Neurog2 controls the leading edge of neurogenesis in the mammalian retina

Abstract

In the mammalian retina, neuronal differentiation begins in the dorso-central optic cup and sweeps peripherally and ventrally. While certain extrinsic factors have been implicated, little is known about the intrinsic factors that direct this process. In this study, we evaluate the expression and function of proneural bHLH transcription factors during the onset of mouse retinal neurogenesis. Dorso-central retinal progenitor cells that give rise to the first postmitotic neurons express Neurog2/Ngn2 and Atoh7/Math5. In the absence of Neurog2, the spread of neurogenesis stalls, along with Atoh7 expression and RGC differentiation. However, neurogenesis is eventually restored, and at birth Neurog2 mutant retinas are reduced in size, with only a slight increase in the retinal ganglion cell population. We find that the re-establishment of neurogenesis coincides with the onset of Ascl1 expression, and that Ascl1 can rescue the early arrest of neural development in the absence of Neurog2. Together, this study supports the hypothesis that the intrinsic factors Neurog2 and Ascl1 regulate the temporal progression of retinal neurogenesis by directing overlapping waves of neuron formation.

Figure 1

Onset of Neurog2 protein, Neurog2^GFP and Atoh7^LacZ expression in the mouse retina. A-B) Whole mount micrographs of Atoh7^LacZ/+ and Neurog2^GFP/+ embryos at E11.5, demonstrated retinal expression for each reporter (arrows). C-D) Immunolabeling for GFP and βgal showed no detectable optic cup expression at E10.75, although coexpressing cells were present in the diencephalon (arrow in D). E-H) Labeling of Neurog2 protein and GFP at onset of neurogenesis. GFP versus βgal at E11.0 (F) and E12.5 (H) in Neurog2^GFP/+; Atoh7^LacZ/+ mice showed consistent overlap of reporters at these ages. G,I) Neurog2^GFP/+;Atoh7^LacZ/LacZ embryos double-labeled for GFP and βgal at E11.0 and E11.75, respectively, demonstrated the temporal progression of each expression domain. Arrows point to double-labeled cells, and brackets show Neurog2^GFP expression peripheral to the Atoh7^LacZ domain. J) There was no expression of Atoh7 mRNA (arrowheads point to cells with purple in situ reaction product) observed in BrdU pulse-labeled retinal (red) cells. K) However, Neurog2 protein was clearly detected in many BrdU+ S-phase cells at E11.75 and E14.5 (arrows). L) Most βgal+ cells were Neurog2+/βgal+ (fuchsia arrows). Yellow arrowheads mark Neurog2+/βgal− cells peripheral to the Atoh7^LacZ domain. M) The overlap of Neurog2 and GFP (fuchsia arrows) showed GFP+/Neurog2− cells (yellow arrowheads). Scale bars: 50 μm in C,G,H,L,J; 25 μm in F. L = lens.

Figure 2

Initiation of both retinal neurogenesis and Neurog2^GFP expression are coincident in the mouse eye. (A-C) Pax2 and Neurog2^GFP co-labeling. At E11.0-E11.5, Pax2+ cells are evident in the optic cup (A). When Neurog2^GFP+ cells first appeared in the central retina, the Pax2 domain was restricted to the optic stalk and nasal retina (B,C). Arrow in C marks a very rare co-labeled Neurog2^GFP+/Pax2+ cell. (D) Extensive Neurog2^GFP coexpression with Pax6 protein. (E-H) Time course of the onset and expansion of neurogenesis and Neurog2^GFP expression. (E) In an E11.25 retina, Neurog2^GFP+/Tubb3+ cells were detected in the diencephalon, but not in the optic cup when Neurog2^GFP is not present. (F) The first neurons appear from E11.0-E11.5 in Neurog2^GFP+ cells. (G) Neurog2^GFP+/Tubb3+ cells are present in the nasal (arrows) and temporal retina at E12.0. (H) By E13.5, the GFP and Tubb3 expression domains had reached the peripheral retina. (I-K) Dorsal, central, and ventral sections from the same eye at E11.75, demonstrating Neurog2^GFP+/Tubb3+ cells in the dorsal and central (I,J), but not ventral retina (K). (L) Tubb3 and p27/Kip1 were extensively coexpressed in cells exiting the cell cycle. (M) Pou4f2/Brn3b expression onsets at E11.75 in Neurog2^GFP+ cells. (N-P) GFP+ cells coexpress markers of other embryonic fates: RXRγ+ cones (N), AP2α+ amacrines (O), and Prox1+ horizontal and amacrine interneurons (P). Scale bars: 50 μm in A,C,H,N,O. Insets in F-P are 8X magnifications of boxed area in each panel. L = lens.

Figure 3

Delay of early neurogenesis in Neurog2 mutants. (A-D) Tubb3 and Neurog2^GFP labeling of double-heterozygote controls (A), Neurog2^GFP/GFP;Atoh7^LacZ/+ (Neurog2 mutants) (B), Neurog2^GFP/+; Atoh7^LacZ/LacZ (Atoh7 mutants) (C), and double mutant (D) embryos at E11.75. (A’-D’) Insets show higher magnification of the peripheral extent of Tubb3 expression, brackets mark GFP+/Tubb3− domain. (E) Measurement scheme for retinal circumference, Tubb3 and Neurog2^GFP expression domain widths. (F) Compared to controls, the Tubb3 domain was diminished relative to the Neurog2^GFP domain in Neurog2 mutants and double mutants, but not in Atoh7 mutants. (G) The percentage of Tubb3+ cells per total DAPI+ nuclei in the Tubb3 domain indicated that both Neurog2 and Atoh7 mutants had diminished neural differentiation. (H) The distal extent of GFP expression was the same in Neurog2^GFP/+ or Neurog2^GFP/GFP eyes. (I-J’) Neurog2 mutants also exhibited a reduction of the p27/Kip1 (I,I’) and Pou4f2/Brn3b (J,J’) domains. (K-L’) Atoh7 (K,K’) and Neurod1 (L,L’) mRNA expression in Neurog2^+/+ and Neurog2^GFP/GFP retinas, indicated a smaller Atoh7 domain, while Neurod1 expression was unaffected. Scale bars: 50 μm in A,I,K. *p<0.05, ***p<0.001; n=6 eyes (3 embryos) per genotype.

Figure 4

Arrested neurogenesis in the absence of Neurog2 is temporary. (A,A’) At E12.0, the peripheral extent of the Tubb3 domain is reduced in Neurog2^GFP/GFP retinas compared to controls (brackets, A,A’). (B,B’) At E13.5, the peripheral extent of Tubb3 domain is reduced only on the nasal side of the optic cup, compared to the Neurog2^GFP domain (brackets). (C,C’) By E15.5, the peripheral extent of Tubb3 expression was indistinguishable between Neurog2 mutants and heterozygous controls. Scale bars: 50μm in A,B,C; n=8 eyes (4 embryos) per genotype.

Figure 5

Comparison prenatal retinal cell types in Neurog2, Atoh7 and Neurog2;Atoh7 double mutants. (A-D) Retinal thickness was measured as the vitreal-scleral width of DAPI+ nuclei in the NBL and GCL at P0.5. (E-H) Cone precursors were assessed by RXRγ labeling in the outer NBL. (I-L) AP2α+ amacrines in the forming INL and GCL. (M) Compared to wild type controls, Neurog2 mutants and Atoh7 mutants had reduced retinal thickness, and double-mutants were significantly thinner than either single mutant. (N) No significant change in RXRγ+ cells was found in any genotype, although there was a trend towards increased cones in Atoh7 mutants and double-mutants. (O) Neurog2 mutants had a small increase in Pou4f2+ RGCs. (P-R) AP2α+ amacrines amacrines were unaffected in Neurog2 mutants, but significantly increased in Atoh7 mutants and double mutants in both the GCL and NBL. (S-U) Prox1+ retinal cells we significantly increased in the GCL of Neurog2;Atoh7 double-mutants, but unaffected in Neurog2 or Atoh7 single mutants. Scale bar: 50 μm in A, I. *p<0.05, **p<0.01, ***p<0.001; n=6-8 eyes (3-4 P0.5 pups) per genotype.

Figure 6

Cell -proliferation and apoptosis in the absence of Neurog2. (A-D) The percentage of BrdU+ S-phase cells at E15.5 was normal in Neurog2 mutants (Neurog2^GFP/GFP;Atoh7^LacZ/+) and double-mutants (Neurog2^GFP/GFP;Atoh7^LacZ/LacZ). (E-H) The percentage of activated Caspase-3+ apoptotic cells (arrows in E-G) was significantly increased in double mutants, but not in Neurog2 single mutants. Scale bar: 50 μm in A. ***p<0.001; n=6 eyes (3 embryos) per genotype.

Figure 7

Ascl1 rescue of delayed neurogenesis in Neurog2 mutants. (A,A’) At E12.0, only rare Ascl1+ cells are present in Neurog2^GFP/+ eyes. The Ascl1+ cell in A coexpresses GFP, thus is in the Neurog2 lineage (arrow in A’. and higher magnification in A”). (B,B’) Neurog2^GFP/Ascl1KI retinas have many more Ascl1+GFP+ cells (arrows in B,B’ and higher magnification in B”). (C-E) Tubb3 and GFP co-labeling at E12.0. Delayed progression of Tubb3+ neurons in Neurog2^GFP/GFP mutants (D) was not found in Neurog2^GFP/Ascl1KI eyes (E), which had an identical Tubb3 domain to controls (C). (F-H) Immunolabeling for Pou4f2/Brn3b and GFP showed that differentiated RGCs were also normal in Neurog2^GFP/Ascl1KI mice (H), compared to Neurog2^GFP/GFP mutants (G). (I-L) Ectopic Ascl1 expression is delayed in Neurog2 mutants, relative to endogenous Neurog2 expression (compare to Figure 1), with a smaller domain of Atoh7 mRNA expression in Neurog2^GFP/Ascl1KI retinas (K,L). (M-P) At E12.5, ectopic Ascl1 and normal Atoh7 mRNA expression patterns are observed. Scale bars: 75 μm in A,C; 50 μm in I,M; n= 3-4 embryos per genotype; L = Lens.