Abstract
The function of the lung is closely coupled to its structural anatomy, which varies greatly across vertebrates. Although architecturally simple, a complex pattern of airflow is thought to be achieved in the lizard lung due to its cavernous central lumen and honeycomb-shaped wall. We find that the wall of the lizard lung is generated from an initially smooth epithelial sheet, which is pushed through holes in a hexagonal smooth muscle meshwork by forces from fluid pressure, similar to a stress ball. Combining transcriptomics with time-lapse imaging reveals that the hexagonal meshwork self-assembles in response to circumferential and axial stresses downstream of pressure. A computational model predicts the pressure-driven changes in epithelial topology, which we probe using optogenetically driven contraction of 3D-printed engineered muscle. These results reveal the physical principles used to sculpt the unusual architecture of the lizard lung, which could be exploited as a novel strategy to engineer tissues.
INTRODUCTION
Function dictates form, and as a consequence, the physiology of many organ systems across the evolutionary tree shows conservation in underlying anatomy. The lungs, however, are radically different across classes of vertebrates. Both mammals and birds have lungs that are structured anatomically into distinct tissue compartments that physically segregate the conduction of air (through bronchi and air sacs, respectively) from gas exchange (through alveoli and parabronchi), despite the fact that air flows tidally in the mammal but unidirectionally in the bird. In contrast, the lungs of many reptiles are simple sacs in which both conduction and gas exchange take place in the same anatomic chamber. Unidirectional airflow was thought to require the complex anatomy of the parabronchial lung until the recent discovery of unidirectional flow patterns in the simple sac-like lungs of the green iguana lizard (1). This pattern of airflow is hypothesized to depend in part on the unique, honeycomb-shaped structure of the wall of the lizard lung (2), but the biological and physical basis of its morphogenesis has not been reported.
RESULTS AND DISCUSSION
To define the physical mechanisms that sculpt the wall of the lizard lung, we established the brown anole (Anolis sagrei) as a model system. We selected this species because of its small size, short incubation time (~30 days) and high breeding potential (3), and because a reference genome for the Anolis genus has been published (4). The lung of the adult anole is a single-chambered (unicameral) hollow lumen surrounded by a simple cuboidal epithelium. The lizard lung is faveolar, meaning that septae or trabeculae generate shallow primary and secondary corrugations in a hexagonal, honeycomb-shaped pattern that increases the surface area available for gas exchange (Fig. 1, A and B). In the anole embryo, the lung buds into a simple wishbone-shaped structure that separates from the ventral foregut, similar to mammalian and avian lungs (Fig. 1C). Between embryonic days (E) 5 and 6, the lumen of the wishbone inflates (Fig. 1C), resulting in a marked decrease in the aspect ratio of the lung (Fig. 1, C and D, and fig. S1A). Inflation is characterized by high levels of proliferation throughout the entire organ (fig. S1B) as well as apical-basal thinning that increases the cross-sectional area of the epithelial cells (Fig. 1, E and F). At E7, the initially smooth epithelium (fig. S1C) forms into the rounded corrugations (Fig. 1C) that eventually give rise to the faveoli, which are alveolus-like structures lined by a thin, squamous epithelium used for gas exchange (5). The formation of epithelial corrugations coincides with an increase in the aspect ratio of the lung from E6 to E7 (Fig. 1D and fig. S1A).
Smooth muscle shapes the developing lung epithelium of the mouse (6, 7) and chicken (8) and also drives epithelial morphogenesis in other organs including the mouse gut (9, 10) and prostate (11). Mature trabeculae in adult lizard lungs contain a smooth muscle core (5), contraction of which maintains the lung structure and promotes airflow (12). We therefore mapped the spatial pattern of α-smooth muscle actin (αSMA)–expressing cells over developmental time in the embryonic anole lung. During the inflation stage at E6, αSMA+ cells are spread uniformly over the basal surface of the epithelium (Fig. 1G). As epithelial corrugations appear at E7, the αSMA+ cells form a hexagonal meshwork of thick bundles (Fig. 1, H and I) that creates a contractile cage around the entire epithelium (movie S1), somewhat reminiscent of the myofibroblast network around the alveoli in the mammalian lung (13) but at a much larger length scale. Epithelial corrugations only emerge from the gaps within the hexagonal mesh (Fig. 1, I and J), which is localized immediately adjacent to the regions of the highest epithelial curvature (fig. S1D), suggesting that the differentiation of this contractile tissue might be linked to morphogenesis of the epithelium.High-magnification imaging revealed that the initially uniform αSMA+ cell population contains predominantly isotropic actin filaments (Fig. 2, A and F). As development proceeds, the actin filaments align as the αSMA+ cells coalesce into horizontal bundles along the medial-lateral axis of the lung (Fig. 2, B, C, and F, and fig. S2A). These horizontal bundles then thicken (Fig. 2D and fig. S2A) and are later joined by thin connective bundles along the anterior-posterior axis (Fig. 2E and fig. S2A), thus generating the smooth muscle meshwork….
