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Fig. 5. Normal expression of stem cell (rx1, pax6, six3), neural identity (notch1, sox2) and differentiation (ngn1, p27, neuroD, Xath5) markers in control embryos. Embryos injected with β-gal mRNA were processed by in situ hybridization to determine the normal expression pattern of each marker gene. All stages 22–27 embryos are external views of whole-mount preparations [bars = 100 μm]; all stage 38 views are from whole mount preparations that were thickly sectioned to reveal internal expression patterns [bar = 20 μm]. (A) Summary of the expression domains of marker genes at stage 38 in the different layers of the retina, based on data from Perron and Harris (1999), Ohnuma et al. (1999) and our results. All genes except ngn1 are expressed in some part of the ciliary marginal zone (CMZ). Only pax6, rx1, six3 and sox2 are expressed in all zones of the CMZ. Other genes are expressed only in the zones of the CMZ (2, 3) closest to the central retina, which are thought to contain RPCs. Within the stage 38 central retina, these genes are differentially expressed in the three cellular layers (GCL, INL, ONL). L, lens. (B) rx1 is expressed throughout the optic cup (arrow). (C) At stage 38, rx1 is expressed in all zones of the CMZ and weakly in the ONL (arrow). (D) pax6 is expressed throughout the optic cup (arrow). (E) At stage 38, pax6 is expressed in the CMZ and throughout the GCL and INL. (F) six3 is expressed throughout the optic cup (arrow). (G) At stage 38, six3 is expressed throughout the CMZ, and in the GCL and INL; the faint staining in the ONL is due to the thickness of the section. (H) notch1 is expressed throughout the optic vesicle (arrow). (I) At stage 38, notch1 is expressed in zones 2 and 3 of the CMZ and throughout the GCL and INL. (J) sox2 is expressed throughout the optic vesicle (arrow). (K) At stage 38, sox2 is expressed in all zones of the CMZ, strongly in the GCL and is barely detected in the INL. (L) ngn1 is expressed in the central part of the optic cup (arrow). (M) At stage 38, ngn1 is expressed in all layers of the central retina, and is absent from the CMZ. (N) p27 is expressed broadly in the optic cup (arrow). (O) At stage 38, p27 is expressed in zones 2 and 3 of the CMZ and throughout the GCL and INL. (P) neuroD is expressed broadly in the optic cup (arrow). (Q) At stage 38, neuroD is expressed in zone 3 of the CMZ and throughout the GCL and INL. (R) Xath5 is expressed broadly in the optic cup (arrow). (S) At stage 38, Xath5 is expressed in zone 3 of the CMZ and throughout the GCL and INL.
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rax ( retina and anterior neural fold homeobox ) gene expression in Xenopus leaves embryo, NF stage 20 embryo, assayed by in situ hybridization, anterior view, dorsal up.
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Xenopus notch1 expression in stage 22 embryo.
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six3 (SIX homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view dorsal up.
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Fig. 1. Altered levels of rx1 expression affect blastomere D1.1.1 contributions to retinal cell types. (A–C) Representative cell types from each cell layer were labeled by immunofluorescence for: (A) calbindin-positive cone photoreceptors (red) in the ONL [bar = 40 μm]; (B) GABA-positive (red) horizontal cells in the OINL (note that GABA also stains amacrine cells in the IINL) [bar = 30 μm]; (C) DAPI-positive nuclei (blue) of ganglion cells in the GCL [bar = 30 μm]. For panels A–C, arrowheads note GFP-labeled (green) descendants of blastomere D1.1.1. L, lens. (D–F) Activation of Rx1-GR protein at early (St. 12) and late (St. 16) neural plate stages decreased cell numbers significantly (*p < 0.05 cf. GFP-injected hormone treated controls) for all three cell types. (G–I) Activation of Rx1EnR-GR protein at St. 12 significantly decreased ganglion cells. Activation at St. 16 significantly increased cone and ganglion cell numbers, but horizontal cells were unchanged. (*p < 0.05 cf. GFP-injected hormone treated controls; **p < 0.05 cf. stage 12). Bars indicate SEM.
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Fig. 3. Activation of Rx1-GR or Pax6-GR protein at stage 12 does not cause cells to contribute to alternate cell fates. Cells descended from the mRNA-injected D1.1.1 blastomere (red) contribute to head tissues in a pattern identical to normal fate maps (cf. Moody, 1987), including diencephalon (d), lateral epidermis (e), retina (r; outlined), and branchial arch mesoderm (ba). These patterns are consistent regardless of which mRNA was injected (βGal, rx1, pax6), and whether hormone (dex) was added to the culture medium. The expression domains of Nkx2.4 in the ventral diencephalon and pax2 in the optic stalk did not change after any of the treatments. Bar = 100 μm.
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Fig. 4. Changing Rx1 and Pax6 levels/activity at stage 12 alters proliferation and the size of the retina. (A) rx1-GR (rx1) and pax6-GR (pax6) expressing clones contained a significantly larger proportion of mitotic cells than control (β-Gal) clones, whereas loss-of-function of rx1 (rx1EnR) or pax6 (dnpax6) resulted in a significantly decreased mitotic index. The mitotic indices of clones at stage 20 (optic vesicle) that descended from blastomere D1.1.1 were calculated by counting the number of cells in the clone (D–F, blue cytoplasm) that expressed phosphohistone-H3 (D–F, brown nuclei), a marker specific to mitotic prophase. Arrowheads note representative double-labeled cells. Bar = 10 μm. (B) The width of the optic cup at stage 25 was significantly larger when Rx1 or Pax6 levels were increased and significantly smaller when rx1EnR or dnpax6 mRNAs were expressed. (C) Retinal volume at stage 44/45 was significantly larger when Rx1 or Pax6 levels were increased and significantly smaller when rx1EnR or dnpax6 mRNAs were expressed. (*p < 0.05 cf. β-Gal or GFP dex-treated controls). Bars indicate SEM.
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Fig. 6. Increasing Rx1 levels at stage 12 represses stem cell, neural identity and differentiation marker genes. (A) pax6 expression domain in the optic vesicle is smaller (arrow), and repressed in β-Gal labeled cells (inset, arrowheads). (B) In a small percentage of stage 38 embryos, pax6 expression is repressed in β-Gal labeled cells (arrowheads). (C) six3 expression domain in the optic vesicle is smaller (arrow). (D) In most stage 38 embryos, six3 expression is normal in β-Gal labeled cells (arrowheads). (E) notch1 expression domain in the optic vesicle is smaller (arrow), and repressed in β-Gal labeled cells (inset, arrowheads). (F) By stage 38 there is no repression surrounding β-Gal-labeled nuclei (arrowheads). (G) sox2 expression domain in the optic vesicle is smaller (arrow). (H) By stage 38 there is no repression surrounding β-Gal-labeled nuclei (arrowheads). (I) ngn1 expression in the optic cup (arrow) is fainter (cf. Fig. 5L) and repressed in β-Gal labeled cells (inset, arrowheads). (J) At stage 38, ngn1 expression continues to be repressed surrounding β-Gal-labeled nuclei (arrowheads). (K) p27 expression in the optic cup (arrow) is fainter (cf. Fig. 5N). (L) At stage 38, p27 expression continues to be repressed surrounding β-Gal-labeled nuclei (arrowheads) in some embryos. (M) neuroD expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5P). (N) At stage 38, neuroD continues to be repressed surrounding β-Gal labeled nuclei (arrowheads). (O) Xath5 expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5R). (P) At stage 38, Xath5 expression continues to be repressed surrounding β-Gal-labeled nuclei (arrowheads) in some embryos. Data for stage 16 activation are similar. Dashed lines indicate the outline of the retinas in the sectioned material. For whole mounts, bars = 100 μm; for sections bars = 20 μm.
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Fig. 7. rx1 is not required after stage 12 for other retinal stem or neural identity marker genes but is necessary for the onset of differentiation markers. (A) pax6 expression domain in the optic vesicle (arrow) is smaller (cf. Fig. 5D). (B) At stage 38, the pax6 expression domain is normal, and no repression surrounds β-Gal-labeled nuclei (arrowheads). (C) six3 expression domain in the optic vesicle (arrow) is smaller (cf. Fig. 5F). (D) At stage 38, there is no repression of six3 surrounding the β-Gal-labeled nuclei (arrowheads). (E) notch1 expression domain in the optic vesicle is smaller (arrow). However, cells with β-Gal labeled nuclei (inset, arrowheads) express notch1. (F) At stage 38, notch1 expression is similar to controls and no repression surrounds β-Gal labeled nuclei (arrowheads). (G) sox2 expression domain in optic vesicle (arrow) is smaller (cf. Fig. 5J). (H) At stage 38, there is no repression of sox2 expression surrounding the β-Gal labeled nuclei (arrowheads). (I) ngn1 expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5L). (J) At stage 38, ngn1 expression continues to be repressed surrounding β-Gal labeled nuclei (arrowheads). (K) p27 expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5N). (L) At stage 38, p27 expression is repressed surrounding β-Gal labeled nuclei (arrowheads) in some embryos. (M) neuroD expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5P). Inset: cells with β-Gal-labeled nuclei (arrowheads) do not express neuroD. (N) At stage 38, neuroD expression continues to be repressed in β-Gal-labeled cells (arrowheads). (O) Xath5 expression in the optic cup (arrow) is greatly reduced (cf. Fig. 5R). Inset: cells with β-Gal-labeled nuclei (arrowheads) do not express Xath5. (P) At stage 38, Xath5 continues to be repressed in β-Gal-labeled cells (arrowheads). Data for stage 16 activation are similar albeit at higher frequencies for differentiation genes (see Table 1). Dashed lines indicate the outline of the retinas in the sectioned material. For whole mounts, bars = 100 μm; for sections bars = 20 μm.
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Fig. 8. Increasing Pax6 levels at stage 12 does not alter stem cell marker genes but represses sox2 and differentiation marker genes. (A) rx1 expression in the optic vesicle (arrowhead) is larger (cf. Fig. 5B). (B) At stage 38, rx1 expression is similar to controls and no repression surrounds β-Gal-labeled nuclei (arrowheads). (C) six3 expression in the optic vesicle (arrowhead) is not different from the control side. (D) At stage 38, no repression of six3 expression surrounds β-Gal-labeled nuclei (arrowhead). (E) notch1 expression in the optic vesicle on the injected side (arrowhead) is similar to the control side. (F) At stage 38, no repression of notch1 expression surrounds β-Gal-labeled nuclei (arrowheads). (G) When Pax6-GR protein is activated at stage 12, the sox2 expression domain in the optic vesicle is smaller (arrow). (H) When Pax6-GR protein is activated at stage 16, sox2 expression in the optic vesicle domain is smaller (arrow), but in the neural plate (np) it is broader and is ectopically expressed (arrow, inset). (I) ngn1 expression in the optic cup (arrowhead) is fainter and reduced in size (cf. Fig. 5L). (J) At stage 38, repression of ngn1 in β-Gal labeled cells persists (arrowheads). (K) p27 expression in the optic cup (arrowhead) is fainter and reduced in size (cf. Fig. 5N). (L) At stage 38, repression of p27 expression in β-Gal labeled cells (arrowheads) persists. (M) neuroD expression in the optic cup is fainter and reduced in size (cf. Fig. 5P). (N) At stage 38, neuroD expression remains repressed (arrowheads) in only a small number of embryos (Table 2). (O) Xath5 expression in the optic cup (arrow) is fainter and reduced in size (cf. Fig. 5R). (P) At stage 38, Xath5 expression remains repressed in β-Gal labeled cells (arrowhead) in a small number of embryos (Table 2). Data for stage 16 activation (except for sox2, see H above) are similar albeit at significantly lower frequencies at stage 38 for ngn1 and p27 and at higher frequencies for neuroD and Xath5 (Table 2). Dashed lines indicate the outline of the retinas in the sectioned material. For whole mounts, bars = 100 μm; for sections bars = 20 μm.
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Fig. 9. pax6 is not required after stage 12 for other retinal stem cell or neural identity marker genes but is necessary for some differentiation markers. (A) rx1 expression domain in the optic vesicle is smaller (arrow). (B) At stage 38, rx1 expression is similar to controls and no repression surrounds β-Gal-labeled nuclei (arrowheads). (C) six3 expression domain in the optic vesicle is smaller (cf. Fig. 5F). (D) At stage 38, no repression of six3 surrounds β-Gal-labeled nuclei (arrowheads). (E) notch1 expression domain in the optic vesicle (arrow) does not change, and β-Gal labeled cells (red) express notch1 (inset). (F) At stage 38, no repression of notch1 expression surrounds β-Gal labeled cells (arrowheads). (G) sox2 expression domain in the optic vesicle is smaller (arrow). (H) At stage 38, no repression of sox2 expression surrounds β-Gal-labeled nuclei (arrowheads). (I) ngn1 expression in the optic cup (arrow) is fainter (cf. Fig. 5L). (J) At stage 38, ngn1 continues to be repressed surrounding β-Gal labeled nuclei (arrowhead). (K) p27 expression in the optic cup (arrow) is fainter (cf. Fig. 5N). (L) At stage 38, p27 expression continues to be repressed surrounding clusters of β-Gal labeled nuclei (arrowheads). (M) neuroD expression domain in the optic cup (arrow) is reduced in size but not expression intensity (cf. Fig. 5P). (N) At stage 38, neuroD expression is not repressed in β-Gal labeled cells (arrowheads). (O) Xath5 expression domain in optic cup (arrow) is fainter in less than half of the embryos (cf. Fig. 5R). (P) At stage 38, Xath5 expression is not repressed in β-Gal labeled cells (arrowheads). Data for stage 16 activation are similar, but at significantly different frequencies for differentiation genes (see Table 2). Dashed lines indicate the outline of the retinas in the sectioned material. For whole mounts, bars = 100 μm; for sections bars = 20 μm.
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