Cepero Malo et al., 2017 - The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages. PLoS One   12:e0170356 Full text @ PLoS One

Fig. 2

Anillin-eGFP marks all cycling cells of the maturing retinal layers.

(A-E) Optical section from a z-stack (frontal view) through the retina of a 48 hpf, anillin:anillin-eGFP/ptf1a:dsRed transgenic zebrafish that have been counterstained with DAPI (A) and pH3-antibody (A,D). (C) Only 34% of the Anillin-eGFP positive cell nuclei are pH3 immunoreactive at 48 hpf (n = 8 retinas). The black horizontal line shows the median while the crosses show the average. The box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, data points are plotted as open circles. Asterisks in (B) and (E) highlight cells that are (ptf1a)dsRed (magenta) and Anillin-eGFP (green) positive, only few of which are also pH3 positive (yellow in D). The arrow in (B) and (E) points at the midbody between dividing daughter cells (asterisk) during late cytokinesis. (F,G) Optical section from a z-stack (frontal view) through the retina of an 60 hpf, anillin:anillin-eGFP/ptf1a:dsRed transgenic zebrafish that have been counterstained with DAPI (grey) and Rx2-antibody (blue). The arrowhead points at the cycling, Anillin-eGFP positive and Rx2-immunoreactive cell located at the base of the outer limiting membrane. ONL, outer nuclear layer; INL, inner nuclear layer; ac, amacrine cell; ph, photoreceptors; mg, Müller glia cell bodies (intermingling with the ac bodies).

Fig. 3

Visualisation of Anillin-eGFP labelled cycling cells in the post-embryonic central retina at 3 dpf.

Top panel: view of the central retina in the sagittal plane (0°, eye facing the viewer). The blue circles represent an optical z-section across the eye corresponding to the blue line in the top panel of Fig 4. (A-F) Optical section from a z-stack through the retina of an 3 dpf, anillin:anillin-eGFP/ptf1a:dsRed transgenic zebrafish taken in the sagittal plane. (B-F) Magnification of the area delineated by the dotted rectangle in (A). The vertical arrows in (B-F) point at dividing horizontal cells that are (ptf1a)dsRed positive (magenta in C,D), Anillin-eGFP positive (green in B) and pH3 positive (yellow in D,E), and at their corresponding nuclei (F). The asterisk in (B-D) highlights an Anillin-eGFP labelled midbody between daughter cells in late cytokinesis, not depicted by pH3. The horizontal arrows in (B) and (E) point at two Anillin-eGFP positive (green in B) and pH3 positive (yellow in E) mitotic nuclei in the ONL. (G-I) and (J-M) represent two different optical sections from a z-stack through the retina of an 3 dpf, anillin:anillin-eGFP/atoh7:gap-RFP transgenic zebrafish in the sagittal plane. The arrows in (G) and (K) point at atoh7:gap-RFP expressing (magenta) photoreceptor and horizontal cell precursors that are also Anillin-eGFP (green) positive, indicating that they are in the cell cycle. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ph, photoreceptor cell; ho, horizontal cell.

Fig. 4

Anillin-eGFP in the CMZ marks transit-amplifying progenitors entering restricted neuronal lineages.

Top panel: view of the retina in the transversal plane (eye position 90° relative to the viewer). The blue line across the retina corresponds to the sagittal view (optical z-section) in the top panel of Fig 3. (A-C) and (E-I) represent two optical sections from confocal z-stacks through the retina of a 48 hpf (A-C) and 3 dpf (E-I) zebrafish taken in the transversal plane. The squared dotted bracket in (A) and (E) delineates the CMZ domains. The squared bracket in (A-C) and (E-H) delineates cells within the CMZ that are both Anillin-eGFP (green) and Rx2 immunoreactive (blue). (D) As the stem cell niche develops, the percentage of Rx2 immunoreative cells that are also Anillin-eGFP positive in the CMZ domain decreases over time from 20% to 11,4%. The arrows in (F-I) point at two dividing, Anillin-eGFP (green) positive cells and at their nuclei (I). (F,G) One of the two cells is also (ptf1a)dsRed positive and non-apically located. Ph, photoreceptors; ac, amacrine cells; hc, horizontal cells; mg, Müller glial cell. ONL, outer nuclear layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Fig. 5

Anillin-eGFP labelled cell division activity is restricted to the CMZ at 3 dpf and at 5 dpf.

(A-C) Optical section from a z-stack taken in the transversal plane (frontal view as represented in the top panel of Fig 4) through the central retina of an anillin:anillin-eGFP transgenic zebrafish fixed at 48 hpf (A), 60 hpf (B) or 3 dpf (C). Counterstaining with DAPI (grey) marks the cell nuclei and the three retinal cell layers. (D) Left: Anillin-eGFP positive cells were counted, which were allocated to the three retinal nuclear layers of the central retina (excluding the CMZ delineated by the dotted line). Anillin-eGFP positive cells were counted per embryo (48hpf, n = 3 apical = 22±4, non-apical = 11±4; 60 hpf, n = 3, apical = 20±4, non-apical = 13±0; 3dpf, n = 3, apical = 3±3, non-apical = 3±1,5). Right: Overlay of a confocal image with a schematic view representing the different locations of Anillin-eGFP positive cells within the central retina: apical, in the outer nuclear layer (ONL) and non-apical in the inner nuclear layer (INL). Error bars represent standard deviation. GCL: ganglion cell layer. (E) Two-photon z-stack projection (z-sections 1 μm apart) of a retina from a 5 dpf zebrafish showing Anillin-eGFP positive cells (green) in the CMZ. The cell nuclei (magenta) were labelled with DAPI. The image represents a dorsal view.

Acknowledgments:
ZFIN wishes to thank the journal PLoS One for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS One