Our lab uses genetics and molecular biology in Drosophila to address two basic questions in cell biology: cell morphogenesis and RNA stability.
For cell morphogenesis, we are studying the development of a specific cell type in the insect respiratory system, the tracheal terminal cell. These remarkable cells each form a complex network of subcellular branches in response to oxygen requirements in underlying tissues. To facilitate the flow of gases each of these branches undergoes further morphogenesis, forming air-filled tubes running through each branch. By identifying mutations affecting branching and/or tube formation, we are learning how cells build complex 3-dimensional architectures required for their functions.
For RNA stability, we are studying the cellular surveillance pathway of nonsense mediated mRNA decay (NMD). NMD functions to identify transcripts that contain certain signals, such as premature stop codons, so as to target these RNAs for rapid destruction. In this way NMD functions to regulate expression of both normal and abnormal genes. We are interested in defining the cis-acting signals and trans-acting components required for NMD and in identifying the endogenous targets of this pathway required for normal development and physiology.
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.
References to Publications:
Ashleigh Long, Cecon Mahapatra, E. A. Woodruff III, Jeff Rohrbough, Hung-Tat Leung, S. Shino, L. An, R. W. Doerge, Mark M. Metzstein, William L. Pak, Kendal Broadie. The nonsense-mediated decay pathway is critical for maintaining synapse architecture and synaptic vesicle cycle efficacy. J Cell Sci. 2010 Oct 1;123(Pt 19):3303-15.
Chung-Yi Nien, Hsiao-Lan Liang, Hsiao-Yun Liu, Mark M. Metzstein, Nikolai Kirov, Christine Rushlow. (2008). The zinc finger protein Zelda is a key activator of the early zygotic genome in Drosophila. Nature, 456, 400-403.
Metzstein MM, Krasnow M. (2006). Functions of the Nonsense-Mediated RNA Decay Pathway in Drosophila Development. PLoS Genet, 2(12), 180.
Metzstein MM, Horvitz HR. (1999). The C. elegans cell death specification gene ces-1 encodes a snail family zinc finger protein. Mol Cell, 4(3), 309-19.
Wen C, Metzstein MM, Greenwald I. (1997). SUP-17, a Caenorhabditis elegans ADAM protein related to Drosophila KUZBANIAN, and its role in LIN-12/NOTCH signalling. Development, 124(23), 4759-67
Metzstein MM, Hengartner MO, Tsung N, Ellis RE, Horvitz HR. (1996). Transcriptional regulator of programmed cell death encoded by Caenorhabditis elegans gene ces-2. Nature, 382(6591), 545-7.
Sulston J, Du Z, Thomas K, Wilson R, Hillier L, Staden R, Halloran N, Green P, Thierry-Mieg J, Qiu L, Dear S, Coulson A, Craxton M, Durbin R, Berks M, Metzstein M, Hawkins T, Ainscough R, Waterston R. (1992). The C elegans genome sequencing project: A beginning. Nature, 356, 37-41.
Waterston R, Martin C, Craxton M, Huynh C, Coulson A, Hillier L, Durbin R, Green P, Showenkeen R, Halloran N, Hawkins T, Metzstein M, Wilson R, Berks M, Du Z, Thomas K, Thierry-Mieg J, Sulston J. (1992). A survey of expressed genes in Caenorhabditis elegans. Nat Genet, 1, 114-123.
Ghabrial A, Luschnig S, Metzstein MM, Krasnow MA. (2003). Branching morphogenesis of the Drosophila tracheal system. [Review]. Annu Rev cell Dev Biol, 19, 623-47.
Metzstein MM, Stanfield GM, Horvitz HR. (1998). Genetics of programmed cell death in C. elegans: past, present and future. [Review]. Trends Genet, 14(10), 410-6.
Aubrey C. Chan, Stavros G. Drakos, Oscar E. Ruiz, Alexandra C. H. Smith, Jing Ling, Samuel F. Passi, Amber N. Stratman, George E. Davis, Mark M. Metzstein, Kevin J. Whitehead, Dean Y. Li. Two hit mechanism of cerebral cavernous malformation pathogenesis. Nat. Medicine (submitted)
Mark Metzstein, Ph.D.
Oscar Ruiz, Ph.D.
Department of Human Genetics
University of Utah
15 N 2030 E RM 6160A
Salt Lake City, Utah 84112-5330