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Inhibition of the TGF β Pathway Enhances Retinal Regeneration in Adult Zebrafish

View Article: PubMed Central - PubMed

ABSTRACT

In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the N-methyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de- and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.

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Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.
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pone.0167073.g001: Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.

Mentions: After inducing retinal degeneration using 150 mg/l MNU, maximal activation of the TGFβ pathway occurred between days 3 and 8 as demonstrated by immunohistochemistry for phosphorylated Smad3 (P-Smad3) (Fig 1; representative immunohistochemistry for day 5 is shown). TGFβ pathway activation was primarily observed in the INL and to some degree in the ONL at the late time points (days 5 and 8). P-Smad3 staining is observed from the central to the peripheral retina, often more pronounced in the peripheral retina towards the ciliary marginal zone (Fig 1). Consistent with the inhibition of the TGFβ receptor by SB431542, partly reduced Smad3 activation was observed in the inhibited group. For both groups, no relevant P-Smad3 was observed at baseline (day 0; uninjured retina), day 1 and between days 15 and 30 after induction of retina degeneration (Fig 1, exemplarily, uninjured retina, day 1, 5 and 30 are shown).


Inhibition of the TGF β Pathway Enhances Retinal Regeneration in Adult Zebrafish
Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.
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pone.0167073.g001: Immunohistological staining for P-Smad3 as an indicator of TGFβ pathway activation.The red channel with P-Smad3 staining is shown in the figures above, whereas overlay with the green (autofluorescence of photoreceptor outer segments) and blue channel (DAPI) is shown below. No relevant staining for P-Smad3 (red) was observed in the uninjured retina and one day after induction of retina degeneration with MNU. Starting at day 3 and until day 8, immunohistochemical staining for P-Smad3 revealed the activation of the TGFβ pathway (exemplarily, day 5 is shown). At day 15 and thereafter, no relevant activation was observed anymore (exemplarily, day 30 is shown). When the TGFβ pathway was inhibited (small molecule inhibitor SB431542), reduced staining for P-Smad3 was observed, when compared to the non-inhibited group in 0.1% dimethyl sulfoxide (DMSO). Lower magnification of retina 3 days after MNU treatment, including the peripheral retina is shown on the right. Cell nuclei are stained with DAPI (blue). The scale bar indicates 50 μm. GC: ganglion cells; INL: inner nuclear layer; ONL: outer nuclear layer.
Mentions: After inducing retinal degeneration using 150 mg/l MNU, maximal activation of the TGFβ pathway occurred between days 3 and 8 as demonstrated by immunohistochemistry for phosphorylated Smad3 (P-Smad3) (Fig 1; representative immunohistochemistry for day 5 is shown). TGFβ pathway activation was primarily observed in the INL and to some degree in the ONL at the late time points (days 5 and 8). P-Smad3 staining is observed from the central to the peripheral retina, often more pronounced in the peripheral retina towards the ciliary marginal zone (Fig 1). Consistent with the inhibition of the TGFβ receptor by SB431542, partly reduced Smad3 activation was observed in the inhibited group. For both groups, no relevant P-Smad3 was observed at baseline (day 0; uninjured retina), day 1 and between days 15 and 30 after induction of retina degeneration (Fig 1, exemplarily, uninjured retina, day 1, 5 and 30 are shown).

View Article: PubMed Central - PubMed

ABSTRACT

In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the N-methyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de- and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.

No MeSH data available.


Related in: MedlinePlus