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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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Loss of Vsx2 allows various cell types to develop in the retina. (A-J) Cryosections of 80 hours post-fertilization (hpf) retinas labelled with different cell-specific markers show that Vsx2 morphant retinas have all cell types present: Sox2 labels amacrine and Müller cells (A, B); protein kinase C (PKC)β1 labels some bipolar cells (C, D); Hermes labels ganglion cells (E, F); Islet 1 labels horizontal, bipolar, some amacrine and ganglion cells (G, H); glutamine synthetase (GS) labels Müller cells (I, J). (K-T)Cryostat sections of 80 hpf transgenic retinas similarly reveal presence of all marked cell types in Vsx2 morphants. (K-N) Vsx2 morphant Tg(ath5:Gap-GFP) retinas (double labelled with the ganglion cell marker Zn5 in red) still have green fluorescent protein (GFP)- and Zn5-labelled ganglion cells and form an optic nerve to the tectum. (O-R) Vsx2 morphant double transgenic Tg(vsx1:GFP-vsx2:dsRed) have both GFP- and DsRed-labelled cells, but appear to have comparatively fewer Vsx2:DsRed cells. As in the control transgenics, Vsx1:GFP and Vsx2:DsRed do not co-localise in the same cells. (S, T) Vsx2 morphant Tg(ptf1a:GFP) retinas show GFP labelling for horizontal and amacrine similar to that in controls, although some GFP cells are displaced and can be found closer to the lens in the GCL of the morphant retinas. L, lens. Scale bar: (A-T) 47 μm.
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Figure 13: Loss of Vsx2 allows various cell types to develop in the retina. (A-J) Cryosections of 80 hours post-fertilization (hpf) retinas labelled with different cell-specific markers show that Vsx2 morphant retinas have all cell types present: Sox2 labels amacrine and Müller cells (A, B); protein kinase C (PKC)β1 labels some bipolar cells (C, D); Hermes labels ganglion cells (E, F); Islet 1 labels horizontal, bipolar, some amacrine and ganglion cells (G, H); glutamine synthetase (GS) labels Müller cells (I, J). (K-T)Cryostat sections of 80 hpf transgenic retinas similarly reveal presence of all marked cell types in Vsx2 morphants. (K-N) Vsx2 morphant Tg(ath5:Gap-GFP) retinas (double labelled with the ganglion cell marker Zn5 in red) still have green fluorescent protein (GFP)- and Zn5-labelled ganglion cells and form an optic nerve to the tectum. (O-R) Vsx2 morphant double transgenic Tg(vsx1:GFP-vsx2:dsRed) have both GFP- and DsRed-labelled cells, but appear to have comparatively fewer Vsx2:DsRed cells. As in the control transgenics, Vsx1:GFP and Vsx2:DsRed do not co-localise in the same cells. (S, T) Vsx2 morphant Tg(ptf1a:GFP) retinas show GFP labelling for horizontal and amacrine similar to that in controls, although some GFP cells are displaced and can be found closer to the lens in the GCL of the morphant retinas. L, lens. Scale bar: (A-T) 47 μm.

Mentions: To assess the role of Vsx2 in cell determination, we used either the translation blocking morpholino (MO) or a combination of 2 splicing MOs (SMOs) affecting the splicing in introns 1 and 2, respectively, to knock down Vsx2. It was necessary to inject the two SMOs together to abrogate normal splicing of Vsx2. Morphant embryos, from the translation blocker or the SMOs, are clearly microphthalmic (Figure 11A, B), in accord with what has been previously recorded [25]. We observed changes of expression of several genes in Vsx2 morphants (Figure 12). Comparable results were found when comparing the expression levels of these genes using RNA from only morphant heads or even only eyes (data not shown). As expected from the finding that Vsx2 can bind the ath5 promoter, we found an upregulation of ath5, which fits well with our finding that Ath5:RFP expression is negatively correlated with Vsx2 expression and that Vsx2 binds the ath5 promoter in vitro. foxn4, a gene that has been described as necessary for the mitotic progenitors of amacrine and horizontal cells in the mouse retina [33] (Figure 12), is also upregulated in Vsx2 morphants. As expected, from previous work, mitf, a retinal pigment epithelium -specific gene normally repressed by Vsx2, was also increased [13], while the bipolar-cell-specific gene bhlh4, on the other hand, was downregulated [34]. rx3, a gene whose late period of expression in Medaka fish is in the INL [35], was also downregulated (Figure 12). Surprisingly, there is a strong increase of vsx2 transcription itself in vsx2 morphants, suggesting that Vsx2 represses its own expression. In agreement with this, we found Vsx2 binding consensus sequences in the vsx2 regulatory element, and this element also interacts with Vsx2 in an EMSA assay (Figure 10B). Surprisingly, there is no clear effect on the level of Vsx1 expression at any time point analysed from 31 to 80 hpf (Figure 12). Although the eyes were clearly microphthalmic and poorly formed, ganglion cells (Hermes- and Islet-positive), amacrine cells (Sox2- and 5E11-positive; Ptf1:GFP cells in the inner part of the INL), horizontal cells (Ptf1:GFP cells in the outermost part of the INL near the outer plexiform layer (OPL)), cone and rod photoreceptors (Zpr1- and 3-positive, respectively, data not shown) were detected in Vsx2 morphants (Figure 13A–J). Both Müller glial cells (glutamine synthetase-positive) and PKC-positive bipolars appeared reduced in numbers at 72 hpf (Figure 13D, J). Indeed Vsx2 morphant Tg(vsx2:dsRed;vsx1:GFP) double transgenic embryos showed a reduced density of dsRed compared to GFP-labelled cells in the INL (Figure 13O–R).


Vsx2 in the zebrafish retina: restricted lineages through derepression.

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

Loss of Vsx2 allows various cell types to develop in the retina. (A-J) Cryosections of 80 hours post-fertilization (hpf) retinas labelled with different cell-specific markers show that Vsx2 morphant retinas have all cell types present: Sox2 labels amacrine and Müller cells (A, B); protein kinase C (PKC)β1 labels some bipolar cells (C, D); Hermes labels ganglion cells (E, F); Islet 1 labels horizontal, bipolar, some amacrine and ganglion cells (G, H); glutamine synthetase (GS) labels Müller cells (I, J). (K-T)Cryostat sections of 80 hpf transgenic retinas similarly reveal presence of all marked cell types in Vsx2 morphants. (K-N) Vsx2 morphant Tg(ath5:Gap-GFP) retinas (double labelled with the ganglion cell marker Zn5 in red) still have green fluorescent protein (GFP)- and Zn5-labelled ganglion cells and form an optic nerve to the tectum. (O-R) Vsx2 morphant double transgenic Tg(vsx1:GFP-vsx2:dsRed) have both GFP- and DsRed-labelled cells, but appear to have comparatively fewer Vsx2:DsRed cells. As in the control transgenics, Vsx1:GFP and Vsx2:DsRed do not co-localise in the same cells. (S, T) Vsx2 morphant Tg(ptf1a:GFP) retinas show GFP labelling for horizontal and amacrine similar to that in controls, although some GFP cells are displaced and can be found closer to the lens in the GCL of the morphant retinas. L, lens. Scale bar: (A-T) 47 μm.
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Figure 13: Loss of Vsx2 allows various cell types to develop in the retina. (A-J) Cryosections of 80 hours post-fertilization (hpf) retinas labelled with different cell-specific markers show that Vsx2 morphant retinas have all cell types present: Sox2 labels amacrine and Müller cells (A, B); protein kinase C (PKC)β1 labels some bipolar cells (C, D); Hermes labels ganglion cells (E, F); Islet 1 labels horizontal, bipolar, some amacrine and ganglion cells (G, H); glutamine synthetase (GS) labels Müller cells (I, J). (K-T)Cryostat sections of 80 hpf transgenic retinas similarly reveal presence of all marked cell types in Vsx2 morphants. (K-N) Vsx2 morphant Tg(ath5:Gap-GFP) retinas (double labelled with the ganglion cell marker Zn5 in red) still have green fluorescent protein (GFP)- and Zn5-labelled ganglion cells and form an optic nerve to the tectum. (O-R) Vsx2 morphant double transgenic Tg(vsx1:GFP-vsx2:dsRed) have both GFP- and DsRed-labelled cells, but appear to have comparatively fewer Vsx2:DsRed cells. As in the control transgenics, Vsx1:GFP and Vsx2:DsRed do not co-localise in the same cells. (S, T) Vsx2 morphant Tg(ptf1a:GFP) retinas show GFP labelling for horizontal and amacrine similar to that in controls, although some GFP cells are displaced and can be found closer to the lens in the GCL of the morphant retinas. L, lens. Scale bar: (A-T) 47 μm.
Mentions: To assess the role of Vsx2 in cell determination, we used either the translation blocking morpholino (MO) or a combination of 2 splicing MOs (SMOs) affecting the splicing in introns 1 and 2, respectively, to knock down Vsx2. It was necessary to inject the two SMOs together to abrogate normal splicing of Vsx2. Morphant embryos, from the translation blocker or the SMOs, are clearly microphthalmic (Figure 11A, B), in accord with what has been previously recorded [25]. We observed changes of expression of several genes in Vsx2 morphants (Figure 12). Comparable results were found when comparing the expression levels of these genes using RNA from only morphant heads or even only eyes (data not shown). As expected from the finding that Vsx2 can bind the ath5 promoter, we found an upregulation of ath5, which fits well with our finding that Ath5:RFP expression is negatively correlated with Vsx2 expression and that Vsx2 binds the ath5 promoter in vitro. foxn4, a gene that has been described as necessary for the mitotic progenitors of amacrine and horizontal cells in the mouse retina [33] (Figure 12), is also upregulated in Vsx2 morphants. As expected, from previous work, mitf, a retinal pigment epithelium -specific gene normally repressed by Vsx2, was also increased [13], while the bipolar-cell-specific gene bhlh4, on the other hand, was downregulated [34]. rx3, a gene whose late period of expression in Medaka fish is in the INL [35], was also downregulated (Figure 12). Surprisingly, there is a strong increase of vsx2 transcription itself in vsx2 morphants, suggesting that Vsx2 represses its own expression. In agreement with this, we found Vsx2 binding consensus sequences in the vsx2 regulatory element, and this element also interacts with Vsx2 in an EMSA assay (Figure 10B). Surprisingly, there is no clear effect on the level of Vsx1 expression at any time point analysed from 31 to 80 hpf (Figure 12). Although the eyes were clearly microphthalmic and poorly formed, ganglion cells (Hermes- and Islet-positive), amacrine cells (Sox2- and 5E11-positive; Ptf1:GFP cells in the inner part of the INL), horizontal cells (Ptf1:GFP cells in the outermost part of the INL near the outer plexiform layer (OPL)), cone and rod photoreceptors (Zpr1- and 3-positive, respectively, data not shown) were detected in Vsx2 morphants (Figure 13A–J). Both Müller glial cells (glutamine synthetase-positive) and PKC-positive bipolars appeared reduced in numbers at 72 hpf (Figure 13D, J). Indeed Vsx2 morphant Tg(vsx2:dsRed;vsx1:GFP) double transgenic embryos showed a reduced density of dsRed compared to GFP-labelled cells in the INL (Figure 13O–R).

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