If one wishes to understand how a complete CNS structure like the retina

is formed at a clonal level, it is critical to know that the growth of clones one is studying can fully account for the growth of the structure, as some marking protocols may preferentially label particular cell types or be harmful to the labeled find more cells. To assess whether the MAZe:Kaede retinal clones are accurately representative of retinal growth and differentiation, we first explored the growth kinetics of the whole retina. By fitting a surface to a three dimensional (3D) reconstruction of the retina, we obtained its volume at distinct developmental stages and combined this with measurements of cell density

determined from confocal sagittal sections (Figures 2A–2C, Experimental Procedures, and Supplemental Experimental Procedures) to obtain total retinal cell number as a function of developmental time (Figure 2D). These results revealed that the embryonic retina consists of approximately 1,800 cells at 24 hpf (Figure 2D and Figures S2G and S2H), rising to approximately 11,000 cells at 48 hpf, and 21,000 cells at 72 hpf. This translates to a 6- and 12-fold increase, respectively. Clones derived from single progenitors at 24 hpf, as expected, showed variability in size, both at 48 hpf learn more Temozolomide and 72 hpf (Figure 3A). Yet, the average increase in the size of these clones was strikingly consistent

with the measured increase in total cell number in a normal retina (Figure 2D). Two other independent methods of clone induction, single-cell electroporation and transplantation, gave very similar results (Figures S2A–S2F). Moreover, clones from RPCs at 24 or 32 hpf produced, when pooled, a ratio of cell types that was comparable to the tissue’s composition (Figure 2E). These results indicate that the clones, though individually variable in size and fates, are quantitatively representative of the retina as a whole. To investigate why retinal clones show such striking variability in size, we first looked at their size distribution as a function of time and retinal position. Clones induced from single RPCs at 24 hpf and examined at 72 hpf form a distribution that is both broad in size and independent of nasal/temporal position in the retina (Figure 3B). The distribution of clones induced at 32 hpf is also broad (Figures 3B and 3C), yet at this stage, clones positioned in the temporal zone were on average significantly larger than those derived from the nasal zone. This suggests a relative delay in the developmental program between temporal and nasal parts of the retina.