Fig. 2

Multiple genes in the ER stress and N-linked glycosylation pathways are upregulated in pmm2m/m mutant larvae (A) Heatmaps of transcriptome data show multiple genes involved in the UPR and ER stress pathways are upregulated in pmm2m/m mutant larvae 5 and 8 dpf. n = 5 biological replicates per genotype with 10 embryos per sample. p values are shown within the heatmap. (B) qRT-PCR analyses performed 6 dpf confirm transcript abundance differences in several genes identified by RNA-seq. n = 3 biological replicates per genotype with 10 embryos per sample. (C) Graph of qRT-PCR analyses show both the unspliced and spliced forms of Xbp1 are increased in mutant larvae 6 dpf, with the ratio of the two similar between WT and mutant larvae. (D) Whole-mount in situ hybridization of hspa5 (BIP) show its abundance is particularly increased in cartilage, supporting a role for ER stress in skeletal pathogenesis. n = 35?40 embryos per genotype. Percentage values represent the number of embryos exhibiting the phenotype pictured. Scale bar, 20 ?m. (E) Heatmaps of transcriptome data show multiple genes involved in N-linked protein glycosylation are upregulated in pmm2m/m mutant larvae 5 and 8 dpf. Also shown are a subset of genes (i.e., dpm1?3) involved in O-mannosylation. The OST and TRAP complexes are important for co-translation transfer of N-glycans. n = 5 biological replicates per genotype with 10 embryos per sample. p values are shown within the heatmap. (F) qRT-PCR analyses confirm increased abundance of several genes identified by RNA-seq. n = 4 biological replicates of 10 embryos each per genotype per time point. (G) Western blot analyses comparing protein abundances of the NgBR, Dpagt1, and Alg9 show increased abundance of NgBR and Dpagt1 in WT and mutant larvae with no change in Alg9. n = 3?5 biological replicates per genotype per time point, with each sample containing 10 embryos. Graphs show quantitation of the NgBR (H), Dpagt1 (I), and Alg9 (J) 4?8 dpf. (K) Immunohistochemical stains of the NgBR27 and Dpagt1 (red in fli1a:EGFP-positive cartilage show each protein is locally increased in mutant cartilages 7dpf. Scale bar, 10 ?m. (L) Graphs showing pixel intensity of NgBR or Dpagt1 normalized to EGFP intensity. For all experiments, error = SEM, Student?s t test, ?p < 0.05, ??p < 0.01, ????p < 0.0001. See also Figure S2.