Novel Genes Involved in Mechanosensory Organ Development
The peripheral nervous system (PNS) serves as the sensory input apparatus which relays information from the external environment and physiological status to the central nervous system for further processing and integration. External mechanosensory organs, or bristles, of Drosophila serve as an excellent model system to study how PNS progenitor cells are specified from the epithelial cells and how the fate of each cell comprising the sensory organ becomes specified via intrinsic and extrinsic cues. In collaboration with Hillary
Andrews and Nikolaos Giagtzoglou, we performed an F1 adult mosaic screen on the 2R chromosome arm to uncover novel genes involved in PNS development.
From the screen, we isolated sequoia (Andrews et al., 2009), a zinc finger transcription factor that is essential for specifying the sensory neuronal fate during bristle development. In addition, we identified dEHBP-1, a putative actin binding protein involved in vesicle trafficking, as a crucial regulator of Notch signaling during the cell fate specification in the bristle lineage. Currently, detailed cell biological characterization of dEHBP-1 mutant cell is underway to uncover the precise role in Notch signaling.
Furthermore, from a forward genetic screen on the X chromosome, we identified tempura, which encodes a novel putative lipid modifying enzyme, to be a novel gene required for Notch signaling in the bristle lineage. Together with Wu-Lin Charng, we are investigating the molecular function of tempura to understand its role in PNS development and Notch signaling.
Gene Discovery on the Drosophila X chromosome
The X chromosome of Drosophila harbors approximately 2,200 genes including genes that are critical for development and function of nervous system such as Notch, shibire and dVAP-33-1. However, due to the genetic complexity, mutations in many essential genes on the X chromosome have not been isolated, or have been isolated but have not been mapped. In order to identify novel genes on the X chromosome that play a crucial role during the development, and maintenance of the nervous system, we initiated an F3 forward genetics adult
mosaic screen together with Manish Jaiswal, Vafa Bayat, Ke Zhang, Bo Xiong and Wu-Lin Chang. This "X chromosome screen team" has been growing as the screen proceed, welcoming Hector Sandoval, Gabriela David, Tongchao Li, Kuchuan Chen, Chao Tong, Upasana Gala, Timothy Mahoney as new members, as well as technical support from Karen Schulze, Adeel Jawaid, Stephen Gibbs, Yong Qi Lin, Claire Haueter, Koen Venken and numerous undergraduate/high school students that spent their summer in the Bellen lab.
From 8,600 EMS treated isogenized y w FRT19A male flies, we established 33,887 mutant stocks, of which 5859 lines were hemizygous lethal. We are maintaining and actively mapping ~2,100 lines which exhibit interesting phenotype in nervous system development, function and maintenance as well as mutants that show gross morphological abnormalities. So far, we have isolated >100 complementation groups with multiples alleles, and identified molecular lesions in >50 genes. We estimate that we will end up with identifying >300 essential
genes, many of which are predicted to be novel. In parallel to the gene identification, we are actively performing various secondary screenings and detailed phenotypic characterization of these mutants, in collaboration with various labs around the world.
In vivo Structure Function Analysis of Notch receptor
Notch Signaling is an evolutionarily conserved signaling pathway that is required for various biological processes and involved in diverse human diseases such as CADASIL, Alagille Syndrome, Tetralogy of Fallot and various types of cancer. Notch signaling is used reiteratively during development. Notch signaling can be classified into three conceptually
different modes of signaling: "lateral inhibition", "binary cell fate determination" and "inductive signaling".
Notch receptor is a large type I transmembrane protein with numerous EGF repeats (varying from 11-36 in different species and paralogs), 3 LNR repeats, and an intracellular domain with RAM domains, Ankyrin repeats, multiple nuclear localization signals and a C terminal PEST domain. Precisely how Notch is regulated and whether different domains of Notch may be involved in different modes of signaling is still being investigated. Since Drosophila is the only model organism with a single Notch gene, it is the best suited model organism
to perform an in vivo structure function analysis to understand the function of distinct domains of Notch.
From the X chromosome screen, we identified 38 novel lethal alleles of Notch. We performed molecular characterization of these mutations via Sanger Sequencing and identified many point mutations that cause single aminoacid substitutions, especially in different EGF repeats that have not been studied in vivo. To our surprise, some mutations only show defects in inductive signaling, whereas others only show defects in lateral inhibition and binary cell fate determination, suggesting that different modes of Notch signaling is molecularly separable. I am currently performing detailed phenotypic and molecular characterization of these mutations to provide novel insights into the molecular function of distinct EGF repeats of the Notch receptor.
- Xiong B, Bayat V, Jaiswal M, Zhang K, Sandoval H, Charng W-L, Li TC, David G, Duraine L, Lin YQ, Neely GG, Yamamoto S, Bellen HJ (2012) Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells. PLoS Biology 10:e1001438. [Abstract]
- Yamamoto S, Charng W-L, Rana NA, Kakuda S, Jaiswal M, Bayat V, Xiong B, Zhang K, Sandoval H, David G, Wang H, Haltiwanger RS, Bellen HJ (2012) A mutation in EGF repeat 8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338:1229-1232. [Abstract]
- Giagtzoglou N*, Yamamoto S*, Zitserman D, Graves HK, Schulze KL, Wang H, Klein H, Rogiers F, Bellen HJ (2012) dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions . Journal of Cell Biology 196:65-83. (*equal contribution) [Abstract]
- Yamamoto S*, Charng WL*, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Current Topics in Developmental Biology 92:165-200. (*equal contribution) [Abstract]
- Andrews HK, Giagtzoglou N, Yamamoto S, Schulze KL, Bellen HJ (2009) Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila. EMBO Reports 10:636-641. [Abstract]