Article Figures & Data
Figures
- Fig. 1.
Immunofluorescent and autoradiographic analysis of dissociated retinal cells. A, Cell heavily labeled for [3H]thymidine that was also immunoreactive with Rho4D2, shown in B (large arrow).C, Cell heavily labeled for [3H]thymidine that was also immunoreactive for BrdU incorporation, shown in D (large arrow). Arrowheads mark examples of immunoreactive cells that were not heavily labeled by [3H]thymidine. Small arrows mark examples of cells that were negative for staining with the corresponding primary antibody. Cells in A andC were viewed under bright-field and UV optics. The nuclei were stained by the nuclear stain DAPI. Cells inB and D were viewed under fluorescent light conditions suitable for detecting the Texas Red-conjugated secondary. Note that Rho4D2 staining was membrane-associated, whereas staining for BrdU incorporation was nuclear.
- Fig. 2.
[3H]thymidine birthdating as a method for labeling retinal cells undergoing their terminal S-phase.A, Representative histograms displaying grain distributions in cells of retinae that were administered a pulse of [3H]thymidine at E17 in vivo and harvested at P1 or P15. The criterion for “heavily labeled” (as indicated) is defined as cells containing more than half the grain number of the most heavily labeled cell in an experiment. In the supporting table, the number of heavily labeled cells that survived to P1 and P15 was approximated. B, Cumulative BrdU labeling after [3H]thymidine pulsein vitro. P0 retinal explant cultures were administered a 1 hr pulse of [3H]thymidine in vitro and then cumulatively labeled by addition of BrdU to cultures at 5, 12, 24, or 48 hr after the [3H]thymidine pulse. Retinae were then examined after 4 d of culture for doubly labeled cells. Plotted are the percentages of [3H]thymidine heavily labeled cells that were positive for BrdU incorporation at the different times of addition. Each data point represents the average ± SEM (n = 4). More than 50 heavily labeled cells were scored per trial for each time point.
- Fig. 3.
Overall kinetics of rod precursor genesis correlated with kinetics of rhodopsin onset. A, Rod precursor genesis in developing rat retina. Developing retinae were administered a [3H]thymidine pulse in vivo on E14, E15, E17, E19, E21, P0, P2, or P5. The percentage of surviving cells born on each day of injection that differentiated as rod photoreceptors was assessed using combined immunocytochemical–autoradiographic analysis on mature retinae.B, Kinetics of rhodopsin expression in neonatal retina and kinetics of onset of immunocytochemically detectable rhodopsin expression in the postnatal rat retina. Plotted are the percentages of cells per retina positive for Rho4D2 staining during postnatal development. C, The kinetics of rhodopsin onset closely mimic the kinetics of rod precursor genesis separated by a 6.5–7.0 d lag. We used the data in A and B and the number of total retinal cells and postmitotic cells generated on each day of retinal development (Alexiades and Cepko, 1996) to approximate the cumulative numbers of rod precursors born and rhodopsin-positive cells for each day of retinal development. (The estimation of the cumulative number of rod precursors born neglects cell death and therefore may be a slight overestimate for the earlier time points in particular.) Theplot lines for A and for rod precursor birth in C represent the average of two trials, and the error range extends to the values of each individual trial, with the exception of P5 where a single trial was performed. One hundred or more heavily labeled cells were scored for each data point for these curves. In B and for the number of rhodopsin-positive cells inC, each plotted value represents the average ± SEM. More than 250 cells per trial were scored, with between two and six trials per time point.
- Fig. 4.
Kinetics of onset of rhodopsin expression in retinal cohorts born postnatally. Neonatal litters were administered [3H]thymidine by intraperitoneal injection on P0, P2, or P5 to label retinal precursor cells undergoing terminal S-phases on these days. A developmental series of freshly dissociated retinae from these litters was then analyzed auto-radiographically and for anti-rhodopsin immunoreactivity. A, P0.75; B, P2.5;C, P5.75. D, Co-plot of kinetics for P0.75, P2.5, and P5.75, normalized for the percentages of rods ultimately found in each mature cohort. The plot linerepresents the average of two trials for each time point. ForA and B, the error range extends to the values of each individual trial.
- Fig. 5.
Kinetics of onset of rhodopsin expression in retinal cohorts born embryonically. Pregnant rats were administered single injections of [3H]thymidine on E17, E19, and E21 to label retinal precursor cells undergoing terminal S-phases on these days. A developmental series of freshly dissociated retinae from these litters was then analyzed auto-radiographically and for anti-rhodopsin immunoreactivity. A, E17.5; B, E19.5;C, E21.75. D, Co-plot of kinetics for E17.5, E19.5, and E21.75, normalized for the percentages of rods ultimately found in each mature cohort. The plot linerepresents the average of two trials for each time point. ForA–C, the error range extends to the values of each individual trial.
- Fig. 6.
Culturing embryonically born cells with excess postnatal cells does not alter lag to rhodopsin or change percentage of embryonic cells differentiating as rods. A, Outline of protocol for mixed reaggregate experiments (see Materials and Methods).B, Kinetics of rhodopsin expression in reaggregates of E16-born cells cultured alone (○), P0-born cells cultured alone (□), or E16-born cells cultured with a 20-fold excess of P0 retinal cells (▪). C, D, Confocal images of reaggregates cultured for 1 hr (C) or 5 d (D). E16 cells are labeled with PKH-26 dye and are depicted in red; P0 cells are labeled with PKH-2 dye and are depicted in green. C is one optical slice, and D is a composite of 16 optical slices covering 10 μm in the z-axis.
- Fig. 7.
Two distinct phases of rod development in vivo. Rod genesis is divided into two phases in vivo: an early phase and a late phase. Depicted schematically are the time lags from the terminal mitosis of rod precursors to the 50% final rhodopsin level for cohorts born from E15 through P5. For a majority of rod precursors, those born after E19, the timing of rhodopsin onset is tightly linked to the timing of cell cycle withdrawal. For these cohorts, the steps leading from the terminal mitosis to rhodopsin expression take an average 5.5–6.5 d. These cells constitute the late phase of rod genesis. During the early phase of rod genesis, those cells born before E19 appear to display lags significantly longer than 6.5 d, and commence rhodopsin expression approximately synchronously with the late phase cells in the early postnatal period.
Tables
- Table 1.
The average lag between terminal S-phase and onset of rhodopsin is 6.5–7.0 d for the total population of developing rods
Number of rods × 107 (% final rods) Day by which rods are born1-a Day by which rods are rhodopsinpositive1-1002 Lag time (d) 0.178 (10) E20.2 ± 0.18 P4.9 ± 0.19 6.7 ± 0.37 0.444 (25) E21.3 ± 0.09 P6.2 ± 0.26 6.9 ± 0.35 0.888 (50) P1.1 ± 0.05 P7.8 ± 0.26 6.7 ± 0.31 1.332 (75) P2.6* P9.3 ± 0.20 6.7 ± 0.20 1.599 (90) P3.4* P10.0 ± 0.20 6.6 ± 0.20 The above data are derived from Figure 3C, where the cumulative numbers of rod precursors born and rhodopsin-positive precursors were approximated for each day of retinal development. From these data, the lag between rod precursor genesis and onset of rhodopsin expression was determined. Each data point represents the average ± SEM, except where indicated.
↵F1-a In this column, data were derived fromn = 2, except where indicated by an asterisk (*) where a single trial was performed.
↵F1-1002 In this column, data were derived fromn = 2–6.
- Table 2.
Rhodopsin onset is temporally correlated with terminal mitosis in postnatally born rod precursors
Day of birth of rod precursors % Final rhodopsin Postnatal day Lag time from terminal mitosis (d) P0.75 10 P4.74 ± 0.04 3.99 ± 0.04 50 P6.40 ± 0.27 5.65 ± 0.27 90 P7.74 ± 0.48 6.99 ± 0.48 P2.5 10 P6.50 ± 0.01 4.00 ± 0.01 50 P8.47 ± 0.01 5.97 ± 0.01 90 P9.70 ± 0.03 7.2 ± 0.03 P5.75 10 P9.97 4.22 50 P11.47 5.72 90 P12.36 6.61 Neonatal litters were administered 3H-thymidine on P0, P2, or P5 to label retinal precursor cells undergoing terminal S-phases on these days. A developmental series of freshly dissociated retinae from these litters was then analyzed by combined immunocytochemical–autoradiographic analysis. Each data point represents the average ± SEM where n = 2, except for P5, where a single trial was performed. Data are derived from Figure 4D.
Postnatal day Number Rho4D2+/total heavily labeled scored (%) Trial 1 Number Rho4D2+/total heavily labeled scored (%) Trial 2 P1.75 0 /100 (0) ND P2.5 0 /100 (0) ND P3.75 0 /100 (0) ND P4.75 ND 0/100 (0) P5.5 0 /74 (0) ND P6.5 8 /103 (7.8) ND P10.5 11 /91 (12.1) ND P19.0 11 /100 (11) 10 /100 (10) Pregnant rats were administered single injections of3H-thymidine at E15.75 to label retinal precursor cells undergoing terminal S-phases at this time. A developmental series of freshly dissociated retinae from these litters was then analyzed autoradiographically and for anti-rhodopsin immunoreactivity. ND indicates a trial timepoint that was not scored.
- Table 4.
Timing of rhodopsin onset in early embryonic-born precursor appears approximately synchronous with later-born cohorts
Day of birth of rod precursors % Final rhodopsin Postnatal day Lag time from terminal mitosis (d) E15.75 10 P5.64 11.89 50 P6.21 12.46 90 P8.45 14.7 E17.5 13.4 ± 1.6 P1.5 6.0 50 P3.74 ± 0.30 8.24 ± 0.30 90 P5.74 ± 0.51 10.24 ± 0.51 E19.5 10 P2.13 ± 0.06 4.63 ± 0.06 50 P3.60 ± 0.07 6.1 ± 0.07 90 P6.64 ± 1.68 9.15 ± 1.68 E21.75 10 P4.34 ± 0.83 4.59 ± 0.83 50 P6.29 ± 0.00 6.54 ± 0.00 90 P8.12 ± 0.70 8.37 ± 0.70 Pregnant rats were administered single injections of3H-thymidine on E15, E17, E19, or E21. A developmental series of freshly dissociated retinae from these litters was then analyzed by combined immunocytochemical–autoradiographic analysis. Each data point represents the average ± SEM wheren = 2, except for E15, for which a single trial was performed. Data are derived from Figure 5D and Table3.
