Sonic hedgehog has a dual effect on the growth of retinal ganglion axons depending on its concentration

Abstract

The stereotypical projection of retinal ganglion cell (RGC) axons to the optic disc has served as a good model system for studying axon guidance. By both in vitro and in vivo experiments, we show that a secreted molecule, Sonic hedgehog (Shh), may play a critical role in the process. It is expressed in a dynamic pattern in the ganglion cell layer with a relatively higher expression in the center of the retina. Through gel culture and stripe assays, we show that Shh has a dual effect on RGC axonal growth, acting as a positive factor at low concentrations and a negative factor at high concentrations. Results from time-lapse video microscopic and stripe assay experiments further suggest that the effects of Shh on axons are not likely attributable to indirect transcriptional regulation by Shh. Overexpression of Shh protein or inhibition of Shh function inside the retina resulted in a complete loss of centrally directed projection of RGC axons, suggesting that precise regulation of Shh level inside the retina is critical for the projection of RGC axons to the optic disc.

Figure 1.

An endogenous positive factor was detected inside the retina. Central and peripheral halves of E5, E6, and E8 retinas were embedded separately into 2% low-melting agarose blocks and cocultured with tester retinal explants, as schematically represented in F. After 44 h, axons of the tester explants in different coculture conditions were stained and photographed. Some examples are shown: A, with control empty agarose block; B, with E5 central; C, with E5 peripheral (^* marks retinal tissue embedded in 2% agarose); D, with E8 central; E, with E8 peripheral retina. The data were quantified and are shown in G. C, Center; P, periphery; +, with cyclopamine. Note that a positive activity for RGC axonal growth was detected in E5, E6 central, and E8 peripheral halves of the retinas but not in the other halves of the retina. Furthermore, this positive activity could be abolished by inclusion of cyclopamine. The number of samples assayed was shown in parentheses, and statistical significance compared with the control (CTL) is indicated (^*p < 0.001). Scale bar, 200 μm.

Figure 2.

Expression of Shh mRNA in the chick retina. Retinas were flat mounted (A) or sectioned (B) and processed for in situ hybridization with a DIG-labeled Shh probe. A, E5.5 and E7 retinas were shown on the ganglion side. Note that Shh is expressed in the center and not the periphery of the retina at E5.5 and E7. B, On retinal cross sections, the expression of Shh mRNA was detected in a subset of cells in the GCL (black arrows). Shh mRNA is expressed in the GCL in the center of the retina at early stages such as E4.5. Gradually, the expression domain of Shh (marked with red arrows) moves toward the periphery of the retina. At E6, Shh expression is still higher in the center than periphery. At E9, the Shh mRNA expression is concentrated at the retinal periphery, and the expression in the center is diminished. L, Lens; P, periphery; C, center. Scale bars, 400 μm.

Figure 3.

Shh acts as a positive factor at low concentrations and as a negative factor at high concentrations on RGC axons. Purified Shh-N protein was added to the gel culture at various concentrations with (+cyc) or without (-cyc) cyclopamine. Axons of retinal explants were stained, and some examples are shown in A. Scale bar, 100 μm. The length of the axons was quantified, and all data were compared with the BSA-added control culture to determine whether a positive or a negative effect could be detected (B). The number of samples assayed is shown in parentheses. Statistical significance between the Shh-added and the BSA-control samples was assessed by Student's t test. p values between 0.05 and 0.1 are indicated by ^*, and p values <0.001 are marked by ^**. Note that cyclopamine blocked both the positive and the negative effects of Shh.

Figure 4.

The response of RGC growth cones to low concentration of Shh is rapid. Time-lapse video microscopic studies were performed to record the growth of retinal axons during the addition of control BSA or 0.5 μg/ml Shh-N protein. A, Photographs of the cultures were shown immediately (0:00), 30 min (0:30), and 1 h (1:00) after protein addition. Red, yellow, and green arrowheads mark the axons that have advanced, remained static, and retracted, respectively. B, The growth rate of the axons in control BSA- or Shh-N-added cultures was shown by plotting average cumulative growth of the axons with time. A significant increase was observed in the average growth rate of the axons within 30 min after addition of 0.5 μg/ml Shh protein compared with that in the control BSA-added culture.

Figure 5.

Shh directs the growth of RGC axons in a concentration-dependent manner. Purified Shh-N or BSA control proteins were coated onto glass coverslips in alternating stripes as the first stripes, which were marked by inclusion of a Cy3-conjugated fluorescent antibody. BSA protein was used to fill in as the second stripes. A, Control experiment. RGC axons did not choose between the BSA-coated first and second stripes. B, RGC axons preferred to grow on 0.5 μg/ml Shh-coated stripes over the BSA-coated stripes. C, RGC axons chose to grow on BSA-coated and not high concentration (2.5 or 4.0 μg/ml) Shh-coated stripes. D, Quantification of the stripe assay results.

Figure 6.

Inhibition of Shh function in vivo with injection of cyclopamine at E3.5 caused a severe abnormality in RGC axon projection. Cyclopamine/HBC (B-D, F) or control HBC (A, E) was injected into either the vitreal space next to the retina at E3.5 (A-D) or the optic vesicle at E1.5 (E, F). The injected samples were harvested at E5.5-E6. Axons were visualized by staining with the anti-neurofilament antibody (270.7). B-D, In some areas of the retinas injected with cyclopamine at E3.5, axonal projection appeared grossly disorganized and lacked central orientation toward the optic disc. In some areas, axons converged to form ectopic optic discs (D). E, F, Injection of cyclopamine at E1.5 did not alter the normal honeycomb appearance of the early axonal projections. Scale bar, 50 μm.

Figure 7.

RGC axon projection toward the optic disc was similarly disrupted by misexpression of Shh. A, Optic vesicles were injected with retroviruses expressing either Shh (B, C, E, F) or a control GFP protein (A, D) at HH stage 10-11. The infected retinas were harvested at E5.5-E6, flat mounted, and analyzed by staining with an anti-neurofilament antibody (A-F). As shown in A and D, the RGC axons appeared normal in the control RCAS-GFP-injected retinas, similar to uninjected wild-type retinas (data not shown). However, in the RCAS-Shh-injected samples, the axons appeared grossly disorganized and lacked central orientation (B, C). White arrows indicate the direction toward the optic disc. Another common phenotype caused by misexpression of Shh was the abnormal crossing of RGC axons, forming ectopic optic discs. Retinal axons normally converge only at the optic disc (arrowheads in D) in which they exit the retina, as shown in this control RCAS-GFP-infected sample (D). However, misexpression of Shh resulted in abnormal crossing of RGC axons at ectopic sites before reaching the optic disc (E, F, arrowheads). G-I, RCAS-Shh-injected samples were sectioned and labeled with an anti-neurofilament antibody (G) and an anti-viral GAG antibody (H). Despite the gross mistargeting of RGC axons in the RCAS-Shh-injected retina, the axons were mostly confined to the GCL and were not present in the deeper layers of the retina. Scale bars: A-C, F, 50 μm; D, E, 400 μm.