Additional file 5: of Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis

Figure S4. Efficient ablation of the Gem locus when combined with the inducible Sox10iCreER T2 line. Whole-mount gut preparations of control (A) and Sox10CreER(i8.5)|Gem (B) E12.5 embryos, immunostained for GFP to visualise the distribution of ENCCs within the gut. Red arrows indicate the position of the most caudally located ENCCs in the gut preparations. (C–D), Relative quantitation of Gem transcript levels in the FACS-purified ENCCs of Sox10CreER(i8.5)|Gem and Sox10CreER(i10)|Gem embryos normalised to the levels of b-actin. Unpaired t-test with Welch’s correction, **P value < 0.01, ***P value < 0.001. Scale bar: (A, B) 400 μm. (TIF 1130 kb

Using <i>ret</i>^{<i>hu2846</i>/+} as sensitized background to test role of <i>mapk10</i> as ENS development modifier gene.

<p>(A) RNA <i>in situ</i> hybridization shows <i>mapk10</i> expression correlates with location of the ENS in WT (<i>ret</i>^+/+), <i>ret</i>^{<i>hu2846</i>/+}, and <i>ret</i>^{<i>hu2846</i>/hu2846} 3dpf larvae (asterisks indicate end of gut, filled arrows indicate <i>mapk10</i>⁺ cells, and open arrows denote gut areas lacking <i>mapk10</i>⁺ cells). (B) MO gene knock-down of <i>mapk10</i> (using a splice blocking MO, <i>mapk10</i>MO) performed on embryos from WT x <i>ret</i>^{<i>hu2846</i>/+} cross to allow knock-down in both WT and <i>ret</i>^{<i>hu2846</i>/+} embryos, and compared to a control MO. Immunostaining with HuC/D at 4dpf shows that most severe phenotypes are observed when <i>mapk10</i>MO is injected into <i>ret</i>^{<i>hu2846</i>/+} embryos. Asterisks indicate end of gut tube (anal pore), and arrows denote position of last HuC/D⁺ neuron. (C) Quantification of neuron number in the last 5 somite lengths shows that injection of <i>mapk10</i>MO into WT embryos causes a modest, statistically significant reduction of enteric neurons in the gut relative to WT larvae injected with control MO (one-way ANOVA, p<0.001; Bonferroni’s post-hoc test, p = 0.0033), revealing a role for <i>mapk10</i> in normal ENS development. Injection of <i>mapk10</i>MO into <i>ret</i>^{<i>hu2846</i>/+} embryos causes severe reduction of enteric neurons, which is statistically different from either <i>ret</i>^{<i>hu2846</i>/+} injected with control MO (one-way ANOVA, p<0.001, Bonferroni’s post-hoc test, <i>P</i><0.0001) or <i>mapk10</i>MO injected into WT embryos (one-way ANOVA p<0.001, Bonferroni’s post-hoc test p = 0.006).</p

<i>ret</i>^{<i>hu2846</i>/+} larvae exhibit ENS progenitor migration deficits.

<p> ENS progenitor migration at the front of migration (wavefront) visualized in real time by virtue of the SAGFF234A;UAS:GFP background labeling ENCCs. (A) Representative still images from confocal time-lapse recordings (depth-coded to resolve 2 migratory streams) of WT (<i>ret</i>^+/+) and <i>ret</i>^{<i>hu2846</i>/+} embryos at 0 minutes (48hpf) and 500 minutes (56hpf), show that distance travelled by the migration wavefront is decreased in <i>ret</i>^{<i>hu2846</i>/+} larvae relative to WT larvae. Asterisk denotes a GFP+ cell from outside the ENS linage that remains in the same position. (B) The calculated wavefront migration speed (microns/minute) is significantly reduced in <i>ret</i>^{<i>hu2846</i>/+} larvae relative to WT at 48–56hpf (p = 0.0022), and 72–80hpf (p = 0.0182).</p

A Novel Zebrafish <i>ret</i> Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for <i>mapk10</i> as a Modifier of Enteric Nervous System Phenotype Severity

<div><p>Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and <i>in vivo</i> physiological studies. We show that <i>ret</i> mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in <i>ret</i> heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in <i>ret</i> mutants, and partially impaired in <i>ret</i> heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant <i>ret</i> heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated <i>mapk10</i> loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of <i>mapk10</i> mutations into <i>ret</i> heterozygotes enhanced the ENS deficit, supporting <i>MAPK10</i> as a HSCR susceptibility locus. Our studies demonstrate that <i>ret</i> heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.</p></div

Loss of <i>ret</i> leads to dose-dependent reduction of ENS progenitors and neurons.

<p>(A-B) WT (<i>ret</i>^+/+), <i>ret</i>^{<i>hu2846</i>/+}, and <i>ret</i>^{<i>hu2846/2846</i>} larvae immunostained at 4dpf with HuC/D antibody to visualize enteric neurons (A) and processed at 3dpf by RNA <i>in situ</i> hybridization to detect <i>nadl1</i>.<i>2</i> expressing ENCCs (B). Asterisks indicate end of gut tube (anal pore), filled arrows and arrowheads denote position of last HuC/D⁺ neuron or <i>nadl1</i>.<i>2</i>⁺ ENCCs, and open arrows denote gut areas lacking <i>nadl1</i>.<i>2</i>⁺ ENCCs. (C) Number of HuC/D⁺ neurons in the distal gut was significantly reduced relative to WT counterparts, at 3dpf (WT: 47±3, <i>ret</i>^{<i>hu2846</i>/+}: 29±8, p = 0.0281) and 5dpf (WT: 137±7, <i>ret</i>^{<i>hu2846</i>/+}: 70±14, p = 0.0009). However, <i>ret</i>^{<i>hu2846</i>/+} larvae had a phenotypic range: some <i>ret</i>^{<i>hu2846</i>/+} larvae showed neuron numbers equivalent to WT, and other <i>ret</i>^{<i>hu2846</i>/+} larvae showed comparably fewer neurons than WT. Position of the 10^th most distal enteric neuron was also significantly altered in <i>ret</i>^{<i>hu2846</i>/+} larvae. Y-axis indicates somite lengths from end of the gut (*) (in <i>ret</i>^{<i>hu2846</i>/+} larvae 4.6±0.8 somite lengths from the end of the gut vs. 2.3±0.3 in WT, p = 0.012). Again, <i>ret</i>^{<i>hu2846</i>/+} larvae display a phenotypic range.</p

<i>mapk10</i> as candidate to account for varying expressivity in HSCR.

<p> (A) Enteric neuron number in the distal gut quantified in 4dpf larvae resulting from crosses between <i>mapk10</i>^Δ10/+ and <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+ zebrafish. No difference was detected between WT and heterozygous <i>mapk10</i>^Δ10 larvae, but <i>mapk10</i>^Δ10/Δ10 larvae have a small, but statistically significant reduction in ENS cell number in the gut relative to WT (one-way ANOVA, p = 0.0442, Tukey post-hoc), indicating a role for <i>mapk10</i> in normal ENS development. No significant difference was detected between <i>ret</i>^{<i>hu2846</i>/+} and <i>mapk10</i>^Δ10/+;<i>ret</i>^{<i>hu2846</i>/+} larvae, however, loss of <i>mapk10</i> in the <i>ret</i>^{<i>hu2846</i>/+} background leads to statistically significant reduction in neuron number (Welch’s one-way ANOVA, <i>p</i> = 0.048). (B) To examine phenotype distribution, individual zebrafish were binned according to neuron number in the distal gut. WT, <i>mapk10</i>^Δ10/+ and <i>mapk10</i>^Δ10/Δ10 larvae show normal distribution of phenotypes (blue, red, and green bars, respectively, Shapiro-Wilk normality test, for WT: p = 0.776, for <i>mapk10</i>^Δ10/+: p = 0.910, and for <i>mapk10</i>^Δ10/Δ10: p = 0.1149). Although <i>ret</i>^{<i>hu2846</i>/+} larvae guts are phenotypic, they display a range of neuron numbers in the distal gut region, reflecting normal distribution of mild and progressively more severe colonization phenotypes (Shapiro-Wilk normality test, p = 0.5720, blue bars), counts of neuron number in <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+ (red bars) and <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 larval guts (green bars) exhibit a non-normal distribution pattern (Shapiro-Wilk normality test, for <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/+: p = 0.0054, and for <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 p = 0.0014), and counts of enteric neuron number in <i>ret</i>^{<i>hu2846</i>/+};<i>mapk10</i>^Δ10/Δ10 larvae, showed a statistically significant increase in standard deviation compared with <i>ret</i>^{<i>hu2846</i>/+} larvae (Brown-Forsythe, 0.0445).</p

Intestinal motility is impaired in <i>ret</i>^{<i>hu2846</i>/+} and <i>ret</i>^{<i>hu2846/2846</i>} larvae.

<p>(A) Spatiotemporal maps (STMs) generated to quantify 400 second recordings of gut motility in live 7dpf zebrafish larvae. WT (<i>ret</i>^+/+) larvae exhibit cyclical retrograde (anal to oral) motility patterns in the intestinal bulb (brackets), and anterograde (oral to anal) peristaltic waves in the intestine starting at the intestinal bulb/intestine junction (white arrows); black arrows denote motility end points. STMs of <i>ret</i>^{<i>hu2846</i>/+} larvae with ENS phenotypes and <i>ret</i>^{<i>hu2846/2846</i>} larvae show alterations in cyclical retrograde motility patterns (brackets) and frequency and distance of anterograde motility waves (white to black arrows). (B) Anterograde contraction characterization. Phenotypic (<i>ret</i>^{<i>hu2846</i>/+}P) and non-phenotypic (<i>ret</i>^{<i>hu2846</i>/+}NP) larvae identified immunohistochemically. Reduced peristaltic frequency in <i>ret</i>^{<i>hu2846/2846</i>} larvae and <i>ret</i>^{<i>hu2846</i>/+} P relative to WT (one-way ANOVA, p = 0.0004; Bonferroni’s post-hoc, p = 0.0001 and p = 0.0367), and also reduced in <i>ret</i>^{<i>hu2846/2846</i>} larvae as compared to <i>ret</i>^{<i>hu2846</i>/+} NP (p = 0.0002) and <i>ret</i>^{<i>hu2846</i>/+} P (p = 0.0035). Reduced contraction travel distance in <i>ret</i>^{<i>hu2846/2846</i>} larvae relative to WT (one-way ANOVA, p = 0.0005; Bonferroni’s post-hoc, p = 0.0002), and relative to both <i>ret</i>^{<i>hu2846</i>/+} P (p = 0.0232) and <i>ret</i>^{<i>hu2846</i>/+} NP (p = 0.0237). Reduced contraction velocity in <i>ret</i>^{<i>hu2846/2846</i>} relative to WT (one-way ANOVA, p = 0.0212; Bonferroni’s post-hoc, p = 0.0138). Increased contractions interval in <i>ret</i>^{<i>hu2846/2846</i>} larvae relative to WT and <i>ret</i>^{<i>hu2846</i>/+} NP (one-way ANOVA, p = 0.0078; Bonferroni’s post-hoc, p = 0.0053 and p = 0.0097). (C) Loss of anterograde contractions in <i>ret</i>^{<i>hu2846/hu2846</i>} larvae compared to WT and <i>ret</i>^{<i>hu2846</i>/+} NP (#, Fisher’s exact, p = 0.0198 and p = 0.028). <i>ret</i>^{<i>hu2846/2846</i>} larvae show retrograde motility changes relative to all other genotypes, with contractions losing cyclic patterns (Fisher’s exact, vs. WT: p = 0.0001, vs. <i>ret</i>^{<i>hu2846</i>/+} NP: p = 0.001, and vs. <i>ret</i>^{<i>hu2846</i>/+} P: p = 0.0082). (D) Equivalent analysis in the SAGFF234A;UAS:GFP background, expressing GFP in ENS cells and their processes, enables mapping of contraction endpoints relative to cell body position (GFP⁺HuC/D⁺ cells, r_s = 0.8857, p = 0.0333) and cell process (GFP⁺HuC/D⁺ process, r_s = 0.942, p = 0.0167). Black lines indicate linear fits.</p

Expression of <i>Ret</i> and its signalling partners in adult thymic populations.

<p><b>A.</b> DN, DP, SPCD8⁺ and SPCD4⁺ thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to <i>Hprt1</i>. Error bars show s.e.. Results from three independent measurements are represented. <b>B.</b> RET expression in DN and DP thymocytes was determined by flow cytometry. RET: black bold line; Isotype control: grey line. <b>C.</b> Thymic DN, DP, SPCD8⁺ and SPCD4⁺ thymocytes and CD45⁻ cells were purified by flow cytometry. Quantitative RT-PCR analysis was normalized to <i>Hprt1</i>. Error bars show s.e.. Results from three independent measurements are represented. <b>D.</b> DN1–2 and DN3–4 thymocytes were purified by flow cytometry. Results show quantitative RT-PCR normalized to <i>Hprt1</i>. Error bars show s.e.. Results from three independent measurements are represented.</p

Impact of <i>Ret</i>, <i>Gfra1</i> or <i>Gfra2</i> ablation in embryonic thymocyte development.

<p>E18.5 thymocytes were analyzed by flow cytometry. <b>A.</b> DN thymocytes were gated on CD45⁺Lin⁻CD3⁻CD4⁻CD8⁻ cells. Results show percentage of DN1–DN4 in <i>Ret</i>, <i>Gfra1</i>and <i>Gfra2</i> deficient mice. Null mice: open symbols; WT littermate controls: full symbols; Mean value: dash line. <b>B.</b> Percentage of DN and DP thymocytes gated on CD45⁺Lin⁻γδTCR⁻ analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052949#pone-0052949-g002" target="_blank">Figure 2A</a>. <b>C.</b> Percentage of γδ TCR expressing thymocytes analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052949#pone-0052949-g002" target="_blank">Figure 2A</a>. <b>D.</b> Absolute number of total thymocytes in <i>Ret</i>, <i>Gfra1</i>and <i>Gfra2</i> deficient mice analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052949#pone-0052949-g002" target="_blank">Figure 2A</a>. Two-tailed student <i>t</i>-test analysis was performed between knockouts and respective WT littermate controls. No statistically significant differences were found.</p

<i>Ret</i>^MEN2B gain-of-function mutation in adult thymocyte development.

<p>8 week old <i>Ret</i>^MEN2B/MEN2B (<i>MEN2B</i>) and their WT littermate controls were analyzed by flow cytometry. <b>A.</b> Left: representative flow cytometry analysis of CD4⁻CD8⁻CD3⁻ thymocytes. Percentages are indicated. Right: Results show percentage of DN1–DN4 in <i>MEN2B</i> (open squares) and WT control (full circle) mice. Mean value: dash line. <b>B.</b> Left: representative flow cytromety analysis of CD4 versus CD8 expression profile. Percentages are indicated. Right: Results show percentage of DN, DP, SP4 and SP4 in in <i>MEN2B</i> (open squares) and WT control (full circle) mice. Mean value: dash line. <b>C.</b> Proportion and absolute numbers of γδ TCR expressing thymocytes in <i>MEN2B</i> (open squares) and WT control (full circle) mice. Mean value: dash line. <b>D.</b> Absolute thymocyte numbers. Two-tailed student <i>t</i>-test analysis was performed between knockouts and respective controls. No statistically significant differences were found.</p