Fig. 4

Using ret^hu2846/+ as sensitized background to test role of mapk10 as ENS development modifier gene.

(A) RNA in situ hybridization shows mapk10 expression correlates with location of the ENS in WT (ret^+/+), ret^hu2846/+, and ret^{hu2846/hu2846} 3dpf larvae (asterisks indicate end of gut, filled arrows indicate mapk10⁺ cells, and open arrows denote gut areas lacking mapk10⁺ cells). (B) MO gene knock-down of mapk10 (using a splice blocking MO, mapk10MO) performed on embryos from WT x ret^hu2846/+ cross to allow knock-down in both WT and ret^hu2846/+ embryos, and compared to a control MO. Immunostaining with HuC/D at 4dpf shows that most severe phenotypes are observed when mapk10MO is injected into ret^hu2846/+ 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 mapk10MO 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 mapk10 in normal ENS development. Injection of mapk10MO into ret^hu2846/+ embryos causes severe reduction of enteric neurons, which is statistically different from either ret^hu2846/+ injected with control MO (one-way ANOVA, p<0.001, Bonferroni’s post-hoc test, P<0.0001) or mapk10MO injected into WT embryos (one-way ANOVA p<0.001, Bonferroni’s post-hoc test p = 0.006).