Abstract
Thymosin β4 (Tβ4), the principal G-actin regulating entity in eukaryotic cells, has also multiple intra- and extracellular functions related to tissue regeneration and healing. While its effect in adult organs is being widely investigated, currently, little is known about its influence on embryonic tissues, i.e., in the developing nervous system. The importance of Tβ4 for neural stem cell proliferation in the embryonic chicken optic tectum (OT) has previously been shown by us for the first time. In the present study, using in ovo electroporation, we carried out a quantification of the effects of the Tβ4-overexpression on the developing chicken OT between E4 and E6 at the hemisphere as well as cellular level. We precisely examined tissue growth and characterized cells arising from the elevated mitotic activity of progenitor cells. By using spinning-disk confocal laser scanning microscopy, we were able to visualize these effects across whole OT sections. Our experiments now demonstrate more clearly that the overexpression of Tβ4 leads to a tangential expansion of the treated OT-hemisphere and that, under these circumstances, overall density of tectal and in particular of postmitotic neuronal cells is increased. Thanks to this new quantitative approach, the present results extend our previous findings that Tβ4 is important for the proliferation of progenitor cells, neurogenesis, tangential expansion, and tissue growth in the young embryonic chicken optic tectum. Taken together, our results further illustrate and support the current idea that Tβ4 is widely implicated in shaping and maintenance of the nervous system.
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Abbreviations
- E:
-
Embryonic day
- EP:
-
Electroporation/electroporated
- DCX:
-
Doublecortin
- H3P:
-
Phospho-histone H3
- HH:
-
Developmental stage according to classification of Hamburger and Hamilton
- NSC:
-
Neural stem cell
- NPC:
-
Neuronal progenitor cell
- OT:
-
Optic tecutm
- PBS:
-
Phosphate-buffered saline
- PFA:
-
Paraformaldehyde
- RGC:
-
Radial glial cell
- Tβ4:
-
Thymosin β4
- Tuj-1:
-
ΒIII-tubulin, clone Tuj-1
- VIM:
-
Vimentin
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Acknowledgements
We wish to thank D. Terheyden-Keighley for critical reading of the manuscript, V. Matschke for precious advices concerning statistics, Abdulatif Al-Haj for his help for plasmid production, C. Grzelak, S. Wenderdel, and A. Lodwig for excellent technical assistance, as well as A. Lenz for secretarial work.
Authors’ contribution
ML, GMP, and CT designed and performed experiments, analyzed data. BBS and CT designed the study, provided technical support and gave conceptual advice. All authors discussed the results and implications and commented on the manuscript at all stages.
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Suppl. Fig. 1
Effect of Tβ4 on the total number of H3P(+) cells in the OT at E4 and E6. The graph shows the highly significant increase in H3P expressing cells in the EP hemisphere as compared to the control hemisphere at E4 (** p < 0,01; n = 8) and at E6 (** p < 0,01; n = 5). (TIFF 172 kb)
Suppl. Fig. 2
Effect of Tβ4 on the total number of Tuj-1(+) cells in the OT at E4 and E6. The graph shows the highly significant increase in Tuj-1 expressing cells in the EP hemisphere as compared to the control hemisphere at E4 (** p < 0,01; n = 8) and at E6 (** p < 0,01; n = 5). (TIFF 171 kb)
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Lever, M., Theiss, C., Morosan-Puopolo, G. et al. Thymosin β4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum. Histochem Cell Biol 147, 555–564 (2017). https://doi.org/10.1007/s00418-016-1529-1
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DOI: https://doi.org/10.1007/s00418-016-1529-1