Distinct effects of Hedgehog signaling on neuronal fate specification and cell cycle progression in the embryonic mouse retina

Abstract

Cell-extrinsic signals can profoundly influence the production of various neurons from common progenitors. Yet mechanisms by which extrinsic signals coordinate progenitor cell proliferation, cell cycle exit, and cell fate choices are not well understood. Here, we address whether Hedgehog (Hh) signals independently regulate progenitor proliferation and neuronal fate decisions in the embryonic mouse retina. Conditional ablation of the essential Hh signaling component Smoothened (Smo) in proliferating progenitors, rather than in nascent postmitotic neurons, leads to a dramatic increase of retinal ganglion cells (RGCs) and a mild increase of cone photoreceptor precursors without significantly affecting other early-born neuronal cell types. In addition, Smo-deficient progenitors exhibit aberrant expression of cell cycle regulators and delayed G(1)/S transition, especially during the late embryonic stages, resulting in a reduced progenitor pool by birth. Deficiency in Smo function also causes reduced expression of the basic helix-loop-helix transcription repressor Hes1 and preferential elevation of the proneural gene Math5. In Smo and Math5 double knock-out mutants, the enhanced RGC production observed in Smo-deficient retinas is abolished, whereas defects in the G(1)/S transition persist, suggesting that Math5 mediates the Hh effect on neuronal fate specification but not on cell proliferation. These findings demonstrate that Hh signals regulate progenitor pool expansion primarily by promoting cell cycle progression and influence cell cycle exit and neuronal fates by controlling specific proneural genes. Together, these distinct cellular effects of Hh signaling in neural progenitor cells coordinate a balanced production of diverse neuronal cell types.

Figure 1.

Expression of Chx10-cre and phenotypes of Chx10-cre induced Smo ablation. A–G, Expression of Cre-GFP fusion protein in E12.5 (A–C) and E14.5 (D–G) retinas of the Chx10-cre transgenic mouse. Retinal sections were labeled by DAPI for nuclei (A, D, F), anti-GFP for Cre-GFP fusion protein (B, E, G), and anti-Brn3a for RGCs (C). H–M, Retinal morphology and distribution of Cre-GFP-expressing retinal progenitor cells at P0. Retinal sections from Smo heterozygous (H–J) and Smo cKO mutants (K–M) were labeled by anti-GFP (H, J, K, M) and DAPI (I, L). I and L show the same sections as in J and M, respectively. The white dotted lines (L, M) outline regions that did not express Cre-GFP. gcl, Ganglion cell layer; le, lens; ret, retina; vz, ventricular zone. Scale bars: A (for A–C), D (for D, E), F (for F, G), H (for H, K), I (for I, J, L, M), 100 μm. N, O, Quantification of retinal progenitor cells by flow cytometry. Percentages of PCNA-positive cells among total cells at E15.5 (N) and GFP-positive cells among total cells at E17.5 (O) are shown. P, Real-time PCR quantification of Hh signaling component gene expression at E15.5. Relative transcript levels are presented as ratios of Smo cKO mutants (−/−) versus Smo controls (+/+) normalized according to 18S rRNA (n = 3). Genotypes (+/+, Smo^flox/flox with no cre; +/−, Smo^flox/+ with Chx10-cre; −/−, Smo^flox/flow with Chx10-cre) and numbers (n) of individual retinas analyzed are indicated below the bar graphs. **p < 0.01, ***p < 0.001, and ****p < 0.0001. Error bars indicate SEM.

Figure 2.

Enhanced retinal ganglion cell production in Smo mutant retinas. A–M, Immunofluorescent labeling of E14.5 (A–C), E15.5 (D–I), and P0 (J–M) retinas. Sections of control (+/+) (A, J, K), Smo heterozygous (+/−) (B, D–F), and Smo cKO mutant retinas (−/−) (C, G–I, L, M) were labeled for βTub (A–C), colabeled for DAPI, Brn3a and GFP (D–F; and G–I), and colabeled for DAPI and Brn3a (J, K; and J, M). gcl, Ganglion cell layer; le, lens; ret, retina; vz, ventricular zone. Scale bars: A (for A–C), D (for D–I), J (for J–M), 100 μm. N–P, Quantification of RGCs by flow cytometry. Percentages of marker positive cells among total cells at E15.5 (N), E17.5 (O), and P0 (P) are shown. Genotypes (+/+, Smo^flox/flox with no cre; +/−, Smo^flox/+ with Chx10-cre; −/−, Smo^flox/flox with Chx10-cre), and numbers (n) of individual retinas analyzed are indicated below the bar graphs. *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 3.

Effects of Smo deficiency on photoreceptor precursor and cone cell genesis. A–F, In situ hybridization analysis for homeobox genes Otx2 and Crx at E15.5. Retinal sections of control (+/+) (A, D), Smo heterozygous (+/−) (B, E), and Smo cKO mutant (−/−) (C, F) were hybridized with antisense probes of Otx2 (A–C) and Crx (D–F). G–I, Immunocytochemistry for an early cone cell marker at E15.5. Retinal sections of control (+/+) (G), Smo heterozygous (+/−) (H), and Smo cKO mutant (−/−) (I) were labeled for GγC. gcl, Ganglion cell layer; rpe, retinal pigment epithelium; vz, ventricular zone. Scale bars: A (for A–F), G (for G–I), 100 μm. J, K, Quantification of photoreceptor markers by flow cytometry. Percentages of photoreceptor precursor marker Crx- or GγC-positive cells among total cells at E15.5 or E17.5 are shown. Genotypes (+/+, Smo^flox/flox with no cre; +/−, Smo^flox/+ with Chx10-cre; −/−, Smo^flox/flox with Chx10-cre) and numbers (n) of individual retinas analyzed are indicated below the bar graphs. **p < 0.01; ***p < 0.001. Error bars indicate SEM.

Figure 4.

Effects of Smo deficiency on expression of bHLH and homeobox genes in the retina. A–I, In situ hybridization analysis of expression patterns for bHLH genes at E15.5. Retinal sections of control (+/+) (A, D, G), Smo heterozygote (+/−) (B, E, H), and Smo cKO mutant (−/−) (C, F, I) were hybridized with antisense probes of Math5 (A–C), Ngn2 (D–F), and Math3 (G–I). gcl, Ganglion cell layer; rpe, retinal pigment epithelium; vz, ventricular zone. Scale bar: (in A) A–I, 100 μm. J, K, Real-time PCR quantification of transcript levels for bHLH (J) and homeobox (K) genes expressed in E15.5 retinas. Relative transcript levels are presented as ratios of Smo cKO mutants (−/−) versus Smo controls (+/+) normalized according to 18S rRNA (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 5.

Altered progenitor cell cycle distribution in Smo mutant retinas. A–D, Immunofluorescent labeling for progenitor cells at E16.5. Retinal sections derived from Smo heterozygous (+/−) (A, B) and Smo cKO mutant (−/−) (C, D) retinas were colabeled with DAPI (A, C), BrdU (red) and GFP (green) (merged in B, D). gcl, Ganglion cell layer; vz, ventricular zone. Scale bar: (in A) A–D, 100 μm. E, Flow cytometry profiles of Smo heterozygous (+/−) and Smo cKO mutant (−/−) retinal cells at E17.5 according to GFP labeling intensity (y-axis) and DNA content as indicated by DAPI labeling (x-axis). The gated areas indicate GFP-positive cells used for cell cycle distribution analyses shown in G. F, G, Flow cytometric analyses of cell cycle distribution of GFP-positive progenitor cells at E14.5 (F) and E17.5 (G). GFP-positive cells of Smo heterozygous (+/−) and Smo cKO mutant (−/−) retinas from boxed regions in F were quantified according to their DNA contents. Percentages of cells in the G₁, G₂/M, and S phases of the cell cycle among total GFP-positive cells are shown as pie charts and bar graphs. Genotypes (+/+, Smo^flox/flox with no cre; +/−, Smo^flox/+ with Chx10-cre; −/−, Smo^flox/flox with Chx10-cre) and numbers (n) of individual retinas analyzed are indicated below the bar graphs. *p < 0.05; **p < 0.01, and ***p < 0.001. Error bars indicate SEM.

Figure 6.

Abnormal cell cycle progression of Smo-deficient retinal progenitors. A–D, In situ hybridization analysis for Gli1 and cyclin D1 at E15.5. Sections of Smo heterozygotes (+/−) (A, C) and Smo cKO mutant (−/−) (B, D) retinas were probed with antisense probes of Gli1 (A, B) and cyclin D1 (C, D). gcl, Ganglion cell layer; rpe, retinal pigment epithelium; vz, ventricular zone. Scale bar: (in A) A–D, 100 μm. E, Real-time PCR quantification of transcript levels for Shh, E2F1, and various cyclins expressed in the retina at E15.5. Relative transcript levels are presented as ratios of Smo cKO (−/−) versus Smo controls (+/+) normalized according to 18S rRNA (n = 3). **p < 0.01; ***p < 0.001. F, Quantification of cell cycle regulators by flow cytometry. Percentages of cyclin D1-, p27^Kip1-, and p57^Kip2-positive cells among total cells at E17.5 are shown. G, Graphic illustration of cell cycle progression following a cohort of BrdU-labeled progenitor cells. Wild-type E16.5 retinal explants were labeled with BrdU for 30 min followed by flow cytometric analysis for BrdU-positive cells in various phases of the cell cycle for up to 21 h. H, Comparison of cell cycle progression between Smo heterozygous (+/−) and Smo cKO mutant (−/−) retinal cells at E17.5. The bar graphs show the distribution of BrdU-positive cells among different phases of the cell cycle at 9 and 18 h after BrdU pulse labeling. Genotypes (+/+, Smo^flox/flox with no cre; +/−, Smo^flox/+ with Chx10-cre; −/−, Smo^flox/flox with Chx10-cre) and numbers (n) of individual retinas analyzed are indicated below the bar graphs. **p < 0.01; ***p < 0.001. Error bars indicate SEM.

Figure 7.

Effects of Smo and Math5 double mutations on RGC and cone cell production. A–P, Immunofluorescent labeling of cell markers in control and different mutant retinas at E17.5. Sections from the control double heterozygous (Smo^+/flox; Math5^+/Cre with Chx10-Cre) (A, E, I, M), Smo single mutants (Smo^flox/flox; Math5^+/Cre with Chx10-Cre) (B, F, J, N), Math5 single mutants (Smo^+/flox; Math5^Cre/Cre with Chx10-Cre) (B, F, J, N), and Smo Math5 double mutants (Smo^flox/flox; Math5^Cre/Cre with Chx10-Cre) (D, H, L, P) were labeled for DAPI (A–D), GFP (E–H), Brn3a (I–L), and GγC (M–P). gcl, Ganglion cell layer; vz, ventricular zone. Scale bar: (in A) A–P, 100 μm. Q–T, Quantification of RGC and photoreceptor marker-positive cells by flow cytometry in single- and double-mutant retinas at E17.5. Q–S, Bar graphs show percentages of marker-positive cells among total cells. Genotypes of the retinas are (1) double heterozygous (Smo^+/flox; Math5^+/Cre with Chx10-Cre), (2) Smo single mutants (Smo^flox/flox; Math5^+/Cre with Chx10-Cre), (3) Math5 single mutants (Smo^+/flox; Math5^Cre/Cre with Chx10-Cre), and (4) Smo Math5 double mutants (Smo^flox/flox; Math5^Cre/Cre with Chx10-Cre). The numbers (n) of individual retinas analyzed are indicated below the bar graphs. T, A table lists p values for different markers according to statistical analyses among various genotypes. N.S., Not significant. Error bars indicate SEM.

Figure 8.

Effects of Smo and Math5 double mutation on progenitor cell expansion and cell cycle regulation. A, Quantification of GFP-positive cells by flow cytometry in single- and double-mutant retinas at E17.5. Percentages of GFP-positive cells among total cells are shown. B, Flow cytometric analyses of progenitor cell distribution in different phases of the cell cycle. Percentages of GFP-positive cells in the G₁, S, and G₂/M phases of the cell cycle among total GFP-positive cells are shown as bar graphs. The numbers (n) of individual retinas and their genotypes are indicated below the bar graphs. Error bars indicate SEM. C, A table lists p values for comparisons among various genotypes for proportions of progenitor cells and cell cycle phase distribution. N.S., Not significant.

Figure 9.

Hh signaling effects in neurogenic retinal progenitors. A proposed model illustrates the distinct Hh signaling effects in uncommitted neural progenitors. On receiving Hh signals, Gli effectors directly or indirectly enhance the expression of G₁-phase cyclins including cyclin D1 and cyclin E to promote G₁ to S phase transition. Hh signals may also facilitate G₂/M progression by upregulating cyclin A and/or cyclin B (data not shown). The Hh effect on cell cycle progression occurs in all proliferating progenitors in the embryonic retina. In addition, Hh-triggered Gli activation directly or indirectly suppresses the bHLH factor Math5 and other proneural gene(s), which cooperate with specific CDK inhibitors such as p27^Kip1 to facilitate cell cycle exit and specify RGC or cone cell fate. Hh signaling also promotes expression of Hes1, which may participate in cell cycle regulation and/or proneural gene suppression. Hh signaling affects cell cycle withdrawal and neuronal fate choice by impacting the dynamics of proneural gene expression during the neurogenic cell cycle when one or two postmitotic neurons are produced.