Perry KJ , Johnson VR , Malloch EL , Fukui L , Wever J , Thomas AG , Hamilton PW , Henry JJ .

EY09844 NEI NIH HHS , R01 EY009844-15 NEI NIH HHS , R01 EY009844 NEI NIH HHS

	Figure 4. Developmental expression of X. laevis GPR84. The lane to the far left is the 1-kb plus DNA ladder, (+) represents the positive RT-PCR control lane using the full-length GPR84 containing plasmid and (−) represents the negative RT-PCR control lane lacking template DNA. A: RT-PCR analysis of GPR84 reveals detectible expression beginning at gastrula stage 10 and continuing through larval stage 45. Expected size of GPR84 fragment is 1.2 kb. B: RT-PCR analysis of GPR84 in isolated retinal and lens tissues. Developing retinas were excised from stage-29 embryos (R29) and stage-41 larvae (R41), and lenses were excised from stage-41 larvae (L41). C: PCR analysis showing presence of GPR84 transcripts in both control cornea and transdifferentiating cornea epithelium undergoing lens regeneration. (T) represents RT-PCR using transdifferentiating cornea RNA and (C) represents RT-PCR using control cornea RNA. D: Sagittal section showing examination of GPR84 within lens fiber cells at stage 36. E–G: In situ hybridization of GPR84 transcripts. Anterior is to the right in all cases. E: Representative stage-19 embryo with no visible expression. F: Stage-28 embryo with expression detectable only in the lens placode. G: Detectable lens expression at stage 33. H: Sense probe control at stage 27 with no detectable expression. cmz, ciliary marginal zone; le, lens epithelium; lf, lens fiber cells; ln, lens; lp, lens placode; nr, neural retina; vc, vitreous chamber. Scale bar in D = 50 and 400 μm in E–H.
	Figure 5. Effects of GPR84 morpholino-mediated knockdown and RNA rescue on eye development. Dorsal is toward the top in each figure. A–F: Typical eye defects following unilateral injection of lissamine-tagged H127MO into single blastomeres at the two-cell stage. All larvae shown are stages 37–39. A: Normal uninjected (CON) side. B: Corresponding H127MO injected side (5.24 ng) to that shown in A. Minor eye defect is observed on this side, as indicated by arrowhead. C: Normal uninjected side. D: Corresponding H127MO injected side (6.5 ng) to that shown in C. Severe eye defect is observed on this side, as indicated by arrowhead. E: Normal uninjected side. F: Corresponding H127MO-injected side (10.74 ng) to that shown in E. Severe eye defect is observed on this side, as indicated by arrowhead. G: Normal uninjected side. H: Corresponding injected side to that shown in G. Representative normal eye development follows co-injection of H127MO (6.5 ng/blastomere at the two-cell stage) and 800 pg synthetic altGPR84 mRNA. I: Normal uninjected side. J: Corresponding injected side to that shown in I. Typical normal morphological development following co-injection of H127MO (6.5 ng/blastomere at the two-cell stage) and 1,200 pg synthetic altGPR84 mRNA. K: Normal uninjected side. L: Normal eye development following injection of 1,200 pg synthetic altGPR84 RNA diluted with fluorescent dextran. The scale bar in L = 400 μm.
	Figure 7. Sagittal sections showing eye defects associated with H127MO (GPR84) knockdown. Hematoxylin/eosin stained specimens were fixed at stage 41. A: Representative mild defect resulting from unilateral injection of 6.5 ng H127MO at the two-cell stage. This eye has a small, disorganized lens with normal polarity and a recognizable lens epithelium and primary lens fiber cells. Note the neural retina is somewhat disorganized. The retinal pigmented epithelium is also intact, but is thinned near the ventral portion of the eye located towards the bottom of the figure. B: Corresponding uninjected eye of the embryo shown in A revealing normal development of the lens and the retinal layers. Note the presence of numerous primary and secondary lens fiber cells. C: Representative severe defect resulting from unilateral injection of 6.5 ng H127MO at the two-cell stage. Note the presence of a small lentoid body without obvious polarity and the absence of a clearly differentiated lens epithelium or lens fiber cells. Additionally, the neural retina has not differentiated the proper layers and the pigmented retinal epithelium is missing on the ventral portion of the eye. D: Corresponding uninjected eye of the embryo shown in C, showing normal development of both the lens and the retinal layers. bc, bipolar cell layer; dn, disorganized neural retina; gc, ganglion cell layer; ip, inner plexiform layer; lb, lentoid body; le, lens epithelium; lf, lens fiber cells; oc; outer cornea; op, outer plexiform layer; pc, photoreceptor cell layer. Scale bar in D = 100 μm.
	Figure 8. Immunohistochemical analysis of lens defects associated with H127MO (GPR84) knockdown. All specimens were fixed at stage 41 and stained with anti-lens crystallin polyclonal antibodies. A: DIC image showing severe eye defect and small lens after unilateral injection of 6.5 ng H127MO at the two-cell stage. This lens exhibits some polarization with a thickened epithelium and some primary fiber cells. Note also the disorganized neural retina, the lack of a defined pigmented retinal epithelium, and a thickened cornea epithelium. B: Corresponding anti-lens crystallin fluorescence image to that shown in A showing the presence of crystallin proteins (green) in primary fiber cells, but not the lens epithelium. C: Fluorescence image showing distribution of lissamine-tagged H127 morpholino throughout the tissues (red). D: DIC image showing representative rescue phenotype observed after co-injection of 6.5 ng H127MO and 1,200 pg altGPR84 mRNA into a single blastomere at the two-cell stage. The normal-appearing eyecup consists of distinct layers of differentiated retinal cells and the lens also has normal morphology. E: Corresponding anti-lens crystallin fluorescence image to that shown in D showing presence of crystallin proteins (green) in primary and secondary fiber cells. F: Fluorescence image showing distribution of lissamine-tagged H127 morpholino throughout the tissues (red). G: DIC image showing uninjected eye (opposite injected side of animal shown in A–C). A normal eye is seen with well-differentiated lens, and neural retina. Note also the normal thin appearance of the cornea epithelium. H: Corresponding anti-lens crystallin fluorescence image to that shown in G showing the presence of crystallin proteins (green) in primary and secondary fiber cells. I: Fluorescence image showing absence of lissamine-tagged red fluorescence in this uninjected side. dn, disorganized neural retina; le, lens epithelium; lf, lens fiber cells; oc, outer cornea; rp, retinal pigmented epithelium. Scale bar in I = 100 μm.
	Fig. 6. Summary of the effects of H127MO and altGPR84 mRNA injections on eye development. Shading represented in the key denotes categories of normal, minor and severe eye defect phenotypes (defined in the text). A: Uninjected embryos and control morpholino (CONMO) injected embryos typically exhibit normal development. B: Embryos injected with lissamine-tagged H127MO, show dose dependent phenotypes. Increasing severity of eye defects are seen with an increase in the amount of H127MO injected. C: Embryos co-injected with H127MO and altGPR84 mRNA exhibit a rescue phenotype with reduction in eye defects. D: Embryos injected with up to 1200pg altGPR84 mRNA alone typically exhibit normal development. Error bars indicate standard error.
	Figure 9. Effects of H127MO injections on cell proliferation in the retina and lens. A–D: Transverse sections of eyes from uninjected and H127MO-injected embryos showing corresponding pairs of DIC and fluorescence micrographs. A and B: Normal, control eye derived from the uninjected side of one example is displayed. C and D: Opposite, defective eye derived from the H127MO-injected side of the same embryo shown in A–B. E: Graphical depiction of the levels of cell proliferation in the retina and lens are shown. Bars represent the mean fraction of histone H3 S10P labeled cells, depicted as a percentage along the Y-axis. Different tissues and conditions are examined and depicted along the X-axis, as indicated. Error bars representing standard deviation are shown. Scale bar in D equals 50μm.
	Figure 10. Effects of H127MO injections on the levels of apoptosis (TUNEL assay). Dorsal is up for images A and B. A and B: Transverse sections of eyes from uninjected and H127MO-injected embryos. A: DIC image from a uninjected embryo with apoptotic cells indicated by the purple/blue colored NBT-BCIP precipitate. B: DIC image from an H127MO injected embryo. Note the increased level of apoptosis in the dorsal retina and lens. C: A graphical depiction of the levels of apoptosis in the retina and lens is displayed. Bars represent the mean fraction of apoptotic cells and are depicted as a percentage along the Y-axis. Different tissues and conditions are examined along the X-axis, as indicated. Error bars represent the standard deviation. Scale bar in B equals 50μm.
	Figure 11. Reciprocal presumptive lens ectoderm (PLE) transplants. See the Experimental Procedures section for details. PLE transplants were performed at stage 14. The arrowhead points to the eye. Note contralateral uninjected sides were completely normal (data not shown here). A: Uninjected host specimen with transplanted H127MO knockdown PLE that has normal morphology. B: Fluorescence image illustrating the lissamine-labeled H127MO PLE transplant in the uninjected host specimen. C: Whole eye section from an uninjected host animal with H127MO knockdown PLE transplant. Note the normal appearance of the lens with well-differentiated neural retina layers. D: H127MO injected host specimen with transplanted PLE obtained from an uninjected control embryo that shows defects in the ventral retina. E: Fluorescence image illustrating the lissamine-labeled H127MO larvae with the uninjected PLE transplant (not labeled red). F: Whole eye section from the H127MO host animal with PLE transplant obtained from an uninjected embryo. Note the presence of a smaller lens displaying a defect in fiber cell differentiation. The neural retina is also displays poorly differentiated retinal layers and has a thinned dorsal pigmented retinal epithelium. G–H. Reciprocal transplant results. G: Lens and retina phenotypes observed with H127MO (GPR84) knockdown presumptive lens ectoderm (PLE) donor and uninjected host specimens at stage 41. H: Lens and retina phenotypes observed with uninjected PLE donor and H127MO knockdown host specimens. Error bars indicate standard error. dn, disorganized neural retina; le, lens epithelium; lf, lens fiber cells; nr, neural retina; oc, outer cornea; rp, retinal pigmented epithelium. Scale bar in E is equal to 400μm and in F represents 100μm.

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