Branched tubular organs, such as the lung and vascular system fulfill the respiratory needs of most animals. Optimal tissue function relies on the uniform sizes and shapes of the constituting branches in each organ. The Drosophila tracheal airways provide a recognized genetic model system for identification and characterization of tube size regulators. We found that the programmed secretion and assembly of the apical extracellular matrix (ECM) is required for the expansion of the trachea and salivary glands (SG) tubes. We have characterized Vermiform (Verm) and Serpentine (Serp), two chitin-binding proteins with predicted polysaccharide deacetylase domains (ChLDs). Verm and Serp mutants show overelongated tubes, suggesting that luminal ECM modification restricts tracheal tube elongation. The luminal deposition of ChLDs, but not other secreted components, depends on paracellular septate junction integrity (SJs) in the tracheal epithelium. Deletion of the deacetylase domain renders Serp-GFP intracellular, arguing that the deacetylase domain harbors uncharacterized secretion signals. To explore this possibility we transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The Gasp-Deac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To identify such signals we characterized conserved sequence motifs in the Serp deacetylase domain. Mutations of the N-glycosylation sites gradually reduced Serp-GFP luminal deposition suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in three conserved amino acid stretches completely blocked the ER-exit of Serp-GFP. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors.

Tubes are a fundamental unit of organ design. Most of our major organs like the lung, kidney and vasculature are composed primarily of tubes. To identify fundamental biological principles of tubular organ formation we used the respiratory organ of Drosophila melanogaster, the trachea.This work dissects embryonic trachea maturation. Three precise epithelial transitions occur during airway maturation. A secretion burst deposits proteins into the lumen; then luminal material is cleared and finally liquid is removed. We identified the cellular mechanisms behind these transitions. Sar1 and γCOP are required for protein secretion, matrix assembly and tube expansion. Rab5-dependent endocytic activity internalizes and clears luminal contents. The data show how programmed transitions in cellular activities form functional airways, and may reflect a general mechanism in respiratory organ morphogenesis.We further focused on tube size regulation. We identified Melanotransferrin, a new component of septate junctions that limits tracheal tube elongation. MTf is a lipid- modified, iron-binding protein attached to epithelial cell membranes, similarly to its human homologue. We show that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. This provides the first genetic model for the functional dissection of MTf in epithelial morphogenesis. In the last part, we describe two genes, which are selectively involved in tube diameter expansion. Obst-A and Gasp are closely related proteins with characteristic chitin-binding domains. They are strongly expressed in the trachea at the time of lumen expansion. The single and double mutants cause a tube diameter reduction, whereas their overexpression leads to its increase. We propose that Obst-A and Gasp organize luminal matrix assembly and thereby regulate the extent of tube diameter expansion.