Genetic diagram of Bad-mediated radiosensitization of zebrafish neural tissue.

<p>IR activates the pro-apoptotic activity of Bad in a pathway that is either downstream of or parallel to p53 (dotted lines indicate that it is unclear whether this step occurs in a p53-dependent or –independent manner). Bad and Puma are dependent upon each other to promote IR-induced apoptosis. However, based on Bad’s established role as a sensitizer BH3-only protein and Puma’s reported role as an activator BH3-only protein, Bad likely functions upstream of Puma to induce IR-mediated apoptosis through the mitochondrial pathway.</p

Bad is required for IR-induced apoptosis in zebrafish embryonic neural tissue.

<p>(A) Shown are lateral views of 27-hpf embryos (head is top left in each panel) either uninjected or injected with 200 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mismatch (mm) MO. Half of each group of embryos were exposed to 15 Gy IR, and all were analyzed by the Casp3 assay. In control embryos (no inj and mm), IR-induced apoptosis occurs predominantly in the brain and all along the spinal cord (white arrowheads), whereas in <i>bad</i>-deficient embryos (ATG and e2i2), residual apoptosis is only observed in the head (arrowheads). (B) Fluorescence intensity, reflecting level of Caspase 3 activity, was measured in the spinal cords of at least 10 embryos from each group in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. The fluorescence intensity in irradiated mismatch-MO-injected embryos was normalized to 1. (C) One-cell stage zebrafish embryos were injected with 100 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mm MO as indicated (“++” indicates that 200 nmol was injected to keep total concentration of MO constant between experimental groups) and irradiated and analyzed as in (A-B). Data represent one experiment, and the experiment was independently performed three times with similar results.</p

Bad does not augment p53 transcriptional activity.

<p>p53 transcriptional activity was analyzed in embryos injected with 50<i>mcherry</i> (cntl) or <i>hBAD</i>. Embryos were exposed to 8 Gy (or not) at 24 hpf. RNA was harvested from each group at 27 hpf and analyzed for gene expression changes by qPCR. Expression of the <i>gapdh</i> gene was measured to normalize <i>puma</i> and <i>p21</i> mRNA levels. All data was compared to unirradiated wild-type control-mRNA-injected data, which was adjusted to a value of 1. Control-injected <i>p53</i> mutant embryos irradiated at 24 hpf and harvested at 27 hpf were included as a negative control for p53-mediated transcriptional induction. Data represent one experiment, but the experiment was independently performed three times with similar results.</p

p53 is required for Bad-mediated sensitivity to IR but not wortmannin.

<p>(A) Shown are lateral views of representative tails from 27-hpf wild-type or <i>p53</i> mutant embryos injected with 50 pg of <i>mcherry</i> (cntl) or <i>hBAD</i> mRNA. Embryos were exposed (or not) to 8 Gy IR at 24 hpf and analyzed three hours later by the Casp3 assay. Apoptosis was observed in the spinal cord after <i>hBAD</i> mRNA was injected into wild-type (arrowheads), but not mutant, <i>p53</i> embryos. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results. (C) One-cell stage wild-type or <i>p53</i> mutant embryos were injected with 50 pg of mRNA encoding either <i>mcherry</i> control (cntl) or the constitutively active mutant <i>zbad 2SA</i>. At 8 hpf, embryos were analyzed for survival (defined by a beating heart) as performed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. Data represent one experiment, but the experiment was independently performed three times with similar results. (D) One-cell stage wild-type embryos were injected with 50 pg of mRNA encoding either zebrafish <i>bad</i> or the apoptotically-inactive <i>zbad bh3 mut</i>. At 8 hpf, embryos were treated with increasing concentrations of wortmannin, or DMSO vehicle alone. At 48 hpf, embryos were examined for survival. At least 10 embryos were analyzed per group in three independent experiments. E) Shown are lateral views of tails from <i>p53</i> wild-type (left) or mutant (right) embryos after injection with 25 pg of mRNA encoding either <i>mcherry</i> (cntl), <i>zbad</i>, or <i>zbad bh3 mut</i>. Embryos were split into two groups and treated with either 0.3 µM wortmannin or DMSO vehicle beginning at 8 hpf and analyzed at 24 hpf by the Casp3 assay. Wild-type Bad synergizes with wortmannin to induce apoptosis in multiple tissues in a <i>p53</i>-independent manner (arrowheads).</p

Puma is required for Bad-mediated radiosensitization.

<p>(A) Shown are lateral views of representative tails from 27-hpf embryos exposed to 8 Gy IR after injection at the one-cell stage with 50 pg of mRNA encoding <i>mcherry</i> (cntl) or <i>hBAD</i> in addition to either 100 nmol of <i>puma</i> MO or mismatch (mm) control MO. Analysis of Caspase 3 activity shows that <i>hBAD</i>-mediated radiosensitivity (arrowheads) is dependent on <i>puma</i> expression. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results.</p

The <i>rs7</i> phenotype is caused by a mutation in the <i>ccdc94</i> gene.

<p>(A) The <i>rs7</i> mutation was localized to a 2.1 cM region (compared to the Massachusetts General Hospital genetic map for linkage group 2) as defined by flanking markers z58296 and z7358. (B) Sequencing of candidate genes (based on gene expression profiles) revealed a premature stop codon in the <i>ccdc94</i> gene. Sequence from the <i>ccdc94</i> gene exon 4 is shown for an <i>rs7</i> mutant, an unrelated wild-type AB embryo, and a heterozygous parent of the <i>rs7</i> mutant. Asterisk indicates the <i>rs7</i> point mutation, and box illustrates mutation to a TGA nonsense codon. (C) Wild-type AB strain or <i>rs7</i> heterozygous fish were incrossed, and the progeny were injected with mRNA encoding the indicated genes at the one-cell stage of development. At 24 hpf, embryos were exposed to 8 Gy IR and visualized by brightfield microscopy 6 hours later. (D) Embryos were injected similar to (C) but were not irradiated. Instead, they were left to develop until 48 hpf. (C and D) Because <i>rs7</i> mutant and sibling embryos injected with wild-type <i>ccdc94</i> mRNAs were morphologically indistinguishable, pictures were taken first, and the embryos were subsequently genotyped to identify mutants. (E) Wild-type embryos were injected with 400 µM mismatch morpholino (mm) as a negative control, or a translation-blocking <i>ccdc94</i> morpholino (<i>ccdc94 atg</i>). Embryos were then irradiated at 24 hpf with 8 Gy IR and analyzed three hours later by immunofluorescence to detect activated Caspase-3. (F) Embryos in (E) were quantified similar to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g001" target="_blank">Figure 1D</a>. Comparisons between mismatch morpholino-injected embryos plus and minus IR generated a <i>p</i> value of 0.0114. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s002" target="_blank">Figures S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s003" target="_blank">S3</a>.</p

Ccdc94 interacts with core members of the Prp19 complex.

<p>(A) TAP/MS analysis shows that human CCDC94 binds to core members of the PRP19 complex. “Total Peptide #” indicates the number of peptides present in the analysis that are derived from the indicated protein. “Coverage Percentage” refers to the percent of protein sequence for a given protein that is represented by the peptides. “Unique # Peptide” refers to the number of unique peptides found for the indicated protein. (B, C) Wild-type embryos were injected with the <i>ccdc94 atg</i> morpholino (400 µM), <i>prp19 e3i3</i> morpholino (200 µM), or mismatch morpholino (as a negative control and to keep the total concentration of morpholino injections at 600 µM) and grown to 22 hpf. A range of phenotypes were observed in the embryos as shown in (B). Embryos showing the different phenotypes from (B) were quantified in (C). (D) Wild-type embryos were injected with the <i>ccdc94 atg</i> morpholino (400 µM), <i>plrg1 atg</i> morpholino (20 µM), or mismatch morpholino (as a negative control and to keep the total concentration of morpholino injections at 420 µM), grown to 22 hpf, and analyzed as in (C). Data in (C) and (D) is derived from one representative experiment; however, three independent experiments were qualitatively similar for each panel. Number (n) of embryos analyzed for each group is indicated. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s007" target="_blank">Figure S7</a>.</p

Loss of <i>prp19</i> or <i>plrg1</i> phenocopies, loss of <i>ccdc94</i>.

<p>(A) Wild-type embryos were injected with the indicated morpholinos. RNA was harvested at 30 hpf, reverse transcribed using oligo-dT primers and analyzed for the expression of <i>p53</i> mRNA by qPCR. Expression of the <i>gapdh</i> gene was also analyzed to normalize <i>p53</i> mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (B) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the <i>p53</i> gene was analyzed by qPCR to determine levels of <i>p53</i> pre-mRNA. Expression of <i>28S</i> RNA was also analyzed to normalize <i>p53</i> pre-mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (C, D) Siblings and mutants from the <i>plrg1(hi3174aTg)</i> line were distinguished by morphology at 30 hpf and collected for analysis. RNA was harvested and analyzed by qPCR as in (A, B). For panels (A–D), error bars represent the standard error of the mean from at least three independent experiments. (E) Wild-type embryos were injected with the indicated morpholinos and analyzed at 30 hpf for p53 protein similar to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g003" target="_blank">Figure 3E</a>. (F) Wild-type embryos were injected with the indicated morpholinos, irradiated (or not) at 24 hpf with 8 Gy and analyzed three hours later by whole-mount immunofluorescence to detect activated Caspase-3. Three independent experiments showed that knockdown of <i>prp19</i> or <i>plrg1</i> radiosensitized the embryos. (G) Activated-Caspase-3-specific immunofluorescence from (F) was quantified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g001" target="_blank">Figure 1D</a>. (H) Genetic diagram showing that the Ccdc94 and core members of the Prp19 complex inhibit the transcription of <i>p53</i>, and therefore p53-mediated induction of <i>puma</i> expression, and normally restrict IR-induced mitochondrial apoptosis, upstream of Bcl-2. For all relevant panels in this figure, morpholinos were injected at the same concentrations as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g004" target="_blank">Figure 4B-4D</a>. RT; reverse transcriptase, mm; mismatch, <i>ccdc; ccdc94, prp; prp19</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s008" target="_blank">Figure S8</a>.</p

The <i>rs7</i>-mediated radiosensitizing phenotype is caused by an increase in <i>p53</i> mRNA expression.

<p>(A) Thirty hpf <i>rs7</i> siblings and mutants were identified based on morphology (similar to 1C). RNA was isolated, reverse transcribed using oligo-dT primers and analyzed for the expression of <i>p53</i> mRNA by qPCR. Expression of the <i>gapdh</i> gene was also analyzed to normalize <i>p53</i> mRNA levels. All data was then compared to sibling data, which was adjusted to a value of one. (B) <i>rs7</i> siblings and mutants were grown to 24 hpf and analyzed by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>p53</i> mRNA. High levels of <i>p53</i> mRNA expression are evident in neural tissue (arrowheads) and the ICM (arrows). Pictures were taken first, and the embryos were subsequently genotyped to identify mutants and heterozygous or wild-type siblings. (C) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the <i>p53</i> gene was analyzed by qPCR to determine levels of <i>p53</i> pre-mRNA. Expression of <i>28S</i> RNA was also analyzed to normalize <i>p53</i> pre-mRNA levels. Similar results were obtained from an analysis of intron 4 (data not shown). All data was then compared to sibling data, which was adjusted to a value of one. (D) <i>rs7^+/−;p53^e7/e7</i> fish were incrossed to analyze <i>rs7</i> siblings and mutants in a <i>p53</i> homozygous mutant background. <i>Rs7</i> mutants and siblings were distinguished by morphology since loss of p53 does not prevent the <i>rs7</i>-mediated “curly-up” tail phenotype. RNA was harvested at 30 hpf and analyzed as in (C). (E) Protein was harvested from <i>rs7</i> siblings and mutants, and p53 and control morphants (injected at 400 µM) at 30 hpf and analyzed for p53 and Tubulin (as a loading control). ImageJ software was used to quantify band intensity from film. Shown below the blot are values for p53 divided by values for Tubulin (corresponding to above lanes) with <i>rs7</i> siblings normalized to one. (F) <i>rs7</i> sibling or mutant embryos were irradiated at 24 hpf with 8 Gy IR and analyzed 2 h later by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>puma</i> mRNA. Arrowheads point to <i>puma</i> expression in neural tissue. (G) <i>rs7</i> sibling or mutant embryos in the <i>p53</i> wild-type or homozygous mutant background were irradiated at 24 hpf with 8 Gy IR and analyzed three hours later by immunofluorescence to detect activated Caspase-3. For panels (A), (C), and (D), error bars represent the standard error of the mean from at least three independent experiments. Panels (B) and (F–G) show representative data from at least three independent experiments. RT; reverse transcriptase. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s004" target="_blank">Figures S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s005" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s006" target="_blank">S6</a>.</p

Identification of <i>radiosensitizing mutation #7</i>.

<p>(A) Wild-type AB strain embryos were exposed to the indicated amount of IR at 24 hpf and visualized by brightfield microscopy 6 hours later. A threshold of IR exists between 8 and 15 Gy that gives rise to obvious cell death (seen as opaque tissue, marked by arrowheads) in the brain. (B) Wild-type embryos (derived from crossing wild-type parents) or <i>rs7</i> mutant embryos (derived from crossing <i>rs7</i> heterozygous parents) were exposed to 8 Gy IR at 24 hpf and visualized by brightfield microscopy 6 hours later. Arrowheads in the <i>rs7</i> mutant mark cell death reminiscent of exposure of wild-type embryos to 15 Gy (as shown in A) (C) Embryos in (B) were sorted by phenotype, fixed at 6 hpIR and analyzed by immunofluorescence to detect activated Caspase-3. The “curly-up” tail phenotype and opaque tissue in the head were used to identify <i>rs7</i> mutants. These phenotypes are present (to different degrees of severity) in both unirradiated and irradiated mutants such that they can be readily distinguished from siblings and wild-type at 30 hpf. After fixation of mutants, tails were clipped to distinguish them from wild-type embryos, which were analyzed in the same tube for Caspase-3 activity. Embryos were grouped for analysis according to whether embryos were irradiated or not, and <i>rs7</i> mutants were identified based on the presence of tail-clips. Arrowheads point to the enhanced apoptosis in the spinal cord of an <i>rs7</i> mutant, and arrows point to increased apoptosis in the ICM region of an <i>rs7</i> mutant. (D) Immunofluorescence in the spinal cords from at least 10 embryos per group in (C) was quantified. wt; wild-type. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s001" target="_blank">Figure S1</a>.</p