Simultaneous Requirements for Hes1 in Retinal Neurogenesis and Optic Cup-Stalk Boundary Maintenance

Abstract

The bHLH transcription factor Hes1 is a key downstream effector for the Notch signaling pathway. During embryogenesis neural progenitors express low levels of Hes1 in an oscillating pattern, whereas glial brain boundary regions (e.g., isthmus) have high, sustained Hes1 levels that suppress neuronal fates. Here, we show that in the embryonic mouse retina, the optic nerve head and stalk express high Hes1, with the ONH constituting a boundary between the neural retina and glial cells that ultimately line the optic stalk. Using two Cre drivers with distinct spatiotemporal expression we conditionally inactivated Hes1, to delineate the requirements for this transcriptional repressor during retinal neurogenesis versus patterning of the optic cup and stalk. Throughout retinal neurogenesis, Hes1 maintains proliferation and blocks retinal ganglion cell formation, but surprisingly we found it also promotes cone photoreceptor genesis. In the postnatal eye, Hes1 inactivation with Rax-Cre resulted in increased bipolar neurons and a mispositioning of Müller glia. Our results indicate that Notch pathway regulation of cone genesis is more complex than previously assumed, and reveal a novel role for Hes1 in maintaining the optic cup-stalk boundary.SIGNIFICANCE STATEMENT The bHLH repressor Hes1 regulates the timing of neurogenesis, rate of progenitor cell division, gliogenesis, and maintains tissue compartment boundaries. This study expands current eye development models by showing Notch-independent roles for Hes1 in the developing optic nerve head (ONH). Defects in ONH formation result in optic nerve coloboma; our work now inserts Hes1 into the genetic hierarchy regulating optic fissure closure. Given that Hes1 acts analogously in the ONH as the brain isthmus, it prompts future investigation of the ONH as a signaling factor center, or local organizer. Embryonic development of the ONH region has been poorly studied, which is surprising given it is where the pan-ocular disease glaucoma is widely believed to inflict damage on RGC axons.

Keywords: Hes1; Notch signaling; bHLH; gliogenesis; neurogenesis; retina.

Figure 1.

Hes1 protein and Cre driver spatiotemporal expression domains. A, Anti-Hes1 labeling delineates low, nonuniform retinal and high, sustained ONH Hes1 expression. B, Hes1 and Tubb3 colabeling clearly shows high Hes1 in ONH and OS cell nuclei, adjacent to RGC axons of the optic nerve. C, D, G, H, Lateral view of live mouse embryos showing Cre-activated Ai9 reporter activity. Rax-Cre is expressed earlier than Chx10-Cre (C, G, arrows). Both drivers express robustly in E11.5 optic cup (arrows in D, H), with Rax-Cre also seen in the developing forebrain. E, F, I, J, E13.5 horizontal sections containing the ONH labeled with anti-DsRed to better show spatial differences in the two Cre transgenes. Chx10-Cre mediated activity is restricted to the neural retina, but the long half-life of tdTomato expression also lineage traces nascent RGC axons (E). Antibody colabeling for Cre-GFP fusion and Pax2 proteins show absent Chx10-Cre expression in the OS or ONH (arrows in F–F′′). I, Rax-Cre activity in the neural retina, RPE, ONH, OS and lineage-traced RGCs axons. J, Comparison of tdTomato (anti-DsRed) and Pax2 highlights Rax-Cre activity within ONH cells. L, lens; NR, neural retina; ONH, optic nerve head; OS, optic stalk; RPE, retinal pigmented epithelium Scale bars: 50 μm in A, B, E, and F; 500 μm in C and D.

Figure 2.

Hes1 conditionally mutant ocular defects. A, E, I, Lateral views of E13.5 live embryos. Chx10-Cre;Hes1^CKO/CKO eyes (E) are indistinguishable from control littermates (A). However Rax-Cre;Hes1^CKO/CKO eyes have fully penetrant colobomas (arrow in I). B, F, J, Anti-Hes1 labeling of E13.5 cryosections indicates the identical loss of low, nonuniform expression from the retina of both Cre-mutants. However, Chx10-Cre;Hes1^CKO/CKO eyes retain high, sustained Hes1 expression in the ONH (arrows in each panel). Rax-Cre;Hes1^CKO/CKO uniquely exhibited an abnormal elongated shape to the retina (J). C, G, K, Gross examination of P21 eyes suggests microphthalmia in Chx10-Cre;Hes1^CKO/CKO (G) and Rax-Cre;Hes1^CKO/CKO (K) mutants. D, H, L, However, H&E-stained P21 sections at the level of the optic nerve support smaller eyes only for Rax-Cre;Hes1^CKO/CKO. Both mutants have lamination defects and retinal rosettes (H), along with severe thinning of retinal layers (L). D′, H′, and L′ are higher magnifications of boxed areas in D, H, and L, respectively. L, lens; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 μm in B, D, and D′ and 500 μm in A and C. n = 3 biologic replicates per age and genotype were evaluated.

Figure 3.

Specific alteration of the optic cup–stalk boundary in Rax-Cre;Hes1^CKO/CKO and Hes1^−/− embryos. Immunolabeling of E11.5 and E13.5 horizontal sections containing retina, ONH and optic stalk. A–F, Pax6-Pax2 colabeling. These transcription factors are initially coexpressed, but by E13.5, abut each another in nonoverlapping domains, thereby highlighting the NR/OC-ONH boundary (arrow in D). Although the abutting expression domains arise in both Chx10-Cre;Hes1^CKO/CKO (B, E) and Rax-Cre;Hes1^CKO/CKO (C, F) eyes, the position of the boundary is more proximal and highlights an expansion of the retinal territory (F). This defect is phenocopied in Hes1 germline mutants (N, P). G–L, Rax-Mitf colabeling for the retinal-RPE boundary in nearby sections. In both conditional mutants, these tissues are essentially normal, although Rax-Cre;Hes1^CKO/CKO eyes always displayed complete suppression of Mitf in the E11.5 ONH/OS (arrows in G–I and insets of Mitf expression). At E13.5, proximal expansion of RPE matched that of the neural retina. M, O, Normal Pax6, Pax2 (arrow in O) and Atoh7-GFP transgene expression domains. N, P, In Hes1 germline mutants, inappropriate proximal expansion of Pax6+ optic cup cells, accompanied by a proximal shift of the Pax2 ONH domain, which is also misshapen (arrow in P). Atoh7-GFP expression highlights a previously described RGC neurogenic phenotype. L, lens. Scale bars in A, M, O, 50 μm. n > 3 biologic replicates per age and genotype were analyzed.

Figure 4.

Hes1 conditional mutants have expected neurogenic and RPC pool depletion phenotypes. A–F, TUBB3 labeling of nascent retinal neurons at E11.5 and E13.5. Premature neurogenesis is in progress at E11, and by E13.5, there is a dramatic increase of Tubb3+ cells in both Chx10-Cre;Hes1^CKO/CKO and Rax-Cre;Hes1^CKO/CKO eyes. Some ectopic neurons are improperly positioned in the outer retina and/or have aberrant trajectories (arrows in E and F). G–I, Precocious Atoh7 expression at E11 (arrows in H, I), also phenocopies Hes1 germline mutants (Takatsuka et al., 2004; Lee et al., 2005). J–L, P, E13.5 sections containing the ON from embryos pulse-labeled with EdU 90 min before dissection. M–O, Q, Anti-cPARP labeling of nearby sections to assess apoptosis. P, Statistically significant loss of EdU⁺ RPCs in both mutants. Q, cPARP⁺ cells (arrows in N, O) were increased, with statistical significance for Rax-Cre;Hes1^CKO/CKO eyes. Both graphs display individual data points, the mean and SEM; n.s., not significant; *p < 0.05; **p < 0.01. L, lens. Scale bars in A and M, 50 μm. n = 3 biologic replicates (2 or more sections/replicate) per age and genotype were quantified.

Figure 5.

RGC patterning defects in Hes1 retinal mutants. A–C, Colabeling for Tubb3 and Mitf highlights patches of abnormal neuronal processes inappropriately on the apical side of the retina (inset boxes). D–F, Pou4f nuclear expression in differentiated RGCs better highlights RGC cell body mispatterning in the patches (arrows in E, F). Previous examination of Hes1 germline mutants showed an early loss of the OLM, a boundary that also confers apical-basal positional information to RPCs (Takatsuka et al., 2004). G–I, P0 H&E sections with retinal rosettes and breaks in the outer retina (arrows in H, I). L, lens. Scale bars: A and D, 50 μm with insets 400× magnified; G, 500 μm. n = 3 biologic replicates per age and genotype analyzed.

Figure 6.

Loss of Hes1 results in failed cone photoreceptor formation. A–C, J, RPCs competent to develop into photoreceptor or bipolar neurons express the Otx2 homeobox protein, as well as early-stage E9–E13 RPE cells (Nishida et al., 2003; Fossat et al., 2007). The percentage of Otx2+ retinal cells was quantified (J), using total DAPI+ nuclei to normalize for the elongated retinal shape of Hes1 mutants (arrows in B, C). D–F, K, Anti-Thrb2 labeling (a cone-specific factor) of E16.5 sections also showed a statistically significant loss of cones in the absence of Hes1 (K). G–I, L, Anti-Arr3 (cone Arrestin) labeling of P21 further highlighted this reduction of cones (L), plus shorter outer segments in the disorganized Arr3+ cones for Rax-Cre; Hes1^CKO/CKO eyes (I). L, lens; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 μm, with boxed insets at 400× magnification. All graphs display individual data points, mean, and SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. n = 3 biologic replicates (2 or more sections/replicate quantified) per age and genotype.

Figure 7.

Evaluation of adult inner retinal cell types in Hes1 retinal mutants. A–C, M, The percentage of Pax6+ INL amacrine cells was equivalent among genotypes. D–F, N, For bipolar neurons, Vsx2+ nuclei were quantified, although some Müller glial cells also express this protein. The Vsx2+ cells were significantly increased in both Hes1 mutants (N). G–L′, O, Two Müller glial markers were evaluated, Sox9 in the nucleus (G–I) and glutamine synthetase in the cell membrane (J–L′). Although the percentage of Sox9+ nuclei was not significantly different among the three genotypes (M), in Rax-Cre;Hes1^CKO/CKO retinas, these cells were frequently misplaced in the ONL (arrows in I) and had abnormal radial processes (L′). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar in A, 50 μm. Graphs display individual data points, mean, and SEM. n.s., not significant; **p < 0.01; ***p < 0.001. n = 3 biologic replicates (2 or more sections/replicate quantified) per age and genotype.