Influences on neural lineage and mode of division in the zebrafish retina in vivo

Abstract

Cell determination in the retina has been under intense investigation since the discovery that retinal progenitors generate clones of apparently random composition (Price, J., D. Turner, and C. Cepko. 1987. Proc. Natl. Acad. Sci. USA. 84:156-160; Holt, C.E., T.W. Bertsch, H.M. Ellis, and W.A. Harris. 1988. Neuron. 1:15-26; Wetts, R., and S.E. Fraser. 1988. Science. 239:1142-1145). Examination of fixed tissue, however, sheds little light on lineage patterns or on the relationship between the orientation of division and cell fate. In this study, three-dimensional time-lapse analyses were used to trace lineages of retinal progenitors expressing green fluorescent protein under the control of the ath5 promoter. Surprisingly, these cells divide just once along the circumferential axis to produce two postmitotic daughters, one of which becomes a retinal ganglion cell (RGC). Interestingly, when these same progenitors are transplanted into a mutant environment lacking RGCs, they often divide along the central-peripheral axis and produce two RGCs. This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

Figure 1.

ath5:GFP is expressed in both mitotic and differentiating retinal progenitors. (A) 3D lateral view of an ath5:GFP transgenic retina at 32 h after fertilization, as seen in all subsequent time-lapse recordings performed in this study (the image is a combination of several stacks). The diagram in B shows the main developmental steps of one retinal neuroepithelial cell. The cell soma undergoes apico-basal interkinetic nuclear movements. Mitosis (M) occurs in proximity of the apical surface (top). (C) Time-lapse series showing an ath5:GFP progenitor dividing at the apical surface (dashed line). Dividing progenitors and daughter cells are highlighted in green or red. The long arrows point at the apical processes that connect the ath5:GFP retinal progenitors to the apical surface. Short arrows point to the apical process of a differentiating ath5-positive cell. This process can be seen retracting in the right-most panel. (D) A single retinal progenitor cell is labeled with GAP-mRFP. The time-lapse series shows that ath5:GFP appears (t = 0) when the cell soma is migrating toward the apical surface (ap). At t = 45 min, the cell divides and both daughter cells migrate basally. White arrows indicate the position of the cell soma of the progenitor or daughter cells. (E) Time-lapse sequence showing the sequential steps of RGC differentiation (also see the diagram in B). The cell highlighted in green is expressing ath5:GFP. At t = 0, the cell is connected to the apical and basal surface with its apical and basal processes, respectively. Later on, the apical process (ap and arrows) retracts from the apical surface (t = 1.50), and the axon starts to elongate and extend from the basal process (bp and arrows). (F–H) Confocal images of an ath5:GFP transgenic retina at 30 h after fertilization, hybridized with ath5 mRNA probe (in red). Each image is a projection of stacks. The white box indicates what is represented in G. Arrows point at two dividing progenitors at the apical surface that are expressing both ath5:GFP and ath5 mRNA, although clearly at different amounts. Arrowheads point at differentiating RGCs. (H) 3D representation of a dividing ath5:GFP cell (outlined) expressing ath5 mRNA. A, anterior; ap; apical process; bp, basal process; D, dorsal; P, posterior; RGC, retinal ganglion cell; RPI, retinal pigmented epithelium; V, ventral.

Figure 2.

ath5:GFP cells become RGCs and other cell types in the zebrafish retina. (A–C) Sections through the central retina of a 5-d ath5:GFP transgenic embryo. (A) The three retinal cell layers are separated by a white dashed line. (B) Retinal ganglion cell (RGC) ath5:GFP progenitors in the ganglion cell layer (GCL) are zn-5+. The white box in B indicates the area shown in C. Some ath5:GFP progenitors (C) become photoreceptors (Ph), amacrines (Am), and horizontal (Ho) cells. (D–F) Sections of 4-d ath5:GFP transgenic embryos immunostained with zpr-1 (D), PKCβ1 (E), and calretinin (F; arrows). White dashed lines separate the three retinal cell layers. ath5:GFP cells colabel with zpr-1 in the outer nuclear layer (ONL) and with calretinin in the inner nuclear layer (INL). ath5:GFP cells in the INL do not colabel with PKCβ1.

Figure 3.

ath5:GFP progenitors appear in G2 and divide once, generating one RGC and one non-RGC daughter cell. (A) Time-lapse series showing the lineage of an ath5:GFP progenitor transplanted in a wild-type environment. Imaging was started 30–32 h after fertilization, and t = 0 corresponds to the time of appearance of ath5:GFP (4 h after the onset of the video recording). Daughter cells have been highlighted with different colors (red or yellow). The apical surface is up, whereas the basal surface is down. After division (t = 45 min), both daughter cells migrate toward the basal surface. At t = 5 h, one daughter cell (red) migrates toward the apical surface, whereas the other one (yellow) retracts the apical process and starts to grow an axon toward the basal surface. The white arrows point at the retracting apical process and the growth cone at the tip of the axon. Every single image is a 3D reconstruction of confocal stacks. AP, apical cell; RGC, retinal ganglion cell. (B) Diagram similar to the one shown in Fig. 1 B illustrating this division lineage, which is schematically represented in C.

Figure 4.

More RGCs are generated at the expense of other retinal cell types by ath5:GFP progenitors in the lakritz mutant environment. Increased number of RGCs generated by ath5:GFP progenitors when transplanted in the lakritz environment. (A and B) Longitudinal sections trough the central retina (at 4 d after fertilization) showing ath5:GFP retinal cells (green) in a wild-type environment (A) or in a lakritz mutant environment (B). RGCs are zn-5+ (red), and nuclei have been stained with DAPI to highlight the retinal cell layers. ath5:GFP retinal cells are found mainly in the GCL in a lakritz environment (B), whereas they are distributed in all three layers in the wild-type environment (A). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. White dashed lines separate the three retinal layers.

Figure 5.

ath5:GFP progenitors generate two RGCs after division in the lakritz environment. (A) The two daughter cells have been highlighted in yellow or red. Time-lapse series showing an example of an ath5:GFP progenitor generating two RGCs in the lakritz environment. Imaging was started at 30–32 h after fertilization, and t = 0 corresponds to the time of appearance of ath5:GFP (4 h after the onset of the video recording). After 11 h, the red daughter cell starts migrating toward the apical surface. Once it has reached the apical surface (t = 15 h), it migrates back again toward the basal surface, where it differentiates in RGCs. The location of both daughter cells after 20 h is outlined by a white dotted line. Both cells were zn-5 positive after immunolabeling of the imaged retina. (B) An example of a time-lapse series showing an ath5:GFP progenitor that divides and generates another dividing progenitor. Imaging was started 30–32 h after fertilization, and t = 0 corresponds to the time of appearance of ath5:GFP (3 h after the onset of the video recording). At t = 9.40 h, the progenitor highlighted in red divides once more at the apical surface, generating one daughter cell (green) that lost its apical process and began to put out an axon and another daughter cell (red) that remained apical. White arrowheads point to the retracting apical process and the forming axon. AP, apical cell; RGC, retinal ganglion cell.

Figure 6.

Comparisons of lineage in a wild-type and lakritz environment. (A) Summary of the lineage patterns of ath5:GFP progenitors observed in the wild-type and lakritz environment. Progenitors have been highlighted in green and RGCs in yellow. The apical cell has been highlighted in red. The fate of the apical cell can be one of a number of cell types, but our data seem to suggest that it is a photoreceptor more often than not. RP, retinal progenitor. From left to right: n = 5, 7, and 2. (B) Using these samples as indicators, one can calculate that in the wild-type environment, ∼50% of the cells generated from ath5:GFP-positive progenitors would be predicted to become RGCs, whereas in the mutant environment, 77% would become RGCs. These differences are compared with the actual percent changes in the production of RGCs from ath5:GFP progenitors transplanted to the wild-type and lakritz environments (see Fig. 4). These are significantly different at the P < 0.005 level determined by the Chi-squared test. For wild-type hosts, n = 795 in nine retinas. For lakritz hosts, n = 1,477 in 11 retinas.

Figure 7.

Analysis of the orientation of cell division in ath5:GFP progenitors cells. (A) 3D representation of the zebrafish retina as an ellipsoid body (Das et al., 2003). Only divisions where the mitotic spindle is oriented perpendicular to the retinal surface (indicated in yellow and red) can be seen in the zebrafish retina. An example of apico-basal division, which is not found in the zebrafish retina, is also indicated. An orientation of 0° represents a cell division along the central-peripheral (radial) axis, whereas an orientation of 90° represents a division along the circumferential axis (Videos 7 and 8, available at http://www.jcb.org/cgi/content/full/jcb.200509098/DC1). (B) Cumulative distributions of the orientation of cell divisions found in wild-type progenitors when transplanted either into a wild-type host (blue plot) or into lakritz hosts (red plot). The y axis indicates the proportion of cell divisions, whereas the x axis indicates the corresponding angles (n = 20 for both wild-type and lakritz hosts). Divisions that tend to be central-peripheral (<45°) are increased in ath5:GFP progenitors in the lakritz environment. The two distributions differ significantly, as determined by the Kolmogorov-Smirnov test (P = 0.013). (C) Divisions with symmetric outcomes tend to be oriented <45° (radial), whereas divisions with asymmetric outcomes preferentially occur with an angle >45° (circumferential). P = 0.02 by the Chi-squared test; n = 5 in symmetric cases; n = 7 in asymmetric cases.