In vivo Analyse zellulärer Dynamik der renalen Tubulogenese in der Entwicklung und zystischer Nierenerkrankung
Zusammenfassung der Projektergebnisse
The main objective of this project was to gain insight into the molecular mechanisms of renal tubule morphogenesis and inherited renal disease, such as cystic kidney disease and nephronophthisis, using two model systems: Xenopus and mice. An initial expression screen for acto-myosin-associated proteins enriched in the kidney uncovered genes previously linked to kidney disease and renal specific extracellular matrix components, but did not find obvious modulators of myosin mediated tubule morphogenesis specific to the developing nephron. We invested a substantial amount of effort into adopting state of the art transgenesis and genome editing technology and established CRISPR based genome editing routinely in Xenopus tropicalis. We focused our attention on a protein (Nme3) that strongly interacts with previously investigated nephronophthisis proteins (Nek8, Anks6). Depletion of Nme3 leads to morphogenetic defects of tubule extension. Nme3 was shown to supply dinucleotides in conditions of DNA-replication stress. Because we find that Nme3 localizes to the base of the cilium and its depletion elicits ciliopathy typical phenotypes in Xenopus, our data supports the notion that DNA-damage response is involved in the pathogenesis of nephronophthisis by implicating novel key protein players. The routine employment of genome editing techniques enabled us to investigate molecular mechanisms of related renal anomalies and ciliopathies and facilitated new and fruitful collaboration with human geneticists. Using ex vivo cultures of embryonic mouse kidneys, we found that genetic labeling of the nephron progenitor population can be used to observe all stages of embryonic nephron development, including pretubular aggregate, comma- and s-shape body formation and extension of the early tubules. We optimized live cell imaging conditions over extended periods using 3D printed custom sample holders and automated image acquisition. Efforts to apply these elaborate imaging techniques to a model of genetic renal disease did not result in conclusive findings. In addition, we investigated transcriptional regulatory mechanisms of renal cell type specification. We found a number of transcription factors that would ectopically induce renal tubules in ectodermal explants of Xenopus and identified four factors that in combination can reprogram human and mouse fibroblasts to renal tubule-like cells without undergoing dedifferentiation into a pluripotent or stem-cell like state. This work resulted in a publication in Nature Cell Biology and a number of follow up articles investigating the use of reprogrammed cells for disease modelling, nephrotoxicity testing, and metabolic profiling. We used reprogrammed cells to determine transcriptional differences of patient specific mutations in HNF4A that cause renal Fanconi syndrome. In summary, we gained substantial insights into the molecular mechanisms of tubulogenesis and ciliary related renal disease, propelled our research into new technological fields, including genome editing, direct reprogramming and machine learning.
Projektbezogene Publikationen (Auswahl)
- Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells. The EMBO journal
Funk MC, Bera AN, Menchen T, Kuales G, Thriene K, Lienkamp SS, Dengjel J, Omran H, Frank M, and Arnold SJ
(Siehe online unter https://doi.org/10.15252/embj.201490805) - Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development. American journal of human genetics 97, 291-301 (2015)
Vivante A, Kleppa MJ, Schulz J, Kohl S, Sharma A, Chen J, Shril S, Hwang DY, Weiss AC, Kaminski MM, Shukrun R, Kemper MJ, Lehnhardt A, Beetz R, Sanna-Cherchi S, Verbitsky M, Gharavi AG, Stuart HM, Feather SA, Goodship JA, Goodship TH, Woolf AS, Westra SJ, Doody DP, Bauer SB, Lee RS, Adam RM, Lu W, Reutter HM, Kehinde EO, Mancini EJ, Lifton RP, Tasic V, Lienkamp SS, Juppner H, Kispert A, and Hildebrandt F
(Siehe online unter https://doi.org/10.1016/j.ajhg.2015.07.001) - The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells. The Journal of cell biology 211, 963-973 (2015)
Yasunaga T, Hoff S, Schell C, Helmstadter M, Kretz O, Kuechlin S, Yakulov TA, Engel C, Muller B, Bensch R, Ronneberger O, Huber TB, Lienkamp SS, and Walz G
(Siehe online unter https://doi.org/10.1083/jcb.201502043) - The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin. Development 142, 174-184 (2015)
Epting D, Slanchev K, Boehlke C, Hoff S, Loges NT, Yasunaga T, Indorf L, Nestel S, Lienkamp SS, Omran H, Kuehn EW, Ronneberger O, Walz G, and Kramer-Zucker A
(Siehe online unter https://doi.org/10.1242/dev.112250) - Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors. Nature cell biology 18, 1269-1280 (2016)
Kaminski MM, Tosic J, Kresbach C, Engel H, Klockenbusch J, Muller AL, Pichler R, Grahammer F, Kretz O, Huber TB, Walz G, Arnold SJ, and Lienkamp SS
(Siehe online unter https://doi.org/10.1038/ncb3437) - A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling. Journal of the American Society of Nephrology : JASN (2017)
Vivante A, Mann N, Yonath H, Weiss AC, Getwan M, Kaminski MM, Bohnenpoll T, Teyssier C, Chen J, Shril S, van der Ven AT, Ityel H, Schmidt JM, Widmeier E, Bauer SB, Sanna-Cherchi S, Gharavi AG, Lu W, Magen D, Shukrun R, Lifton RP, Tasic V, Stanescu HC, Cavailles V, Kleta R, Anikster Y, Dekel B, Kispert A, Lienkamp SS, and Hildebrandt F.
(Siehe online unter https://doi.org/10.1681/ASN.2016060694) - (2018) Fabrication of Kidney Proximal Tubule Grafts Using Biofunctionalized Electrospun Polymer Scaffolds. Macromolecular bioscience, e1800412
Jansen, K., Castilho, M., Aarts, S., Kaminski, M.M., Lienkamp, S.S., Pichler, R., Malda, J., Vermonden, T., Jansen, J. & Masereeuw, R
(Siehe online unter https://doi.org/10.1002/mabi.201800412) - (2018) Metabolic characterization of directly reprogrammed renal tubular epithelial cells (iRECs). Scientific reports 8, 3878
Lagies S, Pichler R, Kaminski MM, Schlimpert M, Walz G, Lienkamp SS, and Kammerer B
(Siehe online unter https://doi.org/10.1038/s41598-018-22073-7) - (2018) The nucleoside diphosphate kinase NME3 associates with nephronophthisis proteins and is required for ciliary function during renal development. Journal of Biological Chemistry Sep 28;293(39)
Hoff S, Epting D, Falk N, Schroda S, Braun AD, Halbritter J, Hildebrandt F, Kramer- Zucker A, Bergmann C, Walz G, Lienkamp SS
(Siehe online unter https://doi.org/10.1074/jbc.RA117.000847) - Network for Early Onset Cystic Kidney Diseases (2018) A Comprehensive Multidisciplinary Approach to Hereditary Cystic Kidney Diseases in Childhood. Frontiers in pediatrics 6, 24
Konig, J.C., Titieni, A. & Konrad, M.
(Siehe online unter https://doi.org/10.3389/fped.2018.00024) - (2019). Impact of Diabetic Stress Conditions on Renal Cell Metabolome. Cells, 8(10)
Lagies, S., Pichler, R., Bork, T., Kaminski, M. M., Troendle, K., Zimmermann, S., Huber, T. B., Walz, G., Lienkamp, S. S., & Kammerer, B.
(Siehe online unter https://doi.org/10.3390/cells8101141) - (2019). Molecular Basis for Autosomal-Dominant Renal Fanconi Syndrome Caused by HNF4A. Cell Reports, 29(13), 4407-4421.e5
Marchesin, V., Pérez-Martí, A., Le Meur, G., Pichler, R., Grand, K., Klootwijk, E. D., Kesselheim, A., Kleta, R., Lienkamp, S., & Simons, M.
(Siehe online unter https://doi.org/10.1016/j.celrep.2019.11.066)
