Conserved Mechanisms in the Formation of the Airways and Alveoli of the Lung

Abstract

Branching is an intrinsic property of respiratory epithelium that can be induced and modified by signals emerging from the mesenchyme. However, during stereotypic branching morphogenesis of the airway, the relatively thick upper respiratory epithelium extrudes through a mesenchymal orifice to form a new branch, whereas during alveologenesis the relatively thin lower respiratory epithelium extrudes to form sacs or bubbles. Thus, both branching morphogenesis of the upper airway and alveolarization in the lower airway seem to rely on the same fundamental physical process: epithelial extrusion through an orifice. Here I propose that it is the orientation and relative stiffness of the orifice boundary that determines the stereotypy of upper airway branching as well as the orientation of individual alveolar components of the gas exchange surface. The previously accepted dogma of the process of alveologenesis, largely based on 2D microscopy, is that alveoli arise by erection of finger-like interalveolar septae to form septal clefts that subdivide pre-existing saccules, a process for which the contractile properties of specialized alveolar myofibroblasts are necessary. Here I suggest that airway tip splitting and stereotypical side domain branching are actually conserved processes, but modified somewhat by evolution to achieve both airway tip splitting and side branching of the upper airway epithelium, as well as alveologenesis. Viewed in 3D it is clear that alveolar "septal tips" are in fact ring or purse string structures containing elastin and collagen that only appear as finger like projections in cross section. Therefore, I propose that airway branch orifices as well as alveolar mouth rings serve to delineate and stabilize the budding of both airway and alveolar epithelium, from the tips and sides of upper airways as well as from the sides and tips of alveolar ducts. Certainly, in the case of alveoli arising laterally and with radial symmetry from the sides of alveolar ducts, the mouth of each alveolus remains within the plane of the side of the ductal lumen. This suggests that the thin epithelium lining these lateral alveolar duct buds may extrude or "pop out" from the duct lumen through rings rather like soap or gum bubbles, whereas the thicker upper airway epithelium extrudes through a ring like toothpaste from a tube to form a new branch.

Keywords: airway; alveolus; branching; conserved; morphogenesis.

FIGURE 1

Human lung aged 12 weeks gestation: whole mount specimen stained red for smooth muscle actin marking smooth muscle and green for e-cadherin marking the epithelium and imaged by confocal microscopy. (A) The smooth muscle actin (red) sleeve surrounding the peripheral stereotypically branching peripheral main airway epithelium (green) extending from left to tight across the panel. Round holes in the sleeve can be clearly seen where lateral branches arise (marked as Orifice). The sleeve of smooth muscle (marked as Stalk) extends outward toward the distal tips of the epithelium, whereat pairs of tips (marked as Tip) can be seen to extrude through round holes (Orifice) in the smooth muscle sleeve to make new peripheral epithelial branches. Smooth muscle knot-like structures between and around the peripheral tip branches are marked as Knot. (B) A more peripheral, juxta pleural view of the more peripheral branching process in the same specimen. The more proximal smooth muscle sleeve (Stalk), the peripheral holes in the sleeve (Orifice) where new branches. Also, the knot-like structure (marked as Knot) of smooth muscle that appears to divide and orient the specific planar direction of epithelial branch clefting and hence of tip extrusion. The plane of the pleura is at the top of the image. The images are adapted with kind permission from Danopoulos et al. (2020). Scale bars 100 microns.

FIGURE 2

Mouse lung aged 14 days postnatal, a time by which alveolarization has substantially progressed in mice. A digital 3D reconstruction of a whole mouse lung imaged by Vibra-SSIM technology. Membrane fluorescence is imparted by the tomato transgene. This stereo-optical view shows a broncho-alveolar duct junction (marked as BADJ), viewed en face and identified by the sharp columnar to flattened epithelial transition. Two alveolar ducts are viewed proceeding distally to this BADJ structure, one posteriorly and interiorly while a second duct (marked as Alveolar duct) proceeds toward the left from the BADJ. Many individual alveolar orifices (marked as Alveolar orifices) are seen arising from the lateral surface of this alveolar duct. In this 3-dimensional view the mouth or orifice of each alveolus or air sac is surrounded by a round purse string like structure that circumscribes the entrance to each alveolus. Some of the purse strings can also be viewed in oblique cross section, where they appear as the tips of finger-like structures, previously delineated in the literature as “alveolar septae.” A pulmonary vein is also viewed in 3D. Alveolar capillaries can also be discerned in cross section between alveolar surfaces. Scale bar 100 microns.

FIGURE 3

Some key characteristics of peripheral alveoli of the mouse lung at 14 days postnatal age. (A) Digital reconstruction of a Vibratome serial section imaging microscopy (Vibra-SSIM) image of peripheral air sacs and their subjacent alveolar capillary plexi, looking outward from the mouth of the air sacs toward the pleural surface. The mouse in question has green membrane fluorescence. Scale bar is 50 microns. (B) The family of air sacs surrounded by the dotted box in (A) is shown enlarged with its gas exchange surface digitally rendered in purple to give contrast with the green background image. At this magnification the lumena of alveolar capillaries can be clearly seen in cross section within the lung mesenchyme. There are 5 alveoli connected to a common central lumen and this is typical for alveolar structures adjacent to the pleura. The irregular shape and surface rugosity of individual alveoli are also clearly shown. Scale bar is 25 microns. (C) Shows second harmonic generation (SHG) in white of collagen fibers located within the mesenchyme of alveolar mouths. Several groups of 3 or 5 air sacs are delineated by interconnected SHG collagen purse strings surrounding the mouths of individual air sacs. In some of them a bed spring like structure can be seen to extend proximally up the neck of a group of air sacs, surrounding the most distal portion of the alveolar duct. Scale bar is 25 microns. (D–F) Show 3-dimensional printed views of these air sacs rendered in purple in (B) and shown here as complementary 7 oblique views of a red plastic 3-dimensional solid object. (D) Shows the proximal to distal view of the round alveolar duct leading into a family of 5 alveoli. (E) Shows an oblique view of the irregular and rugose surface of the distal pleural surface of the family of 5 alveoli. (F) Shows a distal to proximal view of the five interconnected air sacs. An elastic band has been placed around the mouth of one air sac to illustrate where the collagen mouth rings shown in (C) lie in relation to the alveolar orifice.

FIGURE 4

Shows a 3D printed construct of the surface of alveoli and distal alveolar ducts in a 28 days postnatal age distal mouse lung acinus. (A) Shows a proximal to distal view down three branches of the same distal alveolar duct looking down through their alveolar orifices into families of distal alveoli. (B) Shows the topography of distal alveolar families adjacent to the pleural surface. (C) Shows an oblique view of the 3D construct with both the pleural surface and the adjacent interior surface alveoli clearly visible.