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Fig. 3. Rx-L function is needed after eye field induction. (A, B) WMISH analysis of staged embryos injected with MoRx-L at 4-cell stage into one of the dorsoanimal blastomeres. (Aa–Af) Frontal views, injected side to the right. At NF stage 15/16, the expressions of Pax6 (Aa), Rx1 (Ab) and Six3 (Ac) are not significantly affected due to suppression of Rx-L function. (Ad–Af) At tailbud stage (NF stage 24), the expressions of Pax6 (Ad), Rx1 (Ae) and Six3 (Af) are all reduced in the injected sides. (Ba–f') Transversal gelatin/albumin sections of injected embryos probed with the markers of different retinal cell types, with dorsal side upward. The staining of each probe in the retina of injected side (Ba'–f') should be compared with that in the corresponding control side of the same embryo (Ba–f) respectively. The red dashes in Ba' mark the presumptive RPE. Suppression of Rx-L function led to a dramatically reduced expression of the photoreceptor markers, Rhodopsin (Ba, Ba') and Arrestin-C (Bb, Bb'), and a broadened expression of the bipolar cell maker, Vsx1 (Bd, Bd') and the amacrine/ganglion cell marker, Pax6 (Be, Be'). The expression levels Prox1 (Bc, Bc') and Brn3.0 (Bf, Bf') remain almost unchanged upon MoRx-L injection. (C) Effects of MoRx-L microinjection on apoptosis and cell proliferation. (Ca, b, d, e) Transversal sections (dorsal sides up and injected sides to the right) of embryos injected with 2.5pmol of MoRx-L (Ca, d) or Contr-Mo (Cb, e) subjected to TUNEL assay (Ca, b) or immunostaining of phosphorylated histone H3 (p-Histone 3) (Cd, e) at NF stage 31/32 to detect apoptotic or proliferating cells, respectively. (Cc, f) Graphs showing the numbers of TUNEL positive cells (Cc) or p-Histone 3 positive cells (Cf) in total or eye area of the non-injected side (green bars), Contr-Mo injected side (green bars), and MoRx-L (orange bars). The average of TUNEL or p-Histone-3 positive cell numbers on per section was determined in each embryo. In the TUNEL assay, for non-injected, n = 5 embryos; for Contr-Mo, n = 2 embryos; for MoRx-L, n = 3 embyros. In the p-Histone 3 detection, for non-injected, n = 5 embryos; for Contr-Mo, n = 2 embryos; for MoRx-L, n = 3 embyros. Values are given as means ± S.E.M. p = 0.28, , p < 0.05, compared with the non-injected side (Student's t-test). Quantification of the counted cells and sections is shown next to the graphs respectively.
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Fig. 4. Effect of Rx-L gain-of-function on the eye development. (A, B) WMISH analysis of embryos Rx-L-GR injected at the 4-cell stage into one of the dorsoanimal blastomeres. (Aa–j) Anterior views of embryos (dorsal up; injected-side to the right) probed with antisense RNA of Rx1 (Aa–e) or Six3 (Af–j). (Aa, f) NF stage 16 embryos not treated with dexamethazone (Dex). (Ab, g) Embryos treated with Dex at NF stage 14, neither Rx1 nor Six3 expression was affected till NF stage 16. (Ac, h) At NF stage 24, Rx-L-GR injected embryos treated with Dex at NF stage 14 show reduced expression domains of Rx1 and Six3. (Ad, i, e, j) Rx-L-GR injected embryos treated with Dex at NF stage 17, here, the domains of Rx1 and Six3 expression were extended at NF stage 19 and 24. (B) Transversal retinal sections of NF stage 36 embryos stained for maker genes of different retinal cell types. (Ba–e) Retinae of Rx-L-GR injected embryo not treated with Dex. (Ba'–e') Retinae of embryos injected with Rx-L-GR mRNA and treated with Dex at NF stage 17/18. Induction of Rx-L-GR function not only led to Rhodopsin expression (Ba,Ba') in folds evaginated from the ONL into the INL, or the GCL (Ba', arrows), but also to the discrete spots of Rhodopsin expression (Ba' arrowhead). Activation of Rx-L-GR reduced the density of the expression of horizontal cell marker, Prox1 (Bb, Bb'), bipolar cell marker, Vsx1 (Bc, Bc'), and amacrine cell marker Pax6 (Bd, Bd'), while the expression of the ganglion cell marker, Brn3.0 (Be, Be') was not significantly changed (Be, Be'). (C) Effects of Rx-L-GR microinjection on apoptosis and cell proliferation. (Ca–c) Transversal sections (dorsal sides up and injected sides to the right) of embryos injected with 50 pg Rx-L-GR (+Dex at NF stage 17) into one of the dorsoanimal blastomeres at the 4-cell stage and subjected to TUNEL assay (Ca) or immunostaining of phosphorylated histone H3 (p-Histone 3, Cb, c) at NF stage 34 and NF stage 24 to detect apoptotic or mitotic cells, respectively. (Ca'–c') Graphs showing the numbers of TUNEL positive cells (Ca') or p-Histone 3 positive cells (C', c') in total or eye area of the non-injected side (green bars), and Rx-L-GR injected side (blue bars). The average of TUNEL or p-Histone-3 positive cell numbers on per section was determined in each embryo. In the TUNEL assay, for non-injected side, n = 5 embryos; for Rx-L-GR injected side, n = 2 embryos. For p-Histone-3 detection, non-injected side, n = 2 embryos; for Rx-L-GR injected side, n = 2 embryos at each stage. Values are given as means ± S.E.M. p = 0.28, , p = 0.07, compared with the non-injected side (Student's t-test). Quantification of the counted cells and sections is shown next to the graphs respectively.
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Fig. 6. Rx-L promotes RPCs to adopt a photoreceptor cell fate. (Aa–g) Transversal sections of NF stage 42 embryos at the level of the eye. (Aa) Section of a control retina stained by hematoxylin-eosin. (Ab–f) Retinas lipofected with eGFP expression plasmid alone (Ab), or co-lipofected with a plasmid mix of either eGFP and Rx-L (Ac), Rx-L-VP16 (Ad), MoRx-L (Ae) or Contr-Mo (Af). (h, i) Retinal cell types (GC, ganglion cells; AM, amacrine cells; BI, bipolar cells; HOR, horizontal cells; PR, photoreceptor cells and MU, Müller cells) were counted from the eGFP-positive population per lipofected retina (n) and the percentage of each cell type was determined. eGFP, n = 43 retinas, 6037 cells; Rx-L + eGFP, n = 22 retinas, 3503 cells; Rx-L-VP16 + eGFP, n = 22 retinas, 3005 cells; MoRx-L + eGFP, n = 16 retinas, 2575 cells; Contr-Mo + eGFP, n = 9 retinas, 1072 cells. The error bars represent the S.E.M. The asterisks indicate significant difference between the control and the experimental by Student's t-test, (), p < 0.01; (), p < 0.001. Lipofection of Rx-L (Ac, h) or Rx-L-VP16 (Ad, h) significantly increased the proportion of the eGFP-positive photoreceptors, compared to the retina lipofected with eGFP alone (Ab, g). Lipofection of MoRx-L (Ae, h) led to a reduced proportion of eGFP-positive photoreceptors, while Contr-Mo lipofected retinae (Af, h) showed a distribution of eGFP-positive retinal cell types similar to that of retina lipofected with eGFP alone (Ab). (B) Cryostat sections (10 μm) of retinas lipofected with eGFP alone (Ba) or Rx-L-VP16 and eGFP (Bb) stained with an antibody against calbindin (anti-Calbindin in red), for cone photoreceptors, and DAPI (in blue). Non-transfected cones appear red (3, only calbindin), and transfected cones appear orange (1, calbindin and eGFP). The sole DAPI-stained photoreceptor cells in the intervals of cones (4) represent non-transfected rods, while cells appear green in the ONL are transfected rods (2). (E) Graph showing the ratios of calbindin-labelled (Cal+, cones) and -unlabelled (Cal−, rods) eGFP-positive photoreceptor cells (n) in eGFP (n = 86) or eGFP and Rx-L-VP16 (n = 392) lipofected retinas. Percentages of both Cal+ and Cal− photoreceptor cells were averaged in all retinas counted. The error bars represent the S.E.M. , p < 0.005 (Student's t-test). (C) The percentage of each cell type in the eGFP-positive population of retinae (n) lipofected with either eGFP and Rx-L, Rx1 or mouse Rax. eGFP, n = 4 retinas, 330 cells; Rx-L + eGFP, n = 5 retinas, 1048 cells; Rx1 + eGFP, n = 7 retinas, 1995 cells; mRax + eGFP, n = 6 retinas, 1145 cells, (), p < 0.01. Lipofection of Rx-L significantly increased the proportion of the eGFP-positive photoreceptors at the expense of amacrine and bipolar cells, but Rx1 and mRax seem to not promote any retinal cell types.
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Fig. 2. Impairment of photoreceptor formation upon Rx-L suppression. (A) The specificity of Rx-L specific antisense morpholino (MoRx-L) was examined by in vitro transcription and translation system. MoRx-L did not affect the translation of Rx-L-ORF, but blocked the translation of Rx-L-5′UTR-ORF, in which the Rx-L-ORF is flanked by 5′- and 3′-UTRs. The standard control morpholino (Contr-Mo) did not affect the translation of either transcript. (B, C) Stage 44/45 embryos which were co-injected with 60 pg β-gal mRNA and 2.5pmol of MoRx-L (B) or Contr-Mo (C) in one of the dorsoanimal blastomeres at the 4-cell stage; injected sides to the right. (B', B") Transversal sections stained by hematoxylin-eosin of the eyes of the non-injected (B') and injected side (B") of an embryo of the same age shown in panel B, at levels indicated by dashed lines. (C', C") Transversal sections of the eye on the non-injected side (C') and injected eye (C") of an embryo shown in panel C, as indicated by dashed lines. Insets show magnified views of the photoreceptor region as shown in the dashed boxes. Compared with the non-injected retina (B'), the outer segments of photoreceptors in the MoRx-L injected retina (B") were arrayed much more loosely (red arrow), while the retina of embryos injected with Contr-Mo (C", green arrow) looked normal. Abbreviations: cmz, ciliary marginal zone; inl, inner nuclear layer; isl, inner synaptic layer; gcl, ganglion cell layer; le, lens epithelium; lf, lens fibers; onl, outer nuclear layer; os, outer segment; rpe, retinal pigment epithelium.
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Fig. 5. Rx-L is a transcriptional activator rather than a repressor. (A) Lateral views of embryos injected with the indicated amounts of synthetic wild-type Rx-L (Aa–a'''), Rx-L-δOAR (Ab–b'''), Rx-L-VP16 (Ac–c''') or Rx-L-EngR (Ad–e''') mRNA and β-Gal mRNA as lineage tracer into one of dorsoanimal blastomeres at 4-cell stage and analyzed at NF stage 42. (Aa–e) Lateral views of non-injected sides, anterior to the right. (Aa'''–e''') Lateral views of injected sides, anterior to the left. Transversal sections at the level of the eye on the non-injected side (Aa'–e') and injected side (Aa"–e") stained with hematoxylin-eosin. The retinae of embryos injected with Rx-L-δOAR (Ab") or Rx-L-VP16 (Ac") show a phenotype quite similar to those injected with wild-type Rx-L (Aa"). The retina of embryos injected with a low dose of Rx-L-EngR (Ad''') exhibits an eye phenotype similar to that caused by MoRx-L. Moreover, high doses of Rx-L-EngR can suppress eye formation completely (Ae–e'''). (B) Schematic diagrams of chimeric Rx-L constructs. Rx-L-δOAR is Rx-L just lacking OAR domain. Rx-L-VP16 is generated by an in-frame fusion of Rx-L with the activator domain of VP16, and Rx-L-EngR is generated by an in-frame fusion of Rx-L with the repressor domain of Drosophila engrailed.
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arrb2 (arrestin, beta 2 ) gene expression in Xenopus laevis embryos, NF stage 35, as assayed by in situ hybridization, section through the developing eye.
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