Mark M. Metzstein, Ph.D.
Assistant Professor, Department of Human Genetics

Contribution to Society

Many organs, such as the lungs, the vasculature (blood system), and the kidneys, are composed of networks of cellular tubes. The correct development of these networks is critical for organ function, as evidenced by the disruption of these networks being the underlying cause of many common diseases, such as cardiovascular disease, polycystic kidney disease and asthma. To better understand how such networks form we are studying the development of the tracheal (respiratory) of the genetically tractable insect Drosophila melanogaster. By isolating genes affecting the development of the Drosophila tracheal system and molecularly characterizing them, we hope to obtain a more general understanding of the processes involved in forming branched cellular networks of cellular tubes, and eventually use this knowledge to address the important human diseases associated with defects in such networks.

Research Summary

The Drosophila tracheal system, like that of all insects, is composed of a branched network of cellular tubes. These tubes lead from specialized openings, called spiracles, on the animal's surface, and ramify into finer and finer tubes that contact most of the internal tissues to supply them with oxygen and remove waste gases. While the cells which make up this network are derived from common populations of precursors, the tubes these cells go on to form are of diverse cellular structures: some tubes are multicellular, some unicellular and some tubes, the tracheal terminal tubes or tracheoles, are subcellular. The formation of the tracheoles is the focus of our research. These tubes are generated by cytoplasmic extension from a specialized type of tracheal cell called terminal cells. The cytoplasmic extensions undergo numerous branching events to form a tree of terminal branches. In terms of cellular complexity these cells rival some of the very complex axonal and dendritic fields observed in some neurons, with some single terminal cells elaborating over 100 branches. However, unlike neurites, in order to be functional these branches must also form an air-filled tube running through each branch. Furthermore, each of these air-filled tubes must connect to form a continuous network to allow air to flow throughout the network.

We are interested in the mechanisms that control subcellular outgrowth, branching and that form the subcellular lumens. To identify genes involved in these processes we have taken advantage of the genetic tools available in Drosophila to screen for mutants defective in terminal cell development. We have used a genetic mosaic approach which has allowed us to specifically assay the function of genes in the terminal cells while bypassing effects these genes have on other tissues. In a large scale screen on the Drosophila X chromosome (chosen as it has been relatively understudied in analysis of tracheal and other developmental processes) we have identified 32 mutant lines in which terminal cells are generated but have developmental defects. Based on the phenotype of the mutant terminal cells we have divided the mutants into different classes which define different steps in terminal cell morphogenesis.

In 15 lines the terminal cell fails to grow or initiate branch outgrowth and in two of these lines the tracheal cells may degenerate to form a small cell remnant. In 9 lines cell growth occurs but the branching patterns or morphology are aberrant. In some of these lines terminal cell growth is directly almost entirely to the cell body at the expense of the branches. In other lines branch outgrowth from the cell body is initiated, but subsequent branching is inhibited, resulting in a cell with a single large branch but almost no side branches. Finally, some lines have essentially normal branching patterns, but the morphology of the individual branches is abnormal.

In the remaining 8 lines cell growth and branch outgrowth and morphology appears to be essentially normal, but the branches do not form an air-filled lumen. We have studied one of these mutant, called out of gas, in detail. Marker and ultrastructural studies indicate that out of gas mutant cells may define a domain within the terminal branches, which would be site of the future lumen, but that there is a subsequent block to lumenal maturation. We have cloned out of gas and found it is the Drosophila homolog of Rabconnectin3-alpha, a mammalian gene which has been implicated in the regulation of vesicle traffic. Our working model is that Out of Gas directs lumen formation by targeting specialized membrane vesicle population to the center of the branch and that subsequent vesicle fusion forms the continuous lumen. We are testing this model by ultrastructural and protein localization studies and by the molecular characterization of other genes with the out of gas phenotype.

We are also studying a process unrelated to tracheal development, but which was also initiated from our tracheal screen: the analysis of nonsense mediated mRNA decay (NMD). In our screen we identified six mutants in which a tracheal GFP reporter expression was greatly increased in mutant cells.  We have found that these mutations are in the Drosophila homologs of three components of the NMD pathway: Upf1, Upf2, and Smg1. The NMD pathway monitors mRNAs to identify transcripts that contain premature stop codons and to target these RNAs for rapid destruction. Ours are the first Drosophila mutants identified that affect this pathway and we are interested in using these mutations to understand the roles that NMD plays in normal development and to identify the cis acting signals that define which transcripts are to be targeted for degradation.

Recent Publications

Metzstein M.M., Kirby A.S. and Krasnow, M.A. Drosophila out of gas encodes a homolog of mammalian Rabconnectin and is required for subcellular tracheal lumen formation. In prep.

Metzstein MM, Krasnow MA (2006) Functions of the Nonsense-Mediated mRNA Decay Pathway in Drosophila Development. PLoS Genet 2(12)

Metzstein, M.M., and Krasnow, M.A., Genetic analysis of nonsense mediated decay in Drosophila. In prep.

Ghabrial, A., Luschnig, S., Metzstein, M.M., and Krasnow, M.A. 2003. Branching morphogenesis of the Drosophila tracheal system. Annu Rev Cell Dev Biol 19: 623-647.

Metzstein, M.M. and Horvitz, H.R. 1999. The C. elegans cell death specification gene ces-1 encodes a snail family zinc finger protein. Mol Cell 4(3): 309-319.

Metzstein, M.M., Stanfield, G.M., and Horvitz, H.R. 1998. Genetics of programmed cell death in C. elegans: past, present and future. Trends Genet 14(10): 410-416.

Metzstein, M.M., Hengartner, M.O., Tsung, N., Ellis, R.E., and Horvitz, H.R. 1996. Transcriptional regulator of programmed cell death encoded by Caenorhabditis elegans gene ces-2. Nature 382(6591): 545-547.