Many organs contain epithelial tubes that transport gases or liquids . Proper tube size and shape is crucial for organ function, but the mechanisms controlling tube diameter and length are poorly understood. Recent studies of tracheal (respiratory) tube morphogenesis in Drosophila show that chitin synthesis genes produce an expanding chitin cylinder in the apical (luminal) extracellular matrix (ECM) that coordinates the dilation of the surrounding epithelium . Here, we describe two genes involved in chitin modification, serpentine (serp) and vermiform (verm), mutations in which cause excessively long and tortuous tracheal tubes. The genes encode similar proteins with an LDL-receptor ligand binding motif and chitin binding and deacetylation domains. Both proteins are expressed and secreted during tube expansion and localize throughout the lumen in a chitin-dependent manner. Unlike previously characterized chitin pathway genes, serp and verm are not required for chitin synthesis or secretion but rather for its normal fibrillar structure. The mutations also affect structural properties of another chitinous matrix, epidermal cuticle. Our work demonstrates that chitin and the matrix proteins Serp and Verm limit tube elongation, and it suggests that tube length is controlled independently of diameter by modulating physical properties of the chitin ECM, presumably by N-deacetylation of chitin and conversion to chitosan.

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

Many organs including the mammalian lung and vascular system consist of branched tubular networks that transport essential gases or fluids, but the genetic programs that control the development of these complex three-dimensional structures are not well understood. The Drosophila melanogaster tracheal (respiratory) system is a network of interconnected epithelial tubes that transports oxygen and other gases in the body and provides a paradigm of branching morphogenesis. It develops by sequential sprouting of primary, secondary, and terminal branches from an epithelial sac of approximately 80 cells in each body segment of the embryo. Mapping of the cell movements and shape changes during the sprouting process has revealed that distinct mechanisms of epithelial migration and tube formation are used at each stage of branching. Genetic dissection of the process has identified a general program in which a fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) are used repeatedly to control branch budding and outgrowth. At each stage of branching, the mechanisms controlling FGF expression and the downstream signal transduction pathway change, altering the pattern and structure of the branches that form. During terminal branching, FGF expression is regulated by hypoxia, ensuring that tracheal structure matches cellular oxygen need. A branch diversification program operates in parallel to the general budding program: Regional signals locally modify the general program, conferring specific structural features and other properties on individual branches, such as their substrate outgrowth preferences, differences in tube size and shape, and the ability to fuse to other branches to interconnect the network.