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Figure 1 – Ten of the genes identified as exhibiting rax-dependent expression are expressed in the developing eye. A – J: Xenopus laevis embryos were fixed and subjected to in situ hybridization using antisense riboprobes specific for 10 of the genes identified as exhibiting rax-dependent expression in the microarray screen described in the text. Genes/riboprobes are identified to the left of each row. Embryos used were late neurula (st 18 – 20, column 1), with the exception of J1 (st 22), early tailbud (st 24 and 25, column 2), mid tailbud (st 25 – 31, column 3), and mid-late tailbud (st 30 – 33, column 4). Lateral views are shown for all embryos except for A1, B1, C1, D1, G1, H1, I1, where anterior views are shown. In all cases, dorsal is towards the top of the figure. Abbreviations and marks: arrowhead – eye; * - brain; B – branchial arches; P – pronephros.
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Figure 2 – Six of the genes identified in the microarray screen are expressed in the ciliary marginal zone. Fixed X. laevis tadpoles (st 41) were paraffinized, sectioned and subjected to in situ hybridization using antisense riboprobes for the 10 genes expressed in the eye (see Figure 1). Genes/riboprobes are identified in the lower left of each panel. Sections are presented with dorsal side towards the top of the figure. The dorsal CMZ is indicated with an oval. Key: green – positive expression in CMZ; red – negative expression in CMZ; yellow – positive expression in CMZ and ubiquitous expression in retina. L – lens.
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Figure 3 – Four of the genes identified in the microarray screen can significantly rescue the rax knockdown phenotype. X. laevis embryos were injected with rax antisense morpholino oligonucleotides (ASMO) with or without mRNA encoding each of the 6 microarray screen genes expressed in the CMZ (Figure 2). RNAs and lissamine-tagged MOs were injected into a dorsal blastomere at the 4-cell stage and embryos were screened for one-sided injection and normal, small, or absent eyes at st 41. A. Comparison of frequency of normal (black bar portion), small (gray bar portion), and absent (white bar portion) eye phenotypes in embryos injected with rax ASMO with or without RNAs encoding microarray genes (marked ―+‖ or ―-―, respectively, and identified at bottom of graph). Statistical significance of rescue was determined (student’s t-test performed using frequency of normal eyes in RNA injected vs uninjected samples); p-values are shown at the bottom of the graph. N.S. = not significant (p>0.05). Number of embryos for each condition (n) is shown below each column. B. Sampling of embryos injected with rax ASMO with or without neil3 RNA (right and left panels, respectively). Embryos were injected with lissamine-tagged rax ASMO with or without neil3 RNA. Shown are injected sides of st 41 embryos scored as having normal (N), small (S), or an absent (A) eye.
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Figure 4 – neil3 and rax are in a common genetic pathway. A. neil3 is expressed in animal caps prepared from rax-injected embryos in a dose-dependent manner. X. laevis embryos were injected with rax RNA (amount shown in graph). Animal caps were explanted when embryos reached early blastula stage and cultured until early gastrula stage. RNA was extracted and neil3 expression was determined by quantitative RT-PCR. Results are normalized to an internal control and then to the neil3 expression level in caps prepared from uninjected embryos. B. The neil3 antisense morpholino oligonucleotide (ASMO) can inhibit expression of a neil3-gfp reporter. The reporter contains a portion of neil3 5’-UTR and coding region, including the ASMO target, fused in-frame with the GFP coding region downstream of the CMV promoter. The reporter was injected alone (top row, ia-c) or with lissamine-tagged COMO (middle row, iia-c) or lissamine-tagged neil3 ASMO (bottom row, iiia-c). Embryos were imaged under bright field (left column, ia-iiia), red fluorescence to detect the lissamine-tagged MOs (middle column, ib-iiib), or green fluorescence to detect GFP (right column, iiia-iiic). C. Injection of a neil3 ASMO results in embryos with small or no eyes. X. laevis embryos were injected unilaterally (one dorsal blastomere at the 4-cell stage) with neil3 ASMO. Left panel: normal eye development on uninjected side. Right panel: small (S) or absent (A) eyes on the injected side. D. Co-injection of neil3 RNA partially rescues knockdown phenotype. X. laevis embryos were injected with neil3 ASMO, RNA, or a mixture of both. Embryos were cultured to st 41 and scored for eye phenotype on the injected side. E. Co-injection of neil3 and rax ASMOs results in a more severe phenotype than injection of either ASMO alone. X. laevis embryos were injected with a low dose of either rax or neil3 ASMO alone or a mixture of both. Embryos were cultured to st 41 and scored for eye phenotype on the injected side. Number of embryos for each condition (n) is shown below each column in D and E.
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Figure 5 – neil3 knockdown embryos exhibit deficits in retinal development. A – C. Knockdown of neil3 results in a disorganized retina. Histological staining of retinas from uninjected (A) or injected (B,C) sides of embryos injected with neil3 antisense morpholino oligonucleotide (ASMO), sectioned, and stained with hemotoxylin/eosin. D – F. Knockdown of neil3 results in aberrant retinal cell differentiation. Embryos injected with neil3 ASMO were fixed and subjected to immunohistochemistry using antibodies against Islet-1 (D,E) or rhodopsin (F,G). Panels D and F show uninjected side and E and G show injected sides. R – retinal pigmented epithelium (RPE); P – photoreceptor layer; I – inner nuclear layer (INL), G – ganglion cell layer; L – lens. Arrows indicate examples of putative photoreceptor rosettes.
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Figure 6 – Molecular markers of retinal progenitor cells (RPCs) are expressed abnormally in neil3 knockdown embryos. Embryos were injected with neil3 antisense morpholino oligonucleotides (one dorsal blastomere at 4-cell stage), cultured, and subjected to wholemount (A-F) or section (G-I) in situ hybridization using the indicated riboprobes. A-D. Early tailbud stage embryos stained for rax (A,B) or pax6 (C,D). Embryos or oriented with dorsal towards the top of the panel and anterior facing the viewer. Eye on uninjected side is indicated with an asterisk (*). E,F. Early tailbud embryos stained for neurod1 expression. Uninjected side is shown in E and injected side is shown in F. G-K. St 38 embryos stained for rax expression (G-I) or BrdU incorporation (J,K). Examples of eyes from uninjected (G,J) or uninjected (H,I,K) embryo sides are shown. Abbreviations: C – ciliary marginal zone; CG – cranial ganglia; E – eye; G – ganglion cell layer; I – inner nuclear layer; L – lens; P – photoreceptor layer; R – retinal pigmented epithelium.
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Figure 7 – Stereotypical peptide domains are required for neil3 function. A. Diagram of neil3 protein highlighting stereotypical peptide domains: NEI domain (blue), RanBP-type zinc finger (yellow), and GRF-type zinc fingers (red). Wild type (top line) and mutated versions are depicted. GRF: both GRF zinc fingers deleted; GRF2: second GRF zinc finger deleted; RBP: RanBP zinc finger deleted; D166N: point mutation resulting in aspartate 166 substitution by asparagine. B,C. RNA encoding neil3 containing the D166N mutation (B) or deletions of RanBP- or GRF-type zinc fingers (C) does not rescue the rax knockdown phenotype to the same extent as wild type neil3. Open/white portion of graph bar: normal eye; gray: microphthalmia; black: anophthalmia. X. laevis embryos were injected with rax antisense morpholino oligonucleotides (ASMO) alone or with mRNA encoding wild type or nutated neil3. RNAs and lissamine-tagged MOs were injected into a dorsal blastomere at the 4-cell stage and embryos were screened for one-sided injection and normal, small, or absent eyes at st 41. Number of embryos for each condition (n) is shown below each column in B and C.
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abcf2 (ATP binding cassette subfamily F member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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bnip3 (BCL2/adenovirus E1B 19kDa interacting protein 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
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cbx5 (chromobox 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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neil3 (nei-like DNA glycosylase 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
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e2f7 (E2F transcription factor 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior left, dorsal up.
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fam50a (family with sequence similarity 50 member A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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LOC100498624 (uncharacterized LOC100498624) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
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