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Vsx2 in the zebrafish retina: restricted lineages through derepression.

Vitorino M, Jusuf PR, Maurus D, Kimura Y, Higashijima S, Harris WA - Neural Dev (2009)

Bottom Line: Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates.Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2.Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK. mspdcv2@cam.ac.uk

ABSTRACT

Background: The neurons in the vertebrate retina arise from multipotent retinal progenitor cells (RPCs). It is not clear, however, which progenitors are multipotent or why they are multipotent.

Results: In this study we show that the homeodomain transcription factor Vsx2 is initially expressed throughout the retinal epithelium, but later it is downregulated in all but a minor population of bipolar cells and all Müller glia. The Vsx2-negative daughters of Vsx2-positive RPCs divide and give rise to all other cell types in the retina. Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates. Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2.

Conclusion: Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.

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Transplantation of wild type, Ath5, and Vsx2 overexpressing cells in wild-type embryos. (A-C)Tg(vsx2:GFP) transplanted into wild-type embryos after injection with H2B-RFP RNA (to mark all transplanted cells) and various constructs for overexpression. (A) Green-fluorescent protein (GFP)-expressing cells from control Tg(vsx2:GFP) become bipolar and Müller cells, whilst all (H2B-RFP-positive) cells become all kinds of cells in the expected frequency. (B) When co-injecting H2B-RFP RNA with ath5, the ganglion cell fate is promoted (more H2B-RFP cells in the GCL), but Vsx2:GFP cells still become bipolar and Müller cells. (C) Co-injection of H2B-RFP RNA with vsx2 promotes bipolar cell fate, seen by (D) a highly significant (p < 0.001) increase of H2B-RFP in the INL bipolar layer at the expense of amacrine cells (asterisks). (E) Overexpression using a heatshock vsx2 construct similarly results in an increased frequency of bipolar cells, this time at the expense of photoreceptors. Error bars indicate Standard Error of the Mean. AC, amacrine cell; BC, bipolar cell; GCL, ganglion cell layer; H, horizontal cell; hs, heat shock; IINL, inner half of inner nuclear layer; MC, Müller cell; OINL, outer half of inner nuclear layer; ONL, outer nuclear layer; PH, photoreceptor; RGC, retinal ganglion cell. Scale bar (A-C) 25 μm.
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Figure 14: Transplantation of wild type, Ath5, and Vsx2 overexpressing cells in wild-type embryos. (A-C)Tg(vsx2:GFP) transplanted into wild-type embryos after injection with H2B-RFP RNA (to mark all transplanted cells) and various constructs for overexpression. (A) Green-fluorescent protein (GFP)-expressing cells from control Tg(vsx2:GFP) become bipolar and Müller cells, whilst all (H2B-RFP-positive) cells become all kinds of cells in the expected frequency. (B) When co-injecting H2B-RFP RNA with ath5, the ganglion cell fate is promoted (more H2B-RFP cells in the GCL), but Vsx2:GFP cells still become bipolar and Müller cells. (C) Co-injection of H2B-RFP RNA with vsx2 promotes bipolar cell fate, seen by (D) a highly significant (p < 0.001) increase of H2B-RFP in the INL bipolar layer at the expense of amacrine cells (asterisks). (E) Overexpression using a heatshock vsx2 construct similarly results in an increased frequency of bipolar cells, this time at the expense of photoreceptors. Error bars indicate Standard Error of the Mean. AC, amacrine cell; BC, bipolar cell; GCL, ganglion cell layer; H, horizontal cell; hs, heat shock; IINL, inner half of inner nuclear layer; MC, Müller cell; OINL, outer half of inner nuclear layer; ONL, outer nuclear layer; PH, photoreceptor; RGC, retinal ganglion cell. Scale bar (A-C) 25 μm.

Mentions: For misexpression, we found that injection of vsx2 mRNA led to embryos that were anophthalmic, making such an analysis problematical. To get around this problem, we either transplanted cells from Vsx2 misexpressing Tg(vsx2:GFP) embryos into wild-type hosts or injected embryos with the pSGH:vsx2 heatshock plasmid and then incubated the injected embryos at 39°C from 26–28 hpf. Given that a decrease in Vsx2 interferes with bipolar fates in favour of other cell types, we wanted to test whether Vsx2 misexpression promotes bipolar fate at the expense of other cell types. Analysis of the resulting fate change was done on 80–85 hpf retinas. In the transplant experiments, H2B:RFP mRNA was co-injected to compare the proportion of each cell type present (Figure 14A–C). Ath5, which promotes ganglion cell fate, was used as a positive control and, indeed, ath5 RNA injections resulted in an increase in the ganglion cell population. In vsx2 RNA injected cells, we observed an increase in cells in the bipolar layer (outer INL) at the expense of cells in the inner INL (Figure 14D). In the heatshock overexpression experiments, there was an increase in the bipolar cells, but this time at the expense of photoreceptors (Figure 14E).


Vsx2 in the zebrafish retina: restricted lineages through derepression.

Vitorino M, Jusuf PR, Maurus D, Kimura Y, Higashijima S, Harris WA - Neural Dev (2009)

Transplantation of wild type, Ath5, and Vsx2 overexpressing cells in wild-type embryos. (A-C)Tg(vsx2:GFP) transplanted into wild-type embryos after injection with H2B-RFP RNA (to mark all transplanted cells) and various constructs for overexpression. (A) Green-fluorescent protein (GFP)-expressing cells from control Tg(vsx2:GFP) become bipolar and Müller cells, whilst all (H2B-RFP-positive) cells become all kinds of cells in the expected frequency. (B) When co-injecting H2B-RFP RNA with ath5, the ganglion cell fate is promoted (more H2B-RFP cells in the GCL), but Vsx2:GFP cells still become bipolar and Müller cells. (C) Co-injection of H2B-RFP RNA with vsx2 promotes bipolar cell fate, seen by (D) a highly significant (p < 0.001) increase of H2B-RFP in the INL bipolar layer at the expense of amacrine cells (asterisks). (E) Overexpression using a heatshock vsx2 construct similarly results in an increased frequency of bipolar cells, this time at the expense of photoreceptors. Error bars indicate Standard Error of the Mean. AC, amacrine cell; BC, bipolar cell; GCL, ganglion cell layer; H, horizontal cell; hs, heat shock; IINL, inner half of inner nuclear layer; MC, Müller cell; OINL, outer half of inner nuclear layer; ONL, outer nuclear layer; PH, photoreceptor; RGC, retinal ganglion cell. Scale bar (A-C) 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683830&req=5

Figure 14: Transplantation of wild type, Ath5, and Vsx2 overexpressing cells in wild-type embryos. (A-C)Tg(vsx2:GFP) transplanted into wild-type embryos after injection with H2B-RFP RNA (to mark all transplanted cells) and various constructs for overexpression. (A) Green-fluorescent protein (GFP)-expressing cells from control Tg(vsx2:GFP) become bipolar and Müller cells, whilst all (H2B-RFP-positive) cells become all kinds of cells in the expected frequency. (B) When co-injecting H2B-RFP RNA with ath5, the ganglion cell fate is promoted (more H2B-RFP cells in the GCL), but Vsx2:GFP cells still become bipolar and Müller cells. (C) Co-injection of H2B-RFP RNA with vsx2 promotes bipolar cell fate, seen by (D) a highly significant (p < 0.001) increase of H2B-RFP in the INL bipolar layer at the expense of amacrine cells (asterisks). (E) Overexpression using a heatshock vsx2 construct similarly results in an increased frequency of bipolar cells, this time at the expense of photoreceptors. Error bars indicate Standard Error of the Mean. AC, amacrine cell; BC, bipolar cell; GCL, ganglion cell layer; H, horizontal cell; hs, heat shock; IINL, inner half of inner nuclear layer; MC, Müller cell; OINL, outer half of inner nuclear layer; ONL, outer nuclear layer; PH, photoreceptor; RGC, retinal ganglion cell. Scale bar (A-C) 25 μm.
Mentions: For misexpression, we found that injection of vsx2 mRNA led to embryos that were anophthalmic, making such an analysis problematical. To get around this problem, we either transplanted cells from Vsx2 misexpressing Tg(vsx2:GFP) embryos into wild-type hosts or injected embryos with the pSGH:vsx2 heatshock plasmid and then incubated the injected embryos at 39°C from 26–28 hpf. Given that a decrease in Vsx2 interferes with bipolar fates in favour of other cell types, we wanted to test whether Vsx2 misexpression promotes bipolar fate at the expense of other cell types. Analysis of the resulting fate change was done on 80–85 hpf retinas. In the transplant experiments, H2B:RFP mRNA was co-injected to compare the proportion of each cell type present (Figure 14A–C). Ath5, which promotes ganglion cell fate, was used as a positive control and, indeed, ath5 RNA injections resulted in an increase in the ganglion cell population. In vsx2 RNA injected cells, we observed an increase in cells in the bipolar layer (outer INL) at the expense of cells in the inner INL (Figure 14D). In the heatshock overexpression experiments, there was an increase in the bipolar cells, but this time at the expense of photoreceptors (Figure 14E).

Bottom Line: Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates.Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2.Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK. mspdcv2@cam.ac.uk

ABSTRACT

Background: The neurons in the vertebrate retina arise from multipotent retinal progenitor cells (RPCs). It is not clear, however, which progenitors are multipotent or why they are multipotent.

Results: In this study we show that the homeodomain transcription factor Vsx2 is initially expressed throughout the retinal epithelium, but later it is downregulated in all but a minor population of bipolar cells and all Müller glia. The Vsx2-negative daughters of Vsx2-positive RPCs divide and give rise to all other cell types in the retina. Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates. Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2.

Conclusion: Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.

Show MeSH
Related in: MedlinePlus