Figure 1.
There is a neuronal birthdate gradient within the chick optic tectum.
Previous birthdating studies [5] revealed a lateral rostroventral to medial caudodorsal birthdate gradient within the optic tectum. The lateral to medial dimension is not represented here. Taking advantage of this birthdate gradient, we looked for FGF2-induced differences in cell cycle exit in the rostroventral tectum at the beginning of tectal neurogenesis and in the caudodorsal tectum at the end of tectal neurogenesis.
Figure 2.
Schematic of the experimental design and predictions.
On either ED5 (A) or ED8 (B), control and FGF2-treated embryos were infused with EdU followed by regular booster shots sufficient to saturate the system. Birds were then sacrificed on ED12 for processing. EdU is taken up by all proliferating cells as they pass through S-phase (shown in green), whereas all cells born prior to infusion are EdU-unlabeled (shown in brown). In the ED5 condition (A), the early born neurons in the deep layers of control tecta have already been born at the time of EdU infusion and so do not take up EdU (brown). In contrast, if FGF2 delays neurogenesis, then all cells should take up the EdU (green) in the treated embryos (C), because no neurons would have been born at the time of EdU infusion. In the ED8 condition (B), only late born neurons in the middle layers of control tecta have yet to be born, and so take up the EdU (green). If FGF2 delays tectal neurogenesis, then one would expect the neurons in the most superficial layers also to incorporated the EdU, because they, too, would not yet have been born before the EdU infusion (C). The ventricular zone (VZ) contains proliferating cells and is, therefore, EdU-positive after the EdU infusions have begun.
Figure 3.
FGF2 treatment increases the absolute number of tectal cells.
Intra-ventricular injections of FGF2 on ED4 decrease the number of cells above a unit area of ventricular surface by 14% compared to controls (A). Taking into account our previous observation that FGF2 increases tectal ventricular surface area by 181% [3], we estimate that FGF2 treatment increases total tectal cell numbers by approximately 140% compared to controls (B). The photographs (C and D) illustrate the dramatic tangential expansion of the tectum in FGF2 treated birds, relative to controls. Scale bar = 1 mm.
Figure 4.
Sample data showing that FGF2 treatment delays tectal neurogenesis.
Using counting boxes that span the radial extent of the tectum (white rectangle in A), we counted the total number of cells (bisbenzimide counterstain shown in red) and the number EdU-positive cells (Alexa Fluor 488 appears yellow) in the deep, middle and superficial layers. Cumulative EdU labeling beginning at ED5 (A and B) revealed fewer unlabeled (EdU-negative) cells in the rostroventral tectum of FGF2 treated birds than in controls (quantitative comparisons are shown in Fig. 5 A, B), which means that fewer cells had exited the cell cycle by ED5. Similarly, cumulative EdU labeling beginning at ED8 (C and D) revealed fewer EdU-unlabeled cells in the caudodorsal tectum of FGF2-treated birds than in controls (quantitative comparisons are shown in Fig. 5 C, D.). Scale bar = 100 µm.
Figure 5.
Quantitative data showing that FGF2 treatment delays tectal neurogenesis.
In birds infused with EdU starting on ED5, the proportion of EdU-unlabeled cells in the early-born rostroventral tectum of FGF2-treated embryos is decreased by 52% relative to controls (A); the absolute number of EdU-unlabeled cells is decreased by 57% (B). Both observations are consistent with a delay in tectal neurogenesis in FGF2-treated birds. In birds infused with EdU starting on ED8, the proportion and absolute number of EdU-unlabelled cells in the caudodorsal tectum are decreased in the FGF2-treated embryos by 18% (C) and 12% (D). Again, both findings are consistent with a delay in neurogenesis. All mentioned differences are statistically significant (see text).
Figure 6.
FGF2 decreases the thickness of early born tectal layers.
By ED12, birds treated with FGF2 showed a 50% decrease in the radial thickness of the deep layers in the lateral tectum compared to controls (A, B, D). In these same lateral areas, despite the presence of “volcano”-like disturbances in the outer laminae (B), there is no statistically significant difference in the thickness of the superficial or the middle layers between FGF2-treated birds and controls (D). The caudal, folded areas of FGF2 treated birds (C) exhibited no significant changes in the thickness of any layers (D).
Figure 7.
FGF2 delays the birthdates of neurons in deep, superficial and middle layers.
In the rostroventral tectum of FGF2- treated embryos infused with EdU starting on ED5, the proportion of EdU-unlabeled cells is decreased by 32, 72, and 57% in the deep, superficial, and middle tectal layers, respectively (A). There were no statistically significant differences between FGF2 treated birds and controls in any layers in the lateral or caudal tectum when EdU was infused on ED5 (A). In embryos that were infused with EdU starting on ED8, the proportion of EdU-unlabeled cells was decreased by 19, 22, and 14% in the deep, superficial, and middle layers, respectively (B). White bars are controls; gray bars are FGF2 treated. Background shading indicates birth order gradients, with darker shades indicating later birthdates.