Fig. s6

A. Semi-quantitative RT-PCR shows transcript levels of foxo3b, eif4a2 and rpl13 (loading control) in rbm8a mutant and wild-type sibling embryos at 21 and 27 hpf. B. Western blots (on the left) show levels of Foxo3b, Rbm8a, Magoh, and Tubulin in rbm8a mutant embryos (lane 2) compared to WT siblings (lane 1) at 27 hpf (N = 20 embryos per genotype). Right: dot plot showing Foxo3b levels normalized to tubulin levels in rbm8a mutant embryos and WT siblings at 27 hpf in three biological replicates. Error bars: standard error of means. Welch’s t-test p-values are indicated at the top. C. Bar graph showing log2 fold changes of known Foxo3b transcriptional targets that show a significant upregulation (FDR < 0.05) in EJC mutant RNA-Seq datasets. Foxo3b targets are from Morris et al. 2015 [64]. A pound symbol indicates statistically non-significant log2 fold change with FDR > 0.05. D-G. Confocal images showing Myh1 immunofluorescence using anti-A4.1025 in somites 12–16 of WT sibling (D), rbm8a-/- mutant (E), rbm8a-/-; foxo3b+/- mutant (F), and rbm8a-/-; foxo3b-/- mutant (G) embryos. (N = 5 embryos/genotype). H-K. Merged confocal images showing motor neurons (red; detected by anti-SV2 staining) and acetylcholine receptors (green; detected by alpha-bungarotoxin staining) in somites 12–16 of WT sibling (H), rbm8a-/- mutant (I), rbm8a-/-; foxo3b+/- mutant (J), and rbm8a-/-; foxo3b-/- mutant (K) embryos. Neuromuscular junctions in the merged image are yellow. White arrowheads point to the distal end of the motor neuron. (N = 5 embryos/genotype). Scalebar in K (for panels D-K) is 100 nm. L. Boxplots showing quantification of motor axon length in embryos of genotypes indicated along the x-axis) (4 motor neurons/embryo and 5 embryos/genotype). Welch’s t-test p-values are indicated at the top.