Differential gene expression and splicing in apc mutant zebrafish

APC is a classical tumor suppressor in humans, and truncating mutations are early somatic events in most cases of sporadic colon cancer. APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and therefore loss of full length APC reduces GSK-3 activity, leading to stabilization of b-catenin protein and activation of downstream Wnt/b-catenin signaling. GSK-3 also phosphorylates multiple core mRNA splicing factors and loss of Gsk3a/b in mouse ES cells and human lymphocytes alters mRNA splicing at a genome wide level. We therefore predict that loss of APC should similarly alter splicing by reducing GSK-3 activity. Here we use RNA-seq to assess differential gene expression and altered splicing in apc-mcr mutant zebrafish embryos at 48hpf. We find robust activation of known Wnt/b-catenin target genes, as expected. Surprisingly, we also find markedly increased expression of multiple genes related to inflammation and cytokine signaling. We also identify 340 mRNA splicing variations in apc mutant zebrafish. Many of these local splice variants (LSVs) occur in mRNAs the regulate cell adhesion, migration, and morphogenesis. Overall design: WT/het and apcmcr/mcr embryos (3 biological replicates; 20 embryos/replicate) were treated with vehicle (0.1% DMSO) or 100nM Rapamycin (beginning at 24hpf) and harvested at 48hpf with Triazol followed by DNaseI treatment and re-purification on RNeasy (RIN > 8). Total RNA was submitted to Genewiz for polyA-selection and generation of stranded Illumina RNA-Seq library. Sequencing was at a depth of =100 million reads per individual replicate. Alternative splicing and differential expression analyses were based on RNA-Seq reads that were mapped to the zebrafish reference genome (GRCz10) using STAR. Differential splicing analysis was performed comparing WT/het and apcmcr/mcr samples using MAJIQ (Modeling Alternative Junction Inclusion Quantification) deltapsi with intron retention quantification enabled. Results were filtered for high confidence changing local splice variants (LSVs) with a change in percent spliced in (dPSI) =20% (in which junctions had a =90% probability of expected dPSI).