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. 2019 Mar 14;9:4420. doi: 10.1038/s41598-019-40734-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Tectal composition, stem cell niches, and stab lesion model. (a) Cross-sectional view of a single adult tectal hemisphere showing a laminar composition consisting of outer tectal superficial layers (TeO) and the deeper, cell-dense, periventricular grey zone (PGZ). The PGZ is subdivided into an upper neuronal layer (Neu-L, cytoplasmic HuC/D labelling; pink) and a quiescent radial-glial layer (qRG-L; Tg(gfap:GFP)^mi2001 labelling; green) populating the roof of the tectal ventricle. (b) High magnification of the PGZ illustrating the 3–4 cell deep structure of the qRG-L (green) abutting the tectal ventricle (TecV) with radial processes extending upwards from qRG cells through the Neu-L (white arrows) and towards the superficial tectal layers. (c) Neuro-epithelial-like amplifying progenitor cells (NE; pink; zone 2) identified by EdU-labelling are located at the internal tectal marginal zone (TMZi; white dashed circle), adjacent to the qRG-L (green; zone 1). (d) Dorsal view of the homeostatic staining pattern of cell proliferation using EdU (pink) in reconstructed whole brains following Optical Projection Tomography (OPT). Image shows constitutively proliferating NE cells extending the length of the tectum from the TMZi where amplifying NE progenitor cells reside (yellow arrows), to the external tectal marginal zone (TMZe; white arrows) where more slowly proliferating NE cells have been identified^48,49. Hb, hindbrain; Ce, cerebellum; TeO, optic tectum; Fb, forebrain. Yellow line depicts cross-sectional level shown in panel (e). (e) Cross-section view of the TMZe (white dashed circle; zone 3) labelled with EdU (pink), where populations of slow cycling NE exist at the posterior aspect of the adult midbrain adjacent to the medially located cerebellum. (f,g) Proliferating Cell Nuclear Antigen (PCNA) immunolabelling confirming the same constitutive pattern of cell division seen with EdU (compare with panel c) in the qRG-L and TMZi. (h) Schematic representing the three stem cell zones (1–3) investigated following tectal stab lesion (lightning bolt) superficial to the underlying qRG layer: 1 – qRG population (orange) lining the tectal ventricle; 2 – proliferating NE amplifying progenitors (green) located at the TMZi (light blue) adjacent the qRG population; 3 – slowly cycling NE cells located in the caudal TMZe (dark blue). Note that NE cells of zones 2–3 proliferate under homeostatic conditions, with zone 2 NE amplifying progenitors producing lifelong newborn neurons (N-Neu). M-Neu, mature neuron; CCe, corpus cerebelli. (i,j) Dorsal (i) and cross-sectional (j) views of the intact control brain depicting the neuroanatomical level (i, black dashed line) and cannula insertion site (red circles; black and yellow arrows) for our tectal stab lesion into the left hemisphere in OPT reconstructed brains (top) and schematic images (bottom). In panels b,c,e–g, DAPI nuclear counterstaining (blue) was performed. In panels c–g, EdU was pulsed twice over a 4-hr chase period prior to sacrifice. In all cross-sectional and whole brain images dorsal is oriented up.