Konfokales Laserscanning-Mikroskopsystem
Final Report Abstract
This confocal microscope was used extensively by the Edgar research group for the study of Drosophila intestinal stem cells, primarily in situ in whole-mount gut preparations. These studies gave rise to numerous publications describing the genetic mechanisms that control intestinal stem cell proliferation, differentiation, and tumorigenic growth. For instance, the critical functions of cytokine and epidermal growth factor (EGF) control of intestinal stem cells were characterized, the mode of intestinal stem cell specification and differentiation were analyzed, and the development of intestinal stem cell-derived tumors was detailed. In each case the microscope was used to image cellular DNA and proteins in 3D within the Drosophila intestine in by immunofluorescence or using proteins tagged with fluorescent moieties such as green fluorescent protein. In many cases the proliferation of intestinal stem cells was assayed, or the expression of marker genes for signaling pathway activity was visualized. Through these studies we determined the molecular mechanisms that control intestinal stem cell proliferation and differentiation during normal gut epithelial maintenance and regeneration, and also during tumorigenesis as driven by orthologs of human oncogenes such as Notch, Ras, Hpo, and Src. Key discoveries were that regenerative growth in the intestine utilizes damage-dependent feedback signaling via Upd, EGF, Hpo, and JNK signaling, and that this same signaling network is harnessed by micro tumors that develop in the stem cell niche. The Edgar group also used the confocal microscope for the characterization of fluorescence ubiquitin cell cycle indicators (FUCCI) in Drosophila cells and organs. These transgenic indicators are a new tool for tracking cell cycle transitions in living cells in real time, both in cell culture and in living animals. The Fly-FUCCI transgenic animals should prove useful for many research groups interested in cell proliferation. In addition, the microscope, which is housed in a shared core facility of the ZMBH on the Heidelberg campus, was used by several other research groups to make discoveries relevant to protein nuclear localization, kinetochore function, microtubule assembly, and to develop a method for mapping 3D image data onto 2D maps for analysis. For instance, one project showed that the nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation. Another project, carried out by the ZMBH imaging core staff, used this machine to develop a new computational method for mapping 3D image data, as acquired by confocal imaging, onto 2D representations that can be used for easily visualizing and analyzing such data.
Publications
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(2013) The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster. PLoS Genetics 9:e1003835
Bandura JL, Jiang H, Nickerson DW, Edgar BA
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(2014) Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells. EMBO J. 33:2967-2982
Korzelius J, Naumann SK, Loza-Coll MA, Chan JS, Dutta D, Oberheim J, Gläßer C, Southall TD, Brand AH, Jones DL, Edgar BA
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(2014) Fly- FUCCI - a versatile tool for studying cell proliferation in complex tissues. Cell Reports 7:588-598
Zielke N, Korzelius J, van Straaten M, Reuter H, Bender K, Schuhknecht G, Pouch J, Dutta D, Edgar BA
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(2014) Src kinase function controls progenitor cell pools during regeneration and tumor onset in the Drosophila intestine. Oncogene PMID: 24975577
Kohlmaier A, Fassnacht CH, Reuter H, Begum J, Jin Y, Dutta D, Edgar BA
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(2014). Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat Commun. 2014 Jul 14;5:4404
Niopek D, Benzinger D, Roensch J, Draebing T, Wehler P, Eils R, Di Ventura B
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(2014). The nucleoporin MEL-28 promotes RanGTP- dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation. Nat Commun. 2014;5:3270
Yokoyama H, Koch B, Walczak R, Ciray- Duygu F, González-Sánchez JC, Devos DP, Mattaj IW, Gruss OJ
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(2015) Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut. Cell Reports 2015 12:346-348
Dutta D., Dobson, AJ., Houtz, P., Gläßer, G., Revah, J., Korzelius, J., Edgar, BA., Buchon, N
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(2015). 2D map projections for visualization and quantitative analysis of 3D fluorescence micrographs. Sci Rep. 2015 Jul 24;5:12457
Sendra GH, Hoerth CH, Wunder C, Lorenz H
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(2015). Niche appropriation by Drosophila intestinal stem cell tumors. Nature Cell Biol. 17:1182-1192
Patel, PH, Dutta, D, Edgar, BA
