation results. While the morphologies obtained vary with the growth response, the initial non-branched tube always develops into a self-avoiding tree. (<b>C</b>) Time lapse sequence of a bud-scale 3D simulation based on the same model. The initial tube branches, while FGF10 flux focuses at bud tips, showing that the model and mechanisms are relevant to 3D geometry.</p

Gradient calculation in 3D reconstructions.

<p>Calculation of FGF10 flux in a 3D geometry reconstructed from embryonic mouse right cranial lobe at E12.5 with the same laplacian model and boundary conditions. The left image presents an upper view while the right image presents a side view. Both epithelial and mesothelial surfaces are displayed. The color code on the epithelial surface stands for the received flux. The same tip-effect is found in this geometry.</p

Branching mechanism and organ-scale self-organization.

<p>(<b>A</b>) The branching mechanism. Consider any prominence on the epithelium (here displayed in red). This bud increases the local gradient of concentration and thus the local flux of FGF10 received by epithelial cells. It will thus grow faster and be amplified. This instability mechanism is balanced by surface tension, which prevents thin branches to form. (<b>B</b>) The mechanism of self-organization. When two branches get too close, the local gradient of concentration in the interstitial mesenchyme tends to zero (red circle). Growth thus tends to zero and prevents branches from any collision. This mechanism, at the organ scale, leads to the self-avoiding bronchial tree.</p

Mesenchyme dynamics effects on the underlying branching architecture.

<p>Series of E11.25-E12 RCr 3-D reconstructions, (A) Upper panel: lateral views, lower panel: dorsal views, (B) Upper panel: ventral views, lower panel: lateral views. The RCr lobe quickly elongates along the anterior-posterior axis, first inducing planar bifurcations. The first side-branches (SB1 and SB2) sprout latter, as the mesenchyme thickness increases on the medial and dorsal sides respectively (A). The mesenchyme growth dynamics (white crosses and white dashed arrows) also induces absolute orientation changes of the previously formed branches (black and white dashed lines), rotation at the branching site (rounded black dashed arrow) and differential rate of bud growth (black dashed arrows) (B). Scale bar 100 µm.</p

Branching stereotypy and variability.

<p>(A) RCr lobe at E12.75. Planar bifurcations fill the angles and occur rigidly in the angle bisector plane (doted line). The side-branches sprout in front of the flat faces (first the medial/dorsal face). They grows in parallel directions and forms rows of bristle (dashed lines), even if they sprout from different parental branches. The side-branches are formed in a proximal-to-distal order (full circles and arrows) from the large perihilar region to the thin edges. Doted circle and arrow depict the next budding sites (see also Figure S1), where mesenchyme is going to enlarge. (B) Branching variations mostly occur in the more open spaces (here the rounded medial face), where a classical rosette (a) co-exist with poorly stereotyped bunches of sprouts (b, c). (C) The sprouting orientation of SB2 (side-branch 2) is highly variable (white arrow). SB2 also originate from variable sites: up, from (dashed white line) or down the PL branching fork. An optional side branch (SB2*) sprouts proximal to SB2 (white star) and modify the bifurcation plane of OB1 and OB2. (D) The PL branch exhibits polymorphic patterns of planar bifurcations or trifurcation (white crosses). The latter originate from large belly also corresponding to an optional-side branch site (white circle). Subtle differences in the mesoderm growth are associated with variable branching rate (white and black arrow). Of interest, the branching pattern also can raise nomenclature confusions: an apparent side-branch (underscored black cross) is indeed generated through end bifurcation (black cross). (E–H) E13.25 RCr lobes showing several morphological branching variants: (E) 3-D trifurcations, (F) rosette directly sprouting from the parental branch, or at the same site, a missing proximal branch (black star) leading to tripod, (G) elbow in the vicinity of another lineage and (H) variable rotation planes twisting the classical rosette (left panel) and the orthogonal bifurcations (right panel). Scale bar 100 µm.</p

Lung lobe packing in the mouse fetus thorax.

<p>Transversal sections of the thorax are performed at the embryonic day indicated and stained with HPS to show the in vivo relationships of the mouse lung. (A) During the pseudoglandular stage, the size and shape of the lung anatomical cavity is mainly constrained by dense tissues (chest wall, liver) or cavities under pressure (heart and larges vessels). RCr develops competing interface with RMd lobe (arrow heads) and slightly imprint the looser tissues of the chest wall (arrows). (B–F) Magnifications show that the RCr lobe surface fits the shape of the surrounding tissues, even if the parietal and visceral mesotheliums are not in strict abutment. The RCr lateral edge is tightly embedded between the chest wall and the heart/RMd. Ep: lung epithelium, Mes: lung mesenchyme, Pl: pleural cavity (outlined by the visceral and parietal mesothelium), Fl: luminal fluid; scale bar: 100 µm.</p

Overall growth coupling of the bronchial tree and the mesenchyme.

<p>Graphs plotting the length against the width (A), the width against the thickness (B) and the length against the thickness (C) of a series of E11.25-E13.5 RCr lobes. Using the same specimens, the surface and the volume of the bronchial tree were plotted against the respective surface and volume of the mesenchyme cell mass (E and D). The main dimensions are strongly correlated showing that slight inter-specimen differences occur while the overall shape of the RCr lobe is conserved. In comparison, the overall growth of the epithelial tree and the mesenchyme compartment are very strongly correlated at the lobe level. Number of analyzed specimens: 20. Distances are denominated in µm, surfaces in µm² and volumes in µm³.</p

Three-dimensional reconstruction of right cranial lobe full structure.

<p>(A) Dorsal view of a whole mount mouse lungs at E13.5 immunostained for E-cadherin (red) and counterstained with DAPI (bleu), showing both the airway epithelium architecture and the shape of the surrounding mesenchyme. Dotted lines show the trachea (Tr), the right (Rmb) and left (Lmb) main bronchi, the right cranial (RCr), right middle (RMd), right accessory (RAc), right caudal (RCd) and left (L) lobes. (B) Ventral view of a whole mount mouse lung immunostained for E-cadherin at E11.5 to show the epithelial tree. Cr, Md, Ac, Cd and L bronchi give rise to the related lobes. The unwanted background around the buds is a limiting factor to perform quantitative analysis. The procedure we developed (see Material and methods) allowed a highly precise 3D visualization of the bronchial tree (C) and the surrounding mesenchymal cell mass (D), with respect to their in vivo relationships. A, anterior; P, posterior; M, medial; L, lateral; D, dorsal; V, ventral; Scale bar: 200 µm.</p