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The laminar arrays of distinct cell types in the vertebrate retina are built by a histogenic process in which cell fate is correlated with birth order. To explore this co-ordination mechanistically, we altered the relative timing of cell cycle exit in the developing Xenopus retina and asked whether this affected the activity of neural determinants. We found that Xath5, a bHLH proneural gene that promotes retinal ganglion cell (RGC) fate, ( Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997) Neuron 19, 981-994), does not cause these cells to be born prematurely. To drive cells out of the cell cycle early, therefore, we misexpressed the cyclin kinase inhibitor, p27Xic1. We found that early cell cycle exit potentiates the ability of Xath5 to promote RGC fate. Conversely, the cell cycle activator, cyclin E1, which inhibits cell cycle exit, biases Xath5-expressing cells toward later neuronal fates. We found that Notch activation in this system caused cells to exit the cell cycle prematurely, and when it is misexpressed with Xath5, it also potentiates the induction of RGCs. The potentiation is counteracted by co-expression of cyclin E1. These results suggest a model of histogenesis in which the activity of factors that promote early cell cycle exit enhances the activity of factors that promote early cellular fates.
Fig. 1. Cell cycle genes are downregulated as neuronal determination genes are upregulated in the CMZ of stage 41 Xenopus embryos. In situ hybridisations to identify expression of the genes indicated on each panel. C,I,O,U,F,L,R and X are overlaid images of the single hybridisations. In A the dashed line indicates the end of the CMZ and the solid lines, the boundaries between differentiated layers. Brackets indicate regions of co-expression.
Fig. 2. Proliferation of precursor cells in the CMZ. (A-C) Double staining for cdk2 expression (A) and BrdU uptake (B). BrdU was injected at stage 41. The embryos were fixed one hour later and then in situ hybridisation and BrdU immunostaining were performed. (C) Overlaid image. (D) Immunostaining of stage 41 CMZ with MPM-2 antibody. Mitotic cells with endfeet are indicated by arrows, and differentiated photoreceptors and RGCs close to CMZ are indicated by arrowheads. (E), Double staining of stage 41 CMZ using phosphohistone H3 antibody (red) and calbindin antibody (green) to identify cone photoreceptors. (F) Immunostaining with cyclin A2 antibody. (G) Immunostaining with cdc2 antibody. The end of the CMZ is indicated by a dashed line in C, D, E, F and G. (H), Schematic model of the expression of cell cycle components and determination genes in the CMZ of the stage 41 embryo.
Fig. 3. Activity of Xath5 as an RGC determinant is affected by modulation of cell cycle activity. (A) Cell cycle exit of RGC precursors is unaffected by Xath5. Xath5 plus GFP or GFP only was lipofected at stage 15, and then BrdU injection started at stage 28 or late stage 32. At stage 41, the ratio of BrdU-positive cells in all lipofected cells or lipofected RGCs was analysed. (B) Cell cycle exit of RGC precursors is affected by p27Xic1 and cyclin E1. p27Xic1 or cyclin E1 was colipofected with Xath5 at stage 15, and then BrdU injection started at early stage 32. At stage 41, the ratio of BrdU-positive lipofected RGCs to all lipofected RGCs was analysed. (C) Activity of Xath5 as a RGC determinant is affected by modulation of cell cycle activity. Xath5 was colipofected with p27Xic1 or cyclin E1 at stage 15, and then their effect on cell fate determination was analyzed. (D) Deleted constructs of p27Xic1 also increase the fraction of cells that become RGCs. (E) Overexpression of cyclin E1 in retinal precursor cells transiently activates the cell cycle. An expression construct of cyclin E1 was lipofected at stage 15 and BrdU injection started at the indicated stages. At stage 42, BrdU incorporation was analyzed. (F-H) Retina lipofected with Xath5 by itself and in combination with cyclin E1. (F) Retina lipofected with GFP. R, RGC; P, photoreceptor cell; H, horizontal cell; A, amacrine cell; B, bipolar cell; M, Müller glial cell. (G) Retina lipofected with Xath5 and GFP. The ratio of RGCs is 50% (12 RGCs out of 24 lipofected cells). (H) Retina lipofected with Xath5, cyclin E1 and GFP. The ratio of RGCs is 22% (11 RGCs out of 50 lipofected cells).
Fig. 4. Effects of XSu(H)Ank and XSu(H)DBM on cell cycle regulation and determination. (A,B) Stage 41 retina lipofected with XSu(H) constructs plus GFP. (A) XSu(H)Ank. (B) XSu(H)DBM. (C) Immunostaining of XSu(H)Ank lipofected retina with R5 antibody (red), a Müller glial cell marker. Cells expressing XSu(H)Ank are green. The processes of the basal side of the lipofected cells show R5 staining. (D) Distribution of retinal cell type in stage 41 retina lipofected with XSu(H) constructs at stage 15. (E) Effect of XSu(H)Ank and XSu(H)DBM on BrdU incorporation into Müller glial cells. The construct was lipofected at stage 15. After injection of BrdU at stage 34, the ratio of BrdU-positive cells in Müller glial cells was analysed at stage 41. XSu(H)Ank stops the cell cycle in the precursors earlier than the natural timing. (F) Effect of XSu(H)Ank on the ratio of phosphohistone H3-positive cells. After lipofection of the construct at stage 15, the ratio of phosphohistone H3-positive cells in the lipofected cell population was measured at stage 36. (G) Model of Müller glial cell generation in retina overexpressing XSu(H)Ank.
Fig. 5. XSu(H)Ank enhances co-expressed Xath5 activity through its effect on the cell cycle. (A-B) Stage 41 retina lipofected with Xath5 alone (A) and in combination with XSu(H)Ank (B). (C) Almost all RGCs induced by overexpression of Xath5 and XSu(H)Ank send axons that exit the eye through the optic nervehead (ONH). (D) Ratio of cells in the retina lipofected with Xath5 and/or XSu(H)Ank. Retina was lipofected with the indicated constructs at stage 15. The effect on cell type distribution was analysed at stage 41. (E) Effect of XSu(H)Ank on RGC versus Müller cell ratios as a function of stage of transfection. XSu(H)Ank plus GFP was lipofected at the indicated stages, and the effects on cell fate determination analyzed at stage 41. In control, GFP was lipofected at stage 15. Control at stage 18, 21, or 24 was similar to that of stage 15. (F) XSu(H)Ank induces earlier cell cycle exit of RGC precursors induced by Xath5. Xath5 was colipofected with XSu(H)Ank or with XSu(H)Ank plus cyclin E1 at stage 15. BrdU injection started at stage 32. At stage 41, the ratio of BrdU-positive cells in the lipofected RGCs was analyzed. (G) Model of RGC generation in retina overexpressing Xath5 alone or XSu(H)Ank plus Xath5.
Fig. 6. Model of interaction among Notch pathway, proneural genes and cell cycle regulation in the Xenopus retinal development. (A,B) The spatial development of retinal cells in the CMZ (A) and the temporal development of retinal cells (B). (1) Stem cells divide slowly with low level of cell cycle activators; (2) retinoblasts divide rapidly as cell cycle activators rise; (3) as cell cycle activators are downregulated, retinoblasts enter their last cell cycles; (4) as expression of proneural genes of the atonal family rise in the presence of Notch, the first cells exit the cell cycle and acquire early neural fates; (5) later neural fates are the result of progressive cell cycle exit and the effect of the Notch pathway on specific proneural genes; (6) in the last phase of generating retinal cells, proneural gene expression has decreased while p27Xic1 expression has increased, favouring a glial fate. (C) Interaction models showing how the same network produces neurons when Notch and proneural levels are high (left), and glia when Notch and p27Xic1 levels are high (right).
