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Figure 1. Recapitulating Floating-Harbor Syndrome SRCAP truncations in Xenopus laevis Affects Neural Crest Derived Craniofacial Structures. (A) Graphical representation of typical (left) and Floating-Harbor syndrome (right) craniofacial features. Characteristic triangular facial shape (most characteristic and diagnostic feature) demarcated in red.
(B) Plot of frequencies of SRCAP mutations in Floating-Harbor syndrome probands. The x axis goes from amino acid 2200 to 2800, and each mutation is denoted on this axis. The most frequent mutation R2444∗ is highlighted in red.
(C) Schematic of WT and the FHS truncated SRCAP proteins. The hot spot for FHS truncating mutations is indicated by red arrowheads. Protein domains are annotated with HSA in green, ATPase in blue, CBP-binding in red, AT-hooks in yellow. The amino acid scale is below the schematic.
(D and E) Ventral (D) and side (E) view of X. laevis head with craniofacial cartilage stained with Alcian blue at stage 40, WT (mock-injected) and SRCAP FHS morphant (5.0 μM MO). Scale bar, 0.5 mm. Animals from n = 3 biologically independent experiments.
(F) Diagram of homology between branchial arch structures in X. laevis to pharyngeal arches of the developing human face, with key homologous structures highlighted in matching colors.
(G) Blinded quantification of rescue of characteristic craniofacial phenotype with co-injection of FHS MO and 200 pg pB CAG GFP-FLAG, or pB CAG WT-SRCAP-GFP-FLAG, or pB CAG FHS-SRCAP-GFP-FLAG. Statistical test used was Fisher’s exact test (FET). FET p value < 0.005 = ∗∗, FET p value < 10e−5 = ∗∗, FET p value > 0.05 = n.s. Animals from n = 4 independent experiments.
(H) Diagram of injection set up at two-cell stage and of asymmetrical FHS SRCAP MO expression at neurula stage. In situ hybridization at neurula stage for neural crest specification genes twist1, slug, and sox9 (abnormal phenotype in 9/11, 5/5, 5/6 embryos, respectively), for neural crest induction and specification gene tfap2a (abnormal phenotype in 11/12 embryos), for neural plate border maintenance genes zic1 and msx1 (abnormal phenotype in 1/10, 0/6 embryos, respectively), for early neural patterning gene otx2 and for neural plate gene sox3 (abnormal phenotype in 0/5, 1/5 embryos, respectively), with 5.0 μM FHS MO injected on right side only, control on left. Ventral side shown, with anterior at top and posterior at bottom. Scale bar, 250 μm for neurula images. In situ hybridization at tailbud stage (stage 28), with each pair of images from same animal (control image flipped in vertical plane). In situ probes twist1 and tfap2a (abnormal phenotype in 8/10 and 11/13 embryos, respectively) visualize neural crest migration. Scale bar, 250 μm for tailbud images. Blue arrows denote normal gene expression pattern, red arrows denote impact on expression for FHS morphant. Image brightness and color adjusted to optimize visualization.
See also Figure S1, Table S1, and Video S1. |
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Figure S3. Characterizing Molecular and Cellular Phenotypes in FHS Mutant Cranial Neural Crest Cells, Related to Figure 3.
A) Confocal microscopy of immunofluorescence staining in WT and FHS MUT SRCAP CNCCs against canonical NCC markers NR2F1 (red), TFAP2A (orange), and p75 (green) with DAPI nuclear stain (blue). 10μm scale bars shown.
(B) Fluorescence activated cell surface marker analysis of WT (WT-V5 lines #1-#3) and FHS MUT (FHS-V5 lines #1-#3) NCCs, with markers CD271 PE-Cy7, CD266 PE, CD105 PacificBlue, CD24 PerCP-Cy5, and CD10 APC. All are previously characterized markers for normal neural crest cells.
(C) Enrichment of genes involved in calcium-dependent cell-cell adhesion (GO:0016339) with gene enrichment in red and associated gene names in blue.
(D) Enrichment of migration genes enriched in HHSt11cluster 1 (Morrison et al., 2017) that are affected in FHS MUT SRCAP CNCCs with gene enrichment in red and associated gene names in blue.
(E) Cell cycle analysis of WT (WT-V5 lines #1-#3) and FHS (FHS-V5 lines #3) NCCs for two distinct differentiations with cell cycle phases identified using Click-iT and FxCycle DNA staining with flow cytometry analysis. No significant differences detected by binomial regression. Confidence intervals obtained using quasi-binomial regression in glm.
(F) CD24 flow cytometry analysis of WT and FHS MUT NCCs. CD24 by CD10 in one representative experiment, with three WT and three FHS MUT lines differentiated to CNCCs.
(G) Fraction of CD24-low CNCCs compared between WT (orange) and FHS MUT (blue) over n=3 independent differentiations. Statistical analysis was analysis of covariance test with p=0.00003.
(H) Imaging time course from migration assay (see Figure 3H) showing embryos selected for correct fluorescence at neurula stage (time=0 hours), migratory stage (time=5 hours) and tailbud stage (time=15 hours). 250μm scale bar shown. White line for each branchial arch and diagram on right shows number and patterning of branchial arches counted for quantifications.
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