Research: How does gene circuitry specify and pattern embryonic development? What is the nature of the events set in motion by the expression of a single developmental master regulator? Is there a common set of optimal regulatory interactions (rules), analogous to evolutionarily conserved master regulatory genes, which operate among the targets of master regulators to enable developmental robustness and evolvability? To address these questions we are studying the activity of pal-1 , the C. elegans Caudal homolog, an ancient homodomain protein that specifies and patterns posterior development in animals. In C. elegans pal-1 is necessary and sufficient to specify the identity of the C blastomere, a posterior cell in the 8-cell embryo, and then to control its subsequent development or cell lineage. Our goals are to generate a systems-level description of the gene circuitry (network topology) initiated by pal-1 activity and then to infer and test functional units (modules) that enable pal-1 to robustly specify posterior development. We have used microarrays, reporter genes, and RNAi to both identify the initial targets of pal-1 activity in the early embryo and to assemble these genes onto a regulatory framework. Many of these genes are expressed in descendants of other blastomeres and the primary cell types, muscle and skin, descended from the C blastomere are not unique to the C lineage. Thus, we are currently employing computational and a myriad of experimental approaches to understand how muscle- and skin-specific networks are established and maintained (insulated from each other). A global question is whether there are predictable (evolutionarily conserved or optimal natural solutions) regulatory interactions or whether every cell/lineage/organism employs a unique solution. Towards this goal we are comparing the topology of the C-lineage regulatory network between morphologically indistinguishable nematodes species whose genomes have been evolving independently for ~100,000,000 years. The origin of pattern through control of mRNA translation and stability Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. In C. elegans , early polarity cues control the expression of genes that pattern the anterior-posterior embryonic axis. We are investigating the post-transcriptional control mechanisms that target expression of the caudal -like gene pal-1 to posterior blastomeres in 4-cell and older embryos. Through genetic studies we have identified proteins required for repression of translation and, importantly for developmental processes, spatially and temporally controlled de-repression. In particular, components of the conserved RNA induced silencing complex (RISC) appear to be required for efficient translational repression. C. elegans is a terrific system for molecular genetic analysis development control pathways, but its potential for traditional biochemical analysis has not been fully exploited. The accessibility of the germline to direct experimental manipulation via microinjection has encouraged us to develop complementary in vitro assays to investigate the activity of these proteins. Our analysis to date provides strong support for post-initiation repression, a mechanism conducive to a rapid transition to productive protein synthesis, which is critical during the rapid phase of early development. Current efforts are focused on elucidating the mechanism(s) that limits accumulation of mature protein from polyribosome associated mRNAs. Systemic RNAi and intercellular RNA transport The discovery of RNAi has generated tremendous excitement in basic research, where RNAi is an important tool, and in medicine, where dsRNAs are being developed as therapeutics to regulate disease causing genes and viruses. The last few years has seen significant progress in deciphering the intricacies of how introduction of dsRNA into cells leads to gene-specific mRNA degradation. Less well understood is how microRNAs (miRNAs) and other endogenous hairpin RNA genes regulate mRNA expression, stability, and translation. A second aspect of RNAi, the ability of the gene-specific signal to spread between cells, has been little studied, but its implications are equally as large, both as a means to introduce therapeutic dsRNA into cells as well as a new mechanism of intercellular gene regulation. Our discovery and characterization of C. elegans genes essential for the spreading of the RNAi signal has provided a mechanistic basis for understanding how gene-specific information is communicated between cells and the means to manipulate this new form of intercellular gene regulation. Current research efforts in the lab are directed at understanding how dsRNA transits the SID-1 dsRNA channel, investigating the function of vertebrate SID-1 homologs, determining how dsRNA is exported from and transported between cells, cloning and characterizing the other components identified in the original screen, and screening for additional genes involved in transporting dsRNA between cells.
Selected Publications: Yanai I., Baugh L.R. Smith J.J., Roehrig C., Shen-Orr S.S., Clagget J.M., Hill A.A., Slonim D.K., Hunter C.P. (2008). Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression. Mol Sys Biol. 4:163 (Published online: 12 February 2008) Jose, A.M. and Hunter, C.P. (2007) Transport of Sequence-Specific RNA Interference Information Between Cells. Annu. Rev. Genet. 2007. 41:305 30. Buttner, E.A. Gil-Krzewska, A.J., Rajpurohit, A.K. and Hunter, C.P. (2007) Progression from mitotic catastrophe to germ cell death in C. elegans lis-1 mutants requires the spindle checkpoint. Dev Biol. 305(2):397-410. Winston W.M., Sutherlin, M., Wright A.J., Feinberg, E.H. and Hunter, C.P. (2007) Caenorhabditis elegans SID-2 is required for environmental RNA interference. PNAS 104: 10565-10570. Hunter, C.P., Winston, W.M., Molodowitch, C., Feinberg, E.H., Shih, J., Sutherlin, M., Wright, A.J. Fitzgerald, M.C. (2006) Systemic RNAi in Caenorhabditis elegans. Cold Spring Harb Symp Quant Biol 71:95-100. Baugh L.R. and Hunter C.P. (2006) MyoD, modularity, and myogenesis: conservation of regulators and redundancy in C. elegans. Genes Dev. 20(24):3342-3346. Evans, T.C. and Hunter, C.P. Translational control of maternal RNAs (November 10, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.34.1 Schott DH, Cureton DK, Whelan SP, Hunter C.P. (2005) An antiviral role for the RNA interference machinery in Caenorhabditis elegans. PNAS 102(51):18420-4. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, Hunter C.P. (2005) The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo. Development 132(8):1843-54. Baugh LR, Wen JC, Hill AA, Slonim DK, Brown EL, Hunter C.P. (2005) Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions. Genome Biology 6(5):R45. Mootz D, Ho, DM and Hunter C.P. (2004) The STAR/Maxi-KH domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1. Development 131: 3263-3272. Wright A. J. and Hunter, C.P. (2003) Mutations in a β-tubulin disrupt spindle orientation and microtubule dynamics in the early C. elegans embryo. Mol Biol Cell. 14: 4512 4525. Feinberg EH, Hunter CP. (2003) Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545-7. Baugh LR, Hill AA, Slonim DK, Brown EL, Hunter CP. (2003) Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900. Winston WM, Molodowitch C, Hunter CP. (2002) Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295, 2456-9. Huang NN, Mootz DE, Walhout AJ, Vidal M, Hunter CP. (2002) MEX-3 interacting proteins link cell polarity to asymmetric gene expression in Caenorhabditis elegans. Development 129, 747-59. Kay AJ, Hunter CP. (2001) CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans. Curr Biol 11, 474-81. Baugh LR, Hill AA, Brown EL, Hunter CP. (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29. Hunter CP. (2000) Gene silencing: shrinking the black box of RNAi. Curr Biol 10, R137-40. Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, Brown EL. (2000) Genomic analysis of gene expression in C. elegans. Science 290, 809-12. Hunter CP. (1999) Genetics: a touch of elegance with RNAi. Curr Biol 9, R440-2. Hunter CP, Harris JM, Maloof JN, Kenyon C. (1999) Hox gene expression in a single Caenorhabditis elegans cell is regulated by a caudal homolog and intercellular signals that inhibit wnt signaling. Development 126, 805-14. Bowerman B, Ingram MK, Hunter CP. (1997) The maternal par genes and the segregation of cell fate specification activities in early Caenorhabditis elegans embryos. Development 124, 3815-26. Hunter CP, Kenyon C. (1996) Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Hunter CP, Kenyon C. (1995) Specification of anteroposterior cell fates in Caenorhabditis elegans by Drosophila Hox proteins. Nature 377, 229-32. |
