Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Abstract

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.

Figure 1. Expression patterns of Hh target genes at E12.5

Gli1 (A, B), Ptc1 (C, D) and Hhip (E, F) mRNA expression was detected in wild type retinas, but not in orJ retinas. Arrows in (A) and (E) demarcate Gli1 and Hhip expression in the dorsal retina, while closed arrowheads indicate areas of signal outside the retina. In situ hybridization for Vsx2 mRNA (G, H) serves as a control. Open arrowheads in (H) indicate reduced expression of Vsx2 in the extreme periphery of orJ retinas. Ventral is to the left in all panels. Scale bar: 200 µm. C, cornea; EOM, extra-ocular mesenchyme; L, lens; NR, neural retina; RPE, retinal pigmented epithelium; D, dorsal; V, ventral.

Figure 2. Expression patterns of Shh and Hh target genes at E15.5

Shh mRNA (A, B) expression extends to the peripheral retina in wild type eyes, but is restricted to the central region in orJ retinas. Areas of retinal expression indicated with arrows. Gli1 (C) and Ptc1 (E) transcripts were also detected in wild type retinas, with expression extending to the peripheral retina. However, their expression in the orJ retina (D, F) was centrally restricted. Hhip mRNA expression was detected in wild type retinas (G), but not in orJ retinas (H). In situ hybridization for Vsx2 mRNA (I, J) serves as a control. Open arrowheads in (J) indicate regions of presumptive transdifferentiated retina. Asterisk in (J) indicates that lack of Vsx2 expression is an artifact due to tissue folding. Ventral is to the left in all panels. Scale bar: 200 µm.

Figure 3. Hh signaling activity correlates with RGC differentiation in both wild type and orJ retinas

(A–I) Solid and dashed lines indicate the extent of acTUBB3 immunoreactivity and Gli1 mRNA expression, respectively. (A–C) In E15.5 wild type retinas, both acTUBB3 immunoreactivity and Gli1 expression extend to the peripheral retina. (D–F) In E15.5 orJ retinas, both acTUBB3 signal and Gli1 expression are more centrally restricted. Note that acTUBB3-positive cells peripheral to the dashed line in (D) are not located in the retinal neuroepithelium. (G–I) Patterns of acTUBB3 and Gli1 in E12.5 wild type retinas are centrally restricted. (J–L) Expression of POU4F2 in the E15.5 orJ retina correlates well with acTUBB3 staining, indicating that many of the acTUBB3-positive cells are RGCs. Ventral is to the left in all panels. Scale bars: 200 µm for (A–I); 100 µm for (J–L).

Figure 4. SHH-N treatment promotes proliferation in embryonic organotypic retinal explant cultures from orJ mice

E12.5 explants were cultured in the absence (A–C) or presence (D–F) of SHH-N for 24 hr with BrdU added for the last 30 min. (A, D) BrdU incorporation. (B, E) PCNA immunoreactivity. (C, F) acTUBB3 immunoreactivity. Retinal tissue is contained within the dashed lines. Scale bar: 100 µm. (G) Quantification of BrdU⁺ cells as a function of retinal area (mm²). Each bar represents the mean ± standard error of the mean (SEM). p values calculated using Student’s paired t-test

Figure 5. Expression patterns of Hh target genes at P0

In situ hybridization for the Hh target genes Gli1 (A^(*), B^(*)) and Ptc1 (C^(*), D^(*)) reveals reduced expression for both genes in orJ retinas at P0. (E^(*), F^(*)) Ptc2 mRNA expression is undetectable in either genotype. (G^(*), H^(*)) Hhip transcript is expressed in wild type retinas, but absent in orJ retinas. (I^(*), J^(*)) In situ hybridization for Vsx2 mRNA serves as a control. Scale bars: 250 µm (A–J); 40 µm (A*–J*). NBL, neuroblast layer; DCL, differentiated cell layer.

Figure 6. Quantification of relative expression levels for Hh network components in P0 wild type and orJ retinas by sqRT-PCR

The mean wild type expression level for each gene was set at 100 percent with the orJ level presented as a percent of wild type expression. Hh target genes are those genes whose expression levels are generally considered as indicators of pathway activity‥ Bars represent the mean ± standard deviation. n refers to the number of independently isolated RNA samples. p values calculated by Student’s unpaired t-test or Welch’s two sample t-test, as appropriate (based on results of an F test of variances). See Supplemental Figure S1 for sqRT-PCR optimization curves.

Figure 7. Shh expression at P0

(A) Relative expression levels of Shh and Pou4f2 mRNAs in wild type and orJ retinas as determined by sqRT-PCR. Bars represent the mean ± standard deviation. n and p values are defined as described in Fig. 6. (B) Relative expression of SHH protein in wild type and orJ retinal protein lysates. Closed arrowhead points to band consistent with size of full-length SHH and open arrowhead points to band consistent with size of the N-terminal fragment of SHH. Blot was reprobed to show ACTB expression as an independent measure for protein loading. (C–F) Spatial expression of SHH protein in wild type and orJ eyes. Scale bars: 200 µm for (C, D); 50 µm for (E, F). See Supplemental Figure S2 for anti-SHH immunoreactivity controls.

Figure 8. SHH-N treatment promotes proliferation in neonatal explants within 24 hr

BrdU (A, D), pHH3 (B, E), and PCNA (C, F) immunoreactivity in wild type explants after 24 hr in culture. Quantification of BrdU⁺ (G), pHH3⁺ (H), and PCNA⁺ (I) cells in wild type explants as a function of retinal area (G, I) and length of the apical surface of the retina (H). BrdU (J, M), pHH3 (K, N), and PCNA (L, O) immunoreactivity in orJ explants after 24 hr in culture. Quantification of BrdU⁺ (P), pHH3⁺ (Q), and PCNA⁺ (R) cells in wild type explants as a function of retinal area (P, R) and length of the apical surface of the retina (Q). Bars represent mean ± SEM. p values calculated using Student’s paired t-test. Scale bar: 50 µm.

Figure 9. CCND1 expression in retinal explants after 8 hr of culture

Western blot of total retinal protein isolated from retinal explants cultured in the presence (+) or absence (−) of SHH-N and probed with anti-CCND1. Blot was reprobed with anti-ACTB.

Figure 10. SHH-N treatment stimulates expression of Hh target genes in explant culture

Relative expression levels of Hh target genes from retinal explants after 8 hr in culture. All data points are relative to the wild type expression level at 0 hr (set at 100%). 0 hr is defined as isolated retinal tissue frozen immediately after dissection. The expression level for each genotype at 0 hr is referred to in the text as the baseline expression level. Data points represent the mean ± standard deviation. p-values are provided in Table 1 and Table 2.

Figure 11. Endogenous SHH promotes proliferation in P0 orJ retinas

orJ retinal explants were cultured in vehicle alone (0.41% v/v DMSO) or 10 µM cyclopamine (in 0.41% v/v DMSO) for 16 hr (A–D) or 24 hr (E–J). (A, C) Gli1 mRNA expression. (B, D) Vsx2 mRNA expression. BrdU (E, H), pHH3 (F, I), and PCNA (G, J) immunoreactivity. Closed arrowheads in (F, I) indicate pHH3⁺ cells at the apical surface of the retina. Quantification of BrdU⁺ (K), pHH3⁺ (L), and PCNA⁺ (M) cells as a function of retinal area (K, M) and length of the apical surface of the retina (L). Bars represent mean ± SEM. p values calculated using the Student’s paired t-test. Scale bar: 120 µm (A–D), 50 µm (E–J).