Following photoreceptor injury, miR-18a is expressed in both the inner and outer nuclear layers, including in Müller glia and Müller glia-derived progenitors
RT-qPCR and in situ hybridization were performed to determine the temporal and spatial expression of miR-18a following photoreceptor injury. Taqman qPCR showed that, compared with control uninjured retinas, miR-18a expression is significantly higher at 3 days (2.15 ± 0.44 SD fold higher, p = 0.003), 5 days (2.06 ± 0.69 SD fold higher, p = 0.0.024) and 7 days (1.54 ± 0.30 SD fold higher, p = 0.045) post-retinal injury (dpi) (Fig. 1a). In situ hybridization using an LNA ribroprobe for mature miR-18a, combined with immunolabeling for green fluorescent protein (GFP) in Tg(gfap:egfp)mi2002 fish, showed that at in control uninjured retinas, there is only very slight expression of miR-18a (Fig. 1b) but by 24 hpi, around the time that Müller glia divide once to produce a neuronal progenitor [see 39], miR-18a is expressed in many cells throughout the inner nuclear layer (INL), including Müller glia (Fig. 1c). Then at 3 dpi, during the peak of progenitor proliferation, miR-18a remains strongly expressed in the INL and ONL, including in Müller glia and MG-derived progenitors (Fig. 1d). Finally, by 7 dpi, when MG-derived progenitors are normally exiting the cell cycle, miR-18a expression is again similar to the expression pattern seen between 0 and 24 hpi (Fig. 1e, f). Together, these results show that miR-18a expression is upregulated in the retina during the time periods when Müller glia and then MG-derived progenitors divide.
During photoreceptor regeneration, miR-18a regulates proliferation among MG-derived progenitors
To determine if miR-18a regulates cell proliferation among MG-derived progenitors, BrdU labeling and immunostaining were used to quantitatively compare the number of proliferating cells in WT and miR-18ami5012 retinas. In the injured WT retina, progenitor proliferation peaks around 3 dpi, some MG-derived progenitors normally stop dividing between 4 and 5 dpi, and the first regenerated photoreceptors can be detected between 5 and 6 dpi [see 40]. By 7 dpi, many new photoreceptors have normally regenerated and fewer progenitors continue to proliferate and, by 10 dpi, very few progenitors normally continue to proliferate. Compared with WT retinas, the number of proliferating progenitors in miR-18ami5012 retinas did not differ at 3 dpi (miR-18ami5012 81.7 ± 7.2 SD, WT 87.6 ± 8.1 SD cells/0.3 mm, p = 0.60) (Fig. 2a, b), indicating that the initial proliferative response is unaltered in miR-18ami5012 retinas. At 7 dpi, however, there were significantly more BrdU-positive cells in the miR-18ami5012 retinas than in WT (miR-18ami5012 64.3 ± 9.8 SD, WT 30.6 ± 7.2 SD cells/0.3 mm, p = 0.001) (Fig. 2c, d). In miR-18ami5012 retinas, there were also significantly more proliferating cells at 10 dpi (miR-18ami5012 45.7 ± 3.2 SD, WT 14.0 ± 3.6 SD cells/0.3 mm, p < 0.001) (Fig. 2e, f). These data show that following photoreceptor injury, in the absence of miR-18a, the timing of the initial proliferative response is unchanged, but that MG-derived progenitors continue to proliferate longer than in WT retinas.
During photoreceptor regeneration, miR-18a regulates the timing, but not the extent, of photoreceptor regeneration
In miR-18ami5012 retinas, the prolonged period of proliferation among MG-derived progenitors suggests that photoreceptor regeneration might be delayed and/or that more photoreceptors might be produced. To determine if miR-18a regulates the timing and/or extent of photoreceptor regeneration, in situ hybridization was used to differentially label mature cones and rods in the retina at 7 dpi, when large numbers of newly differentiated photoreceptors can first be detected, at 10 dpi, when most new photoreceptors have been normally regenerated, and 14 dpi, when photoreceptor regeneration is normally complete. In situ hybridization for mature cones (arr3a) and rods (rho) (Fig. 3a, b) and quantification of these cells (Fig. 3c, d) showed that at 7 dpi, miR-18a retinas have fewer mature cones than WT (miR-18ami5012 32.3 ± 14.7 SD, WT 77.5 ± 4.5 SD cells/0.3 mm, p = 0.001), but the number of mature rods does not differ (miR-18ami5012 98.5 ± 38.6 SD, WT 127.1 ± 72.1 SD cells/0.3 mm, p = 0.51). Hoechst labeling of cone nuclei at 7 dpi also showed that miR-18ami5012 retinas have fewer cone nuclei than WT (miR-18ami5012 86.8 ± 9.2 SD, WT 101.3 ± 5.2 SD cells/0.3 mm, p = 0.033). Together, these data indicate that both cone photoreceptor maturation and regeneration are delayed. At 10 dpi, miR-18a retinas have fewer mature cones and rods than WT (cones miR-18ami5012 76.4 ± 2.5 SD, WT 107.6 ± 6.5 SD cells/0.3 mm, p = 0.001; rods miR-18ami5012 146.4 ± 46.7 SD, WT 275.6 ± 32.5 SD cells/0.3 mm, p = 0.017) but, at 14 dpi, the number of mature photoreceptors does not differ (cones miR-18ami5012 125.7 ± 11.4 SD, WT 120.3 ± 14.7 SD cells/0.3 mm, p = 0.462; rods miR-18ami5012 237.7 ± 28.8 SD, WT 261.3 ± 32.1 SD cells/0.3 mm, p = 0.241) (Fig. 3c, d). Taken together, these data show that in miR-18ami5012 retinas compared with WT, the same overall numbers of photoreceptors are regenerated by 14 dpi, but photoreceptor regeneneration and maturation are delayed.
Following photoreceptor injury, miR-18a regulates the number of neuronal progenitors that are produced
In miR-18ami5012 retinas compared with WT, MG-derived progenitors proliferate for a longer period of time, suggesting that excess neuronal progenitors are produced. However, since extra photoreceptors are not generated, these excess progenitors might either die or migrate to other retinal layers, possibly differentiating into other cell types. To investigate this, TUNEL labeling was used to label dying cells at 10 dpi, when the largest differences in photoreceptor numbers were identified between WT and miR-18ami5012 retinas. This experiment showed that, at 10 dpi, there were no differences in the number of TUNEL-positive cells between WT and miR-18ami5012 retinas (data not shown), indicating that excess progenitors in miR-18ami5012 retinas are not eliminated by cell death. To establish the potential fates of the excess MG-derived progenitors in miR-18ami5012 retinas, fish were exposed to 5 mM BrdU from 3 to 4 dpi during the peak of cell proliferation, and then fish were sacrificed at 14 dpi when photoreceptor regeneration is complete. Immunolabeling for BrdU at 14 dpi shows that compared with WT, miR-18ami5012 retinas have more BrdU+ cells in both the INL (miR-18ami5012 22.7 ± 4.3 SD, WT 11.0 ± 3.2 SD cells/200 mm, p = 0.019) and ganglion cell layer (GCL) (miR-18ami5012 15.1 ± 2.2 SD, WT 6.2 ± 1.9 SD cells/200 mm, p = 0.006) (Fig. 4), indicating that the excess progenitors produced at 3–4 dpi either remain in the INL or migrate to the GCL, suggesting that they may differentiate into a variety of other cell types.
Following photoreceptor injury, miR-18a regulates the extent and duration of the inflammatory response
The effect of miR-18a on the cell cycle during photoreceptor regeneration is strikingly different from embryonic development, in which miR-18a regulates photoreceptor differentiation but not cell proliferation [30]. This indicates that in the injured retina, miR-18a regulates pathways that are specific to the post-injury response. Silva et al. [21] showed that in injured mmp9 mutant retinas, there was increased inflammation resulting in excess proliferation among MG-derived progenitors. This finding helped lead to the rationale that increased or prolonged inflammation might cause the excess proliferation observed in miR-18ami5012 retinas. Indeed, the miRNA target database TargetscanFish (http://www.targetscan.org/fish_62) predicts that miR-18a interacts with mRNA for many molecules involved in inflammatory pathways. To determine if miR-18a regulates inflammation, RT-qPCR was used to compare the mRNA expression levels of key inflammatory molecules at 1, 3, 5 and 7 dpi in WT and miR-18ami5012 retinas. These data show that at 1 dpi, around the time that Müller glia normally begin to divide, expression of tnfα is higher in miR-18ami5012 retinas compared with WT (miR-18ami5012 4.51 ± 1.14 SD, WT 2.31 ± 0.99 SD, p = 0.033) (Fig. 5a). Then at 3 dpi, around the normal peak of MG-derived progenitor proliferation, il1β expression is higher in miR-18ami5012 retinas compared with WT (miR-18ami5012 1.61 ± 0.21 SD, WT 0.97 ± 0.06 SD, p = 0.007) (Fig. 5b). At 5 dpi, when many photoreceptor progenitors normally stop proliferating and begin to differentiate, expression of il6, il1β, and the cytokine regulator Nuclear Factor Kappa B 1 (nfkb1) are higher in miR-18ami5012 retinas compared with WT (il6 miR-18ami5012 1.32 ± 0.26 SD, WT 0.85 ± 0.25 SD, p = 0.042; il1b miR-18ami5012 1.71 ± 0.47 SD, WT 0.97 ± 0.08 SD, p = 0.028; nfkb1 miR-18ami5012 1.68 ± 0.23 SD, WT 1.19 ± 0.17 SD, p = 0.021) (Fig. 5c). Finally, at 7 dpi, when many photoreceptor progenitors have normally stopped proliferating and have fully differentiated, nfkb1 expression is still higher in miR-18ami5012 retinas (miR-18ami5012 1.33 ± 0.22 SD, WT 1.03 ± 0.07 SD, p = 0.044) (Fig. 5d). Taken together, these results indicate that, compared with WT, there is both a higher level of inflammatory pathway activity and a prolonged inflammatory response in miR-18ami5012 retinas following photoreceptor injury and death.
Supressing inflammation in miR-18a mutants rescues both the excess proliferation and delayed photoreceptor regeneration
The increased and prolonged expression of inflammatory genes in the injured miR-18ami5012 retina led to the hypothesis that, in the absence of miR-18a, prolonged and excessive inflammation is causally related to the excess proliferation and delayed photoreceptor maturation observed in miR-18a mutants. To test this hypothesis, dexamethasone was used to suppress inflammation in WT and miR-18ami5012 fish from 2 to 6 dpi, the time range during which miR-18a expression is upregulated in injured retinas (see Fig. 1a, b) and when inflammatory molecules are expressed higher than WT in miR-18ami5012 retinas (see Fig. 5). Fish were then exposed to 5 mM BrdU from 6 to 7 dpi, to label proliferating cells. BrdU immunolabeling was used to label BrdU+ cells in S-phase of the cell cycle, and in situ hybridization was used to label mature rod or cone photoreceptors. Compared with controls, the dexamethasone treatment fully rescued the excess proliferation in miR-18ami5012 retinas, reducing the number of BrdU+ cells to that observed in controls (miR-18ami5012 control 65.0 ± 13.5 cells/0.3 mm vs. WT control 42.5 ± 3.5 cells/0.3 mm p = 0.009; miR-18ami5012 dexamethasone treated 36.2 ± 7.7 cells/0.3 mm vs. WT control 42.5 ± 3.5/0.3 mm p = 0.096). Dexamethasone treatment also reduced the number of BrdU+ cells in WT retinas by a smaller amount (WT control 42.5 ± 3.5 cells/0.3 mm vs. WT dexamethasone treated 29.2 ± 9.0 cells/0.3 mm p = 0.020) (Fig. 6a, b). Further, dexamethasone treatment rescued the delay in cone maturation and regeneration at 7 dpi, increasing the number of arr3a-expressing mature cones to WT control levels (miR-18ami5012 control 41.8 ± 3.1 cells/0.3 mm vs. WT control 57.1 ± 6.6 cells/0.3 mm p = 0.015; miR-18ami5012 dexamethasone treated 63.1 ± 7.6 cells/0.3 mm vs. WT control 57.1 ± 6.6 cells/0.3 mm p = 0.314), and also increasing the number of Hoechst-labeled cone nuclei to WT control levels (miR-18ami5012 control 57.2 ± 11.4 cells/0.3 mm vs. WT control 76.7 ± 9.2 cells/0.3 mm p = 0.021; miR-18ami5012 dexamethasone treated 76.3 ± 4.04 cells/0.3 mm vs. WT control 76.7 ± 9.2 cells/0.3 mm p = 0.451). Dexamethasone treatment had no effect on the number of arr3a-expressing mature cone cells in WT retinas (WT control 57.1 ± 6.6 cells/0.3 mm vs. WT dexamethasone treated 56.5 ± 12.6 cells/0.3 mm p = 0.933) (Fig. 6c, d). Together, these data indicate that in the injured retina, miR-18a regulates MG-derived progenitor proliferation and photoreceptor regeneration by regulating inflammation.