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Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones

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Abstract

We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.

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Abbreviations

DAPI:

4′,6′-diamidino-2′-phenylindole

2D:

Two-dimensional

3D:

Three-dimensional

E6:

Day 6 of embryonic development

FA:

Formaldehyde

FISH:

Fluorescence in situ hybridization

Η4Κ8ac:

Histone H4 acetylated at lysine 8

PBS:

Phosphate-buffered saline

PI:

Propidium iodide

Pol II Ser 2:

RNA polymerase II phosphorylated at serine 2

RS:

Retinospheroid

TT:

0.2% Tween 20 and 0.2% Triton X-100

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Acknowledgments

We thank Jeannette Koch (LMU Munich) for artwork and Steffen Dietzel (LMU Munich) for providing HD12 cells. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (La 379/12-5) to PL and by a grant from the VolkswagenStiftung to DZ.

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Author notes

  1. Joscha Muck & Daniele Zink

    Present address: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore, 138669, Singapore

Authors and Affiliations

  1. Department Biologie II, Ludwig-Maximilians-Universität (LMU) München, Biozentrum, Grosshadernerstr. 2, 82152, Planegg-Martinsried, Germany

    Doris Berchtold, Stephanie Fesser, Alexander Kaiser, Nicolas Sadoni, Joscha Muck & Daniele Zink

  2. Technische Universität Darmstadt, Fachbereich Biologie, Schnittspahnstrasse 13, 64287, Darmstadt, Germany

    Gesine Bachmann, Florian Frohns & Paul G. Layer

  3. BioImaging Zentrum, Grosshadernerstr. 2, 82152, Planegg-Martinsried, Germany

    John-Christian Eilert

  4. Helmholtz Zentrum München, Institut für Molekulare Immunologie, Marchioninistr. 25, 81377, München, Germany

    Elisabeth Kremmer

  5. Helmholtz Zentrum München, Abteilung für Molekulare Epigenetik, Center of Integrated Protein Science, Marchioninistr. 25, 81377, München, Germany

    Dirk Eick

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  1. Doris Berchtold
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Correspondence to Daniele Zink.

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Responsible Editor: Irina Solovei

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Figure S1

Part of a RS (light optical section) stained with an antibody against vimentin (green), which is a prominent component of the cytoskeleton of Müller glia cells. Nuclei were stained with DAPI (blue). Arrows point to elongated Müller glia cells. Scale bar 50 μm (DOC 25 kb)

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Berchtold, D., Fesser, S., Bachmann, G. et al. Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones. Chromosome Res 19, 165–182 (2011). https://doi.org/10.1007/s10577-010-9182-3

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