Fig 2: A loss-of-function mouse model uncovers a specialized role for RPL10A/uL1 in the paraxial mesoderm lineage.a Median relative abundance of RPL10A/uL1 in the polysomes of the mesoderm lineage relative to hESCs. N = 6 for all cell types, except n = 7 for primitive streak and error bars are standard error. Student’s t test P values: anterior primitive streak to early somites 0.006, anterior primitive streak to sclerotome 0.03. b Schematic of the genetic complementation test to identify loss-of-function mutations. c Observed frequencies of Rpl10a null/null homozygotes, Rpl10a deletion/deletion homozygotes, Rpl10a extended/null double heterozygotes, and Rpl10a extended/deletion double heterozygotes. d Lateral views of E8.5, E9.5, E10.5, and E12.5 Rpl10a LOF/LOF and control embryos. The tail bud is traced in white and indicates the posterior trunk truncation; the hindlimb is also traced in red on the E12.5 images. e Quantification of posterior trunk length at E8.5, E9.5, E10.5, and E12.5. Graph shows average length relative to wild-type with SEM error bars. No significant differences were observed between wild-type and Rpl10a LOF/+ embryos; Rpl10a LOF/LOF embryos were significantly different from both wild-type and Rpl10a LOF/+ embryos at all stages except E8.5. For E8.5 wild-type n = 5, Rpl10a LOF/+ n = 7, Rpl10a LOF/LOF n = 8; for E9.5 wild-type n = 13, Rpl10a LOF/+ n = 11, Rpl10a LOF/LOF n = 25; for E10.5 wild-type n = 10, Rpl10a LOF/+ n = 8, Rpl10a LOF/LOF n = 15; for E12.5 wild-type n = 8, Rpl10a LOF/+ n = 13, Rpl10a LOF/LOF n = 19. Wild-type vs. Rpl10a LOF/LOF Student’s t test P values: 0.65 (E8.5), 2.55 × 10-7 (E9.5), 2.45 × 10-5 (E10.5), 1.13 × 10-5 (E12.5); Rpl10a LOF/+ vs. Rpl10a LOF/LOF P values: 0.10 (E8.5), 5.31 × 10-6 (E9.5), 8.89 × 10-7 (E10.5), 1.20 × 10-10 (E12.5). f Schematic of gastrulation at the tail bud to produce the paraxial mesoderm lineage, with the embryonic tissues represented in the in vitro hESC differentiation indicated. APS anterior primitive streak, PXM paraxial mesoderm, ESom early somite, Scler sclerotome, *P value < 0.05; **P value < 0.01, ***P value < 0.001. Source data are provided as a Source Data file.

Fig 3: Rpl10aLOF/LOF embryos exhibit defects in paraxial mesoderm lineage formation.a Whole-mount in situ hybridizations in control and Rpl10a LOF/LOF embryos at E9.5 and E10.5. The region of expression at the tail bud is demarcated with a solid white line. A dashed white line outlines the tail bud in the Shh in situ. b Quantification of the area of staining relative to total tail bud area. For each probe, stage, and genotype n = 3 with the exception of Fgf8 (n = 4 for each genotype) and T (n = 4 for E9.5 Rpl10a LOF/LOF). Data are shown as mean +/- SEM, Student’s t test P values: 0.04 (T E9.5), 0.03 (T E10.5), 0.09 (Msgn1 E9.5), 0.045 (Msgn1 E10.5), 0.005 (Tbx6 E9.5), 0.049 (Tbx6 E10.5), 0.40 (Fgf8 E9.5). c Quantification of the distance between the end of the notochord, as demarcated by Shh in situ, and the tip of the tail bud (n = 3). Distances were normalized to the distance between limb buds and otic placode in each embryo to account for any differences in embryo size. Data are shown as mean +/- SEM, and significance was calculated using Student’s t tests (P value = 0.04). d Whole-mount in situ hybridizations for Uncx4.1 in control and Rpl10a LOF/LOF embryos at E9.5, lateral view (top), and view of the posterior end of the embryo (bottom). The dashed white line demarcates the region of expression; black arrows indicate abnormalities in somite boundaries and spacing. *P value < 0.05; **P value < 0.01. Source data are provided as a Source Data file.

Fig 4: Rpl10a loss-of-function hESCs have defects in paraxial mesoderm production.a Western of whole-cell lysates from Rpl10a LOF/LOF and Rpl10a LOF/+ embryos, unedited hESCs, and hESC clones either homozygous or heterozygous for the Rpl10a LOF insertion. b Cell viability of wild-type, heterozygous, and homozygous Rpl10a LOF/LOF hESCs as measured by Cell Titer Glo (n = 44). Data are shown as mean +/- SEM and significance was calculated using Student’s t tests. c Cell viability of heterozygous and homozygous Rpl10a LOF/LOF hESCs as measured by Cell Titer Glo during differentiation down the paraxial mesoderm lineage (n = 12 for each cell line for anterior primitive streak, n = 10 for each cell line for paraxial mesoderm, n = 5 for each cell line for sclerotome, n = 5 for Rpl10a LOF/+ cells and n = 4 for Rpl10a LOF/LOF cells for early somites). Data are shown as mean +/- SEM. Student’s t test P values: 0.77 (anterior primitive streak), 0.04 (paraxial mesoderm), 0.03 (early somite), 0.002 (sclerotome). APS anterior primitive streak, PXM paraxial mesoderm, ESom early somite, Scler sclerotome. *P value <0.05; **P value <0.01. Source data are provided as a Source Data file.

Fig 5: Rpl10aLOF/LOF embryos exhibit reduced translation of Wnt signaling components.a (Left) Polysome traces of wild-type, Rpl10a LOF/+, and Rpl10a LOF/LOF whole E13.5 embryo extracts show similar distributions of polysomes, monosomes, and free subunits. (Right) Western blots of protein precipitated from each sucrose-gradient fraction show the extended RPL10A/uL1 protein is incorporated into polysomes equivalently to wild-type. b (Left) Fluorescence image of E9.5 Meox1 Cre; Ai9 embryo illustrating the presomitic/somitic mesoderm tdTomato reporter expression. (Right) OP-Puromycin incorporation rates in Meox1 Cre; Ai9; Rpl10a LOF/LOF E9.5 presomitic/somitic mesoderm (tdTom+) and in the rest of the embryo (tdTom-) (n = 3). Values are normalized to OP-Puro incorporation rates of TdTom+ and TdTom– cells of Meox1 Cre; Ai9; Rpl10a LOF/+ E9.5 control littermates. Data shown are mean +/- SEM, and significance was calculated using Student’s t test. c Comparison of RNA-seq and ribosome profiling for whole E8.5 Rpl10a LOF/LOF and wild-type embryos (n = 3 each). Genes changing significantly (false discovery rate (FDR) < 0.1) only in mRNA abundance are blue; genes changing significantly (FDR < 0.1) only in ribosome occupancy are red; genes changing significantly in both datasets are purple. d Selected Wnt signaling gene sets that were significantly (FDR < 0.1) enriched in CAMERA gene set enrichment analysis of genes with altered translation efficiency in Rpl10a LOF/LOF embryos compared to wild-type. All gene sets shown are translationally downregulated. Node size = gene set size; edge size = gene set overlap. e Genes from the “Wnt signaling pathway, planar cell polarity pathway” set with statistically significant changes in ribosome-protected footprints (RPF) but not in total RNA-Seq (RNA). f E9.5 wild-type (n = 2) and Rpl10a LOF/LOF (n = 4) gradient RT-qPCR for Vangl2 showing the fraction of the total mRNA found in each of the five fractions of the 25–50% sucrose gradient demarcated in the schematic. Data shown are mean +/- SEM. Vangl2 is significantly decreased in Rpl10a LOF/LOF medium and heavy polysomes relative to wild-type (Student’s t test P values = 0.04 and 0.008, respectively). *P value < 0.05; **P value < 0.01. Source data are provided as a Source Data file.