Vertebrate lungs and insect tracheal systems are respiratory organs that facilitate gaseous exchange between the air and body tissues. During development, when these organs mature and form a three-dimensional tubular network, the tubes are filled with liquid. However, shortly before birth or hatching, the tubes absorb the liquid and become filled with gas, a process that is known as liquid clearance. The underlying mechanisms of liquid clearance were largely unclear. In a study published recently in Developmental Biology, a team of scientists from Göttingen, Germany, guided by Reinhard Schuh demonstrated that gas-filling of Drosophila tracheal tubes depends on Waterproof, a fatty acyl-CoA reductase, which seems to be required for the production of a hydrophobic coat lining the inner tube wall that reduces the liquid's tensile strength so that gas bubbles are produced to fill the tubes.
The tracheal system of Drosophila is a model for studying the development of tubular networks in animals. Its development starts during mid-embryogenesis with the differentiation of groups of tracheal cells that invaginate from both sites of the embryo's lateral body wall. Without further cell divisions, these cells migrate, extend and form branches that finally fuse and interconnect to establish a liquid-filled tubular network. To prevent tube collapse, the tracheal cells produce a ridged cuticle forming the inner tube wall that, in addition, is covered by a coat of hydrophobic waxes. Shortly before hatching, liquid clearance and gas filling are initiated by a single gas bubble, which forms stochastically in one of the trunks, expands and finally fills the entire tubular lumen within 20 min. To uncover genes that are potentially involved in this process, Schuh's team performed an RNA interference screen. They silenced the expression of various genes and inspected the embryos by light microscopy to determine whether gas filling was impaired. In doing so, they identified a gene termed waterproof, which was specifically expressed in the tracheal system. Loss of function of the gene completely abolished gas filling while tracheal branching and maturation were unaffected.
The scientists were fascinated by this finding, because waterproof encodes a fatty acyl-CoA reductase, which is required for synthesis of the long-chain fatty alcohols that are essential for the production of the waxy coat on top of the tracheal cuticle directly facing the tracheal lumen. Indeed, when they analysed the ultrastructure of the tracheal cuticle by electron microscopy, they observed that in waterproof-deficient flies this innermost layer was damaged. To further analyse the function of waterproof, the researchers designed a set of elaborate genetic experiments that allowed them to direct expression of the waterproof gene in specific regions of the tracheal system. These experiments clearly indicated that regardless of where waterproof is expressed in the tracheal system, it always rescues the loss of the gas-filling phenotype of waterproof-deficient embryos, indicating that Waterproof acts in the entire tubular system regardless of the genotype of individual tracheal cells.
Taken together, these findings suggest that the tracheal gas-filling process depends on the presence of a hydrophobic coat on the surface of the tracheal cuticle. This coat presumably reduces the tensile strength of the liquid inside the tracheal lumen, which triggers the formation of a gas bubble that acts as a nucleation point for gas to permeate the entire tubular network. As Waterproof is also conserved in vertebrates, it is tempting to speculate that orthologues may have a similar function in lung airway clearance.