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Fig 1. Expression of nol11 during vertebrate development.
A) Wild type nol11 expression pattern during Xenopus tropicalis development. Note the strong expression in developing neural folds (NF) and the presumptive CNC at stages 16 and 22 (lateral [left] and dorsal [right] views presented). Expression is strongly associated with the migrating and differentiating CNC at subsequent stages, and is also detected in the region of the ventral blood islands (BI) and isthmus (Is) at stage 28. BA, branchial arch; Ht, heart; Lv, liver region; Op, optic placode. B) and C) Anterior transverse dissections showing expression of nol11 in neural folds and premigratory CNC of stage 14 and 16 embryos respectively. Plane of dissection is represented by the red dotted line marked c in A. D) Horizontal dissection (shown as dotted red line marked d in A) of nol11 expression in the branchial arches of stage 28 Xenopus embryos. E) nol11 expression in E8.5, E9.5 and E10.5 wild type mouse embryos. At E8.5 expression is strongly detected in the neural folds. Transcripts are associated with CNC positive regions at both E9.5 and E10.5. BA2, 2nd branchial arch; FNP, frontonasal prominence; Ht, heart; mdBA1, mandibular BA1; mxBA1, maxillary BA1; Op, optic placode; Ot, otic placode; T, trigeminal region.
doi:10.1371/journal.pgen.1005018.g001
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Fig 2. The nol11 craniofacial phenotype.
A) Gross morphology and cartilage staining of UC, nol11 whole embryo, nol11 one-sided knockdowns and CMO one-sided knockdown embryos. Note the reduced cartilage size and abnormal morphology in nol11 morphants (red arrowheads) while CMO injected embryos are unaffected. B) Craniofacial cartilage size is significantly reduced in nol11 but not CMO morphants. C) Co-injection of human NOL11 RNA can rescue the cartilage phenotype in approximately 75% of treated embryos. Cartilage staining of an RNA rescued embryo; nol11 MO was injected at the one cell stage and human NOL11 RNA was injected into one cell at the two cell stage. Green arrowheads highlight rescued side.
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Fig 3. Knockdown of nol11 disrupts cranial neural crest development.
At stages 14 and 24 neural and CNC development appears normal in one side treated embryos, as assayed by expression of key marker genes including sox3, cytokeratin, twist and slug. By stage 28 however, reductions are apparent in the expression of numerous CNC genes. The branchial arches are also smaller on the MO treated side at stage 28. Graph displays number of embryos exhibiting abnormal gene expression in control and nol11 morphant embryos.
doi:10.1371/journal.pgen.1005018.g003
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Fig 4. Increased apoptosis underlies the nol11 cartilage defects.
A) nol11 knockdown results in a progressive increase in apoptosis. At stage 14 no significant difference was observed in rates of TUNEL staining between knockdown and control halves of the embryo. At stages 18 and 28 increased apoptosis was evident on the treated side of whole mount and sectioned paraffin embedded embryos. Note that this increased apoptosis occurs primarily within the craniofacial ectomesenchyme. The graph represents the relative quantification of apoptosis rates at stages 14, 18 and 28. This stage specific increase in apoptosis was confirmed by a similar increase in p53 protein levels in 1 cell injected embryos as assayed by western blot (lower right panel). Dotted red lines mark the embryonic midline. B) No significant change in proliferation rates was noted following nol11 knockdown. C) Inhibition of apoptosis by p53 MO results in a partial rescue of cartilage size and morphology. Each pair of columns in the graph compares cartilage size measured in bilateral halves of embryos. The blue pair reveals no significant difference in cartilage measurements in the left vs right side of the UC embryonic head. In the second pair (red), cartilage size is seen to be comparable on either side of the nol11 morphant head. The final pair illustrates that cartilage size is significantly improved on the side of nol11 morphants rescued with p53 MO (green) relative to the side that received nol11 MO only (red). D) Western blot demonstrating that the p53 MO efficiently reduces p53 protein levels in nol11 morphants.
doi:10.1371/journal.pgen.1005018.g004
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Fig 5. Nol11 depletion impairs rDNA transcription and pre-rRNA processing in X. tropicalis.
A) Scheme of pre-rRNA processing pathways in X tropicalis. The pre-rRNA is transcribed by RNAPI as a 40S polycistronic precursor. Several cleavages are required to separate the mature rRNAs. The locations of oligonucleotide probes used for northern blots are indicated by lettered lines (a, c) and the cleavage sites indicated. This scheme was adapted from [71–75]. B) Morpholino (MO) depletion of Nol11 impairs pre-rRNA transcription at stage 28. The northern blot was hybridized with probe a (Fig. 5A) and with a probe to the 7SL RNA as a loading control (lower panel). Bands were quantified and analysed by RAMP ([60]; S6A,B Fig) C) Morpholino (MO) depletion of Nol11 impairs pre-rRNA transcription and processing. The northern blot was hybridized with probe c (Fig. 5A) and with a probe to the 7SL RNA as a loading control (lower panel). Bands were quantified and analysed by RAMP ([60]; S6C,D, E, F Fig). D) Depletion of Nol11 leads to increased p53 levels. The expression of p53 from control and nol11 depleted embryos was analysed by western blot with anti-p53 antibodies. GAPDH levels were used as a loading control. Values for p53 expression normalized to GAPDH are represented in the bar graph. E) MO-resistant human NOL11 (hNOL11) mRNA but not p53 depletion rescues pre-rRNA levels. Embryos injected as shown by + and—in the figure at stages 22 and 28. The pre-rRNAs were visualized with probe a on a northern blot; hybridization to the 7SL RNA was used as a loading control.
doi:10.1371/journal.pgen.1005018.g005
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S1 Fig. nol11 in vertebrate development.
A) Whole mount in situ hybridization of digoxigenin labelled Nol11 probe in E9.5 and E10.5 mouse embryos (E10.5 sample shown has been hemisected). BA, branchial arch; FNP, frontonasal prominence; Ht, heart; mdBA1, mandibular BA1; mxBA1, maxillary BA1; Op, optic placode; Ot, otic placode. B) Gross morphology of stage 45 nol11 morphants. Gut morphology is abnormal in morphants, while organ situs appears largely normal relative to wild type controls. Heart (red), gall bladder (green) and gut (yellow) are pseudocoloured in lower right panels. C) Left sided expression nkx2.5 is reduced or absent in the splenic anlage of a subset of nol11 morphants (compare red arrowheads). D) Example of the normal sided pitx2 expression present in nol11 knocked down embryos. E) Kidney development appears largely intact in nol11 MO treated side compared to control side. F) Quantification of number of embryos displaying the described phenotypes.
doi:10.1371/journal.pgen.1005018.s001
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S2 Fig. Efficacy of the nol11 MO.
A) Western blot demonstrating that injection of nol11 MO at the one cell stage reduces Nol11 protein levels at stage 28 in a dose dependent manner. C, control, 2, 2ng nol11 MO, 3, 3ng nol11 MO. B) Injection of 2ng of nol11 MO at the one cell stage results in a 30% reduction of protein level at stage 15 and a 44% reduction at stage 23.
doi:10.1371/journal.pgen.1005018.s002
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S3 Fig. Patterning and Apoptosis in nol11 morphants.
A) Somite, CNC and neural development appear intact in nol11 morphants at stage 14 as assayed by myoD, ap2 and pax2 expression. Expression of xnot appears reduced at stage 28. B) Reduced dlx5 expression and BA hypoplasia on treated side of a stage 28 embryo. Neural patterning is normal at this stage as assayed by expression of pax2, hoxb3 and sox3. C) Whole mount TUNEL staining of treated and untreated sides of a stage 28 embryo. Note the increased staining in the craniofacial regions of the morphant side.
doi:10.1371/journal.pgen.1005018.s003
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S4 Fig. Rates of cell death in the branchial arches.
A) Graph of the number of Tunel positive cells per 100 cells present in sections of the branchial arch region of stage 28 UC, CMO, Nol11 MO, Nol11 MO + p53 MO, and p53 MO only injected embryos (* = P < 0.05). B) Whole mount stage 28 embryo with plane of section represented by the red dotted line. Representative Tunel stained (green) sections from each control and morphants.
doi:10.1371/journal.pgen.1005018.s004
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S5 Fig. The X. tropicalis 5â external transcribed spacer.
The 5âETS sequences for X. laevis (GeneBank ID: X02995.1; nucleotides 318â1029) and X. borealis (GeneBank ID: X00184.1; nucleotides 545â1156) were aligned to X. tropicalis scaffolds 169 and 2385 from the Xentr7.1 database.
doi:10.1371/journal.pgen.1005018.s005
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S6 Fig. Statistical analysis of pre-rRNA levels by Ratio Analysis of Multiple Precursors (RAMP) [60].
RNAs were quantitated from northern blots using a phosphorimager (Bio-Rad Personal Molecular Imager). All ratios are representative of three biological replicates (n = 3). The levels of all RNAs were normalized to the uninjected control (UC). All statistical analyses for significance for Nol11-depleted embryos (NOL11) were performed compared to the control morpholino injected embryos (CMO). A. Pre-rRNA levels in Nol11-depleted embryos are not significantly affected at stage 12 compared to CMO as shown by a probe in the 5’ETS (probe a). B. At stage 28, the levels of 40S and 20S pre-rRNAs, are significantly decreased relative to the loading control 7SL RNA for Nol11-depleted embryos compared to CMO as shown by probe a. This is consistent with decreased pre-rRNA transcription. C. Pre-rRNA levels relative to the 7SL RNA for Nol11-depleted embryos are not significantly affected compared to CMO at stage 12 as shown by a probe in the ITS1 (probe c). D. At stage 28 for Nol11-depleted embryos, the 40S, 36S, and 20S pre-rRNAs are all significantly decreased relative to the 7SL RNA compared to CMO as shown by probe c. This is consistent with decreased pre-rRNA transcription. However, the levels of 19S are significantly increased relative to the 7SL RNA compared to CMO as this precursor accumulates in Nol11-depleted embryos. This is indicative of a pre-rRNA processing defect. E. and F. For both stage 12 and 28 Nol11-depleted embryos, the ratios of 19S/40S, 19S/36S, and 19S/20S are significantly increased compared to CMO indicating accumulation of the 19S pre-rRNA relative to the other three pre-rRNAs that hybridize with probe c. This is indicative of a pre-rRNA processing defect. [ns = p>0.05 (not significant), * = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001]
doi:10.1371/journal.pgen.1005018.s006
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nol11 (nucleolar protein 11) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior left.
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nol11 (nucleolar protein 11) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
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nol11 (nucleolar protein 11) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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nol11 (nucleolar protein 11) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.
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