A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Abstract

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.

Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment

“…1B,C′). The mechanism of gas filling in terminal branches is not well understood, but in terminal cells mutant for genes involved in chitin synthesis, gas filling does not occur (Ghabrial et al, 2011), suggesting that normal cellular architecture is required for either gas production or for supplying gas to the lumen.…”

Compensatory branching morphogenesis of stalk cells in the Drosophila trachea

“…We characterized the role of okg and cnj (Ghabrial et al, 2011) in tube architecture and connectivity in mosaic animals, with a focus on the connection between autocellular and seamless tubes. In wildtype larvae, a gas-filled autocellular tube connects the stalk cell to its terminal cell neighbor.…”

“…EMS alleles of okg and cnj were generated previously (Ghabrial et al, 2011 RNAi, UAS-Tor.TED, Tor HMS01114 RNAi at 29°C. drm-GAL4 drove expression of UAS-GFP and V0a R755A at 22°C.…”

“…To better understand seamed-to-seamless tube connections, we characterized mutations in oak gall (okg) and conjoined (cnj), which confer identical tube connection defects (Ghabrial et al, 2011). Sequence analysis revealed that okg and cnj carry mutations in Vha26 and Vha13, which encode the E and G subunits of the vacuolar ATPase (vATPase).…”

Mechanisms of Lumen Development in Drosophila Tubular Organs

“…The amenability of Drosophila to large-scale genetic screens has allowed the generation of many new mutants affecting TC lumen formation (Ghabrial et al, 2011). Analysis of these new mutants is bound to bring novel insights to lumen formation in the tracheal TCs in the years to come.…”