Transcription of the SCL/TAL1 interrupting Locus (Stil) is required for cell proliferation in adult Zebrafish Retinas

Abstract

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Previously, we identified a homolog of the Stil gene in zebrafish mutant (night blindness b, nbb), which showed neural defects in the retina (e.g. dopaminergic cell degeneration and/or lack of regeneration). In this research, we examined the roles of Stil in cell proliferation after degeneration in adult zebrafish retinas. We demonstrated that knockdown of Stil gene expression or inhibition of Sonic hedgehog (Shh) signaling transduction decreases the rate of cell proliferation. In contrast, activation of Shh signal transduction promotes cell proliferation. In nbb(+/-) retinas, inhibition of SUFU (a repressor in the Shh pathway) rescues the defects in cell proliferation due to down-regulation of Stil gene expression. The latter data suggest that Stil play a role in cell proliferation through the Shh signal transduction pathway.

Keywords: Cell Proliferation; Neurons; Oncogene; Retina; Zebrafish.

FIGURE 1.

Proliferation of Müller glial cells and rod precursor cells in response to 6-OHDA treatment. A, retinal sections of transgenic zebrafish Tg (gfap::GFP) labeled with PCNA antibodies after 3 days of 6-OHDA treatment. The Tg (gfap::GFP) transgene is expressed in Müller glial cells, revealed by the expression of GFP (left panel, arrows). PCNA antibodies labeled proliferating cells (middle panel, arrows). Note that the GFP-positive cells included Müller glial cells that are either proliferating (PCNA positive) or quiescent (PCNA negative). Some of the PCNA-positive cells are likely Müller glial-derived neuronal progenitor cells. The merged image shows that GFP and PCNA co-localized in proliferating cells (right panel, outlined by ovals). B, double labeling of retinal sections with PCNA antibodies (left panel, arrows) and EdU (middle panel, arrows) after 3 days of 6-OHDA treatment. EdU was injected after 1 and 2 days of 6-OHDA injection. Clusters of cells were labeled by PCNA antibodies and EdU. The merged image shows that PCNA immunoreactivity and EdU were co-localized (outlined by ovals). C, EdU and DAPI labeling of retinal sections after 30 days of 6-OHDA treatment. EdU was injected after 4, 5, and 6 days of 6-OHDA injection. EdU-positive cells (green) were found in both the inner and outer nuclear layers. Abbreviations: RPE, retinal pigment epithelia; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.

FIGURE 2.

Cell proliferation in adult wild-type zebrafish retinas in response to 6-OHDA treatment. A, fluorescent images of retinal sections labeled with PCNA antibodies (red; proliferating Müller glial cells) at different days post-6-OHDA injection. Dashed lines outline the outer and inner nuclear layers of the retina (RPE is on the top of the image). Note the increase in PCNA cell number in the first 6 days post-6-OHDA injection. After 6 days, PCNA expression decreased. B, PCNA-positive cell count per retinal section at different days post-6-OHDA injection. Cryostat sections in the nasal retina adjacent to the optic nerve were used for cell counts. Note that PCNA expression peaked at 6 days post-6-OHDA injection. Data represent the mean ± S.E. (n = 5 in each group). dpi, days post-injection. Scale bar, 100 μm.

FIGURE 3.

RT-PCR analyses of the transcription of Stil (A) and Gli1 (B) in wild-type retinas at different days post-6-OHDA injection. Control transcription of Stil and Gli1 measured in PBS-injected wild-type retinas was normalized to 1, respectively. Note the increase of Stil and Gli1 transcription at 5 and 6 days post-6-OHDA injection. Data represent the mean ± S.E. (n = 5 in each group). *, p < 0.01; ns, not significant.

FIGURE 4.

Cell proliferation and gene expression in wild-type and nbb^+/− retinas at 6 days post-6-OHDA injection. A, fluorescent images of retinal sections labeled with PCNA antibody (RPE is on the top of the image). Note the decrease in PCNA cell number in mutants. B, PCNA-positive cell count in wild-type and mutant retinas. Note the decrease in PCNA cell number in mutants. C and D, relative transcription of Stil and Gli1 mRNA in wild-type and nbb^+/− retinas. The transcription of Stil and Gli1 in wild-type retinas were normalized to 1, respectively. Note the decrease in the transcription of Stil and Gli1 in mutants. Data represent the mean ± S.E. (n = 4 in each group). *, p < 0.01. Scale bar, 100 μm.

FIGURE 5.

Effects of cyclopamine treatment on Shh signal transduction and cell proliferation in wild-type retinas. A, RT-PCR analysis of Gli1 gene transcription in control and cyclopamine-treated retinas. The transcription of Gli1 in control retinas was normalized to 1. Note the decrease in transcription of Gli1 in cyclopamine-treated retinas. B, PCNA-positive cell count in control and cyclopamine-treated retinas. Note the decrease in PCNA cell number in cyclopamine-treated retinas. C, fluorescent images of retinal sections labeled with PCNA antibody (RPE is on the top of the image). Note the decrease of PCNA labeling in cyclopamine-treated retinas. Data represent the mean ± S.E. (n = 4 in each group). *, p < 0.01. Scale bar, 100 μm.

FIGURE 6.

Effects of SUFU knockdown on Gli1 gene expression and cell proliferation in nbb^+/− retinas. A, Western blot of retinal lysates probed with anti-SUFU antibody. Note the decrease in SUFU expression in MO-treated retinas. B, relative Gli1 transcription in mutant retinas at 6 days post-6-OHDA injection. The transcription of Gli1 in untreated mutant retinas was normalized to 1. Note the increase in transcription of Gli1 after MO treatment. C, PCNA-positive cell count in untreated and MO-treated mutant retinas at 6 days post-6-OHDA injection. Note the increase in PCNA cell number in MO-treated retinas. D, fluorescent images of mutant retinal sections labeled with anti-PCNA antibody at 6 days post-6-OHDA injection (RPE is on the top of the image). Note the increase in PCNA cell number in MO-treated retinas. Data represent the mean ± S.E. (n = 4 in each group). *, p < 0.01. Scale bar, 100 μm.