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Figure S1. In Vivo Recapitulation of SRCAP FHS Truncation Leads to a Characteristic Craniofacial Phenotype that is Phenocopied by Epistatic Gene H2A.Z.2, Related to Figure 1. (A) Comparison of SRCAP orthologs. Protein domains are annotated with HSA in green, ATPase in blue, CBP-binding in red, AT-hooks in yellow, and SANT domain in purple. Protein name and relevant organism are indicated. (B) Morpholino strategy for generating FHS truncated SRCAP mRNA, with domains defined as in (A). Splice blocking by morpholino denoted by bar-headed line at target region. (C) Western blot of cellular extract from dissected X. laevis at tailbud stage, with WT and 5.0 μM FHS SRCAP MO samples used. Antibodies against C-terminal SRCAP (short and long exposures), N-terminal SRCAP (showing WT and truncated SRCAP), and total histone H3 (loading control). 1X and 2X dilution of each sample. (D) RT-PCR showing successful targeting of final intron-exon junction with FHS SRCAP MO #1 at two concentrations (5.0μM, 20μM) and FHS SRCAP MO #2 (10μM). Primers designed to span exons, with expected products at (i) ∼126 bp. (ii) FHS product with intron incorporated expected to be 844bp. Bands indicated with blue and red arrows, respectively. (E) Diagram of MO targeting and expected protein product based on Sanger sequencing results from RT-PCR products from (i) WT (126 bp band) and (ii) FHS morphant (844 bp band) (from D). (F) Ventral and lateral views of dissected X. laevis cartilage stained with Alcian blue at stage 40, WT (water injected) and SRCAP FHS MO #1 (SRCAP truncation) (5.0 μM). 0.5 mm scale bar shown. Animals from n > 3 biologically independent experiments. (G) Ventral view of FHS dose titration with X. laevis cartilage stained with Alcian blue at stage 40. WT (water injected), SRCAP FHS morphant (SRCAP truncation with FHS MO #1) at 0.1 μM, 1.0 μM, 5.0 μM, 10.0 μM, and 20.0 μM. 0.5 mm scale bar shown. (H) Surface models from 3D Optical projection tomography images of dissected cartilage from WT (blue) and FHS SRCAP MO #1 (green) with ventral views. 3D reconstruction produced using inverse Radon transform in MATLAB and visualized in Slicer. (I) Images of SRCAP gut looping in WT and in FHS MO #1 (5.0 μM) injected morphants, with example diagrams of typical and atypical looping patterns observed on right. 0.5 mm scale bar shown. (J) Quantification of SRCAP gut looping defect. Normal counter-clockwise gut looping is indicated in blue, abnormal gut looping (typically disorganization of loops, definitively no coiling) in red. Statistical test was Pearson's chi-squared 2-sample test for equality of proportions with continuity correction. ∗∗∗ - p-value <2.2e-16. Animals from n=4 independent experiments. (K) Quantitative analysis of craniofacial phenotype due to FHS truncation. WT in light blue, FHS truncated in light green. At top are diagrams of features measured. Nose to tail length in red (p-value not significant), distance between eyes in pink (p-value =8.719e-12), angle between Meckel’s cartilage and ceratohyal cartilage in green (p-value < 2.12e-16), area of ceratohyal cartilage in blue (p-value < 5.046e-12), area of gillrake cartilage in orange (p-value = 1.477e-10), area of entire craniofacial cartilage in yellow (p-value = 0.03523). Statistical analysis by Wilcoxon-Mann Whitney test, n.s. - p-value > 0.05, ∗ - p-value < 0.05, ∗∗∗ - p-value < 0.0005. Further details of how measurements were made can be found in STAR Methods section. (L) Ventral view of dissected X. laevis cartilage from WT embryos and embryos asymmetrically injected with 10 μM of FHS SRCAP MO #1 (injected side shown on the right) stained with Alcian blue at Nieuwkoop and Faber stages 40 and 46. 0.5 mm scale bar shown. (M) WT and SRCAP FHS morphant (5.0 μM) with Alcian staining of Meckel’s cartilage at 12x magnification. WT and SRCAP FHS morphant (5.0 μM) with Alcian staining of Meckel’s cartilage and ceratohyal cartilage. 250 μm scale bars shown. (N) Ventral view of representative tadpoles for SRCAP rescue experiment. Showing WT, and co-injection of FHS MO and 200pg pB CAG GFP-FLAG, or pB CAG WT-SRCAP-GFP-FLAG, or pB CAG FHS-SRCAP-GFP-FLAG. Injection schematic above each image and below normal craniofacial phenotype is indicated in blue, abnormal craniofacial phenotype is indicated in red. 0.5 mm scale bar shown. (O) Ventral view of representative tadpoles of WT frogs and frogs injected with 10 μM FHS MO #2. Injection schematic indicated above each image and below normal craniofacial phenotype is indicated in blue, abnormal craniofacial phenotype is indicated in red. 0.5 mm scale bar shown. (P) Blinded quantification of characteristic craniofacial phenotype caused by FHS MO#2. Statistical test used was Fisher’s Exact Test (FET). FET p-value < 10e-5 = ∗∗∗. Animals from n=3 independent experiments. (Q) Western blot of cellular extract from dissected X. laevis at tailbud stage, with WT and 10 μM FHS MO #2 samples. Antibodies against C-terminal SRCAP and total H2A.Z (loading control). 1X and 2X dilution of each sample. Imaged on LI-COR Odyssey. Image published in: Greenberg RS et al. (2019) Copyright © 2019. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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