Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo Cedric S. Wesley1*, Heng Guo 2 , Kanita A. Chaudhry3, Markus J. Thali4, Jerry C. Yin1, Todd Clason 5 , Umadevi V. Wesley6 1 Departments of Genetics and Medical Genetics, University of Wisconsin, Madison, Wisconsin, United States of America, 2 Department of Biochemistry, Boston University, Boston, Massachusetts, United States of America, 3 Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America, 4 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America, 5 Department of Anatomy and Neurobiology, University of Vermont, Burlington, Vermont, United States of America, 6 Department of Neurological Surgery, University of Wisconsin, Madison, Wisconsin, United States of America Abstract Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso- lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions. Citation: Wesley CS, Guo H, Chaudhry KA, Thali MJ, Yin JC, et al. (2011) Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo. PLoS ONE 6(7): e21876. doi:10.1371/journal.pone.0021876 Editor: Suzannah Rutherford, Fred Hutchinson Cancer Research Center, United States of America Received February 16, 2011; Accepted June 12, 2011; Published July 5, 2011 Copyright: 2011 Wesley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIH 1 R21 HD062928-01 (to CSW), University of Vermont Neuroscience NIH/NCRR COBRE (P20 RR16435), University of Vermont-College of Medicine Bridge Fund, and R01 NS063245-01 (to JCY) provided financial support for the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cswesley@wisc.edu Introduction Polypyrimidine Tract Binding (PTB) protein (also known as Heterogeneous Nuclear RibonucleoProtein I or hnRNP I) plays critical roles in mRNA metabolism. It contains four RNA Reco- gnition Motifs (RRMs) and binds to pyrimidine-rich sequences with motifs such as UCUUC and CUCUCU. Biochemical studies, most of them performed using mammalian in vitro and cultured cell systems, indicate that PTB regulates (often negatively) mRNA splicing, 39 processing, stabilization, nuclear export, subcellular localization, and translation [1–11]. PTB in Drosophila melanogaster (dmPTB) is encoded by the hephaestus locus. Genetic screens and developmental studies performed on post-embryonic stages of this organism indicate that PTB regulates oogenesis in adult females [12], spermatogenesis in adult males [13], and sensory bristle and wing margin development during larval and pupal stages [14–17]. Studies of wing development, which include a large-scale genetic screen and an analysis of somatic clones of mutant cells, indicate that hephaestus is a negative regulator of Notch pathway activities [15,17]. In hephaestus somatic mutant clones the level of active Notch molecule (the Notch intracellular domain, see below) is known to increase [17] but it is not known whether that is a direct or an indirect effect. In other words, neither the Notch pathway target nor the underlying mechanism is known. Our recent studies show that the Notch gene is negatively regulated at the level of mRNA 39 processing during Drosophila embryogenesis [18,19] raising the possibility that PTB regulates Notch mRNA and protein production during development. The Notch protein is a cell surface receptor that is required for the development of all animal tissues. At any particular developmental stage, it is involved in the differentiation of many different tissues in different regions. The mechanism underlying Notch function is generally thought to be the same everywhere: In response to binding of a ligand such as Delta, the Notch receptor is proteolytically cleaved to release the Notch intracellular domain (Nintra/NICD) from the cell surface. Nintra/NICD translocates to the nucleus, associates with transcription factors including Suppre- ssor of Hairless and Mastermind, and turns on transcription of target genes. A Mastermind-dependent process degrades Nintra/ NICD, possibly to keep transcription commensurate with the amount of Nintra/NICD production. This mechanism, called canonical Notch signaling, is used as a binary switch during cell fate specification. Cells that block Nintra/NICD production commit to one fate (generally the default fate) and cells that produce Nintra/NICD acquire the alternative cell fate. Depending on the developmental event or context, Nintra/NICD [20–30] targets different sets of effector genes. There is suggestive evidence in assorted systems that some Notch functions are not based on canonical Notch signaling. PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e21876