Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine-rich motif present in subsets of 5′-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find a transcript specific effect in Eif3c loss-of-function embryos whereby translation of Ptch1 through this pyrimidine-rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts. [Display omitted] • The global translation initiation factor (eIF3c) is required for Shh signaling • eIF3c mutant mice do not show reduced global protein synthesis • Tissue-based eCLIP- and Ribo-seq revealed transcript-specific regulation by eIF3 • Reduced Ptch1 translation may lead to Shh-related phenotypes in Eif3c mutant mice Using tissue-based eCLIP- and Ribo-seq in mouse embryos, Fujii et al. demonstrate that translation initiation factor 3 (eIF3) directly associates with cell signaling transcripts and regulates their translation. Furthermore, they observed a Shh-gain-of-function phenotype in Eif3c mutant mice associated with reduced translation of the eIF3 direct target, Ptch1 mRNA. [ABSTRACT FROM AUTHOR]