Xenbase Image ID: 176852

Figure S4. Characterization of Histone Variant H2A.Z Subtype H2A.Z.2 Dysfunction in X. laevis, Related to Figure 4. (A) In situ hybridization staining for srcap expression at tailbud stage in WT X. laevis tadpoles. 250μm scale bar shown. (B) Quantification of H2A.Z protein levels from immunoblot image (Figure 4B) taken with LI-COR Odyssey. For each sample, H2A.Z signal was normalized to signal from 1X dilution of H3 (loading control). Normalization of both 1X and 2X dilution for each sample shown. (C) RT-PCR showing specific H2A.Z.1 and H2A.Z.2 mRNA targeting. One h2afz primer set and one h2afv primer set was used for RT-PCR. WT – cDNA from control injected embryos; H2A.Z.1 MO– cDNA from H2A.Z.1 MO injected embryos; H2A.Z.2 MO– cDNA from H2A.Z.2 MO injected embryos; Z.1 MO + Z.2 MO - cDNA from embryros co-injected with H2A.Z.1 MO and H2A.Z.2 MO. Expected PCR sizes without targeting are indicated by blue arrows (h2afz – 229bp; h2afv – 200bp), splicing defects affecting PCR product size due to morpholino targeting are indicated by red arrows. (D) Ventral view of tadpoles stained with Alcian blue at stage 40, WT (water injected), SRCAP FHS morphant (SRCAP truncation) (5 μM), H2A.Z.1 morpholino (2.5 μM morpholino), H2A.Z.2 morpholino (2.5 μM morpholino). Scale bar is 0.5 mm for each image. Animals from n = 3 biologically independent experiments. (E) Ventral view of H2A.Z.2 dose titration with X.laevis cartilage stained with Alcian blue at stage 40. WT (water injected), H2A.Z.2 morpholino at 1.0 μM, H2A.Z.2 morpholino at 2.5 μM, H2A.Z.2 morpholino at 5.0 μM. Scale bar is 0.5 mm for each image. (F) H2A.Z.1 (2.5 μM) and H2A.Z.2 morphant (2.5 μM) with Alcian staining of ceratohyal cartilage at 12x magnification. 250μm scale bars shown. (G) Quantitative analysis of craniofacial phenotype for all morpholinos. Normal phenotypes in light blue, abnormal phenotypes in light green. Nose to tail length in red (p-value not significant), distance between eyes in pink, ceratohyal cartilage area in blue. Statistical analysis was ANOVA followed by Tukey HSD test, n.s. - p-value > 0.05, ∗ - p-value < 0.05, ∗∗ - p-value < 0.005, ∗∗∗ - p-value < 0.0005. (H) Ventral view of tadpoles stained with Alcian blue at stage 40, WT, H2A.Z.1 MO (5.0 μM), H2A.Z.1 MO (5.0 μM) with HA-tagged H2A.Z.1 mRNA overexpression, H2A.Z.2 MO (2.5 μM), H2A.Z.2 MO (2.5 μM) with HA-tagged H2A.Z.2 mRNA overexpression, and H2A.Z.1 MO (5.0 μM) with H2A.Z.2 MO (2.5 μM). All mRNA injected at 2.5ng each. 0.5 mm scale bar indicated for each image. Animals from n = 2 biologically independent experiments (except H2A.Z.1 MO with H2A.Z.2 MO, as only one experiment had any survival). (I) Blinded quantification of rescue capacity for developmental defects with for H2A.Z.1 MO and H2A.Z.2 MO with overexpression of H2A.Z.1 or H2A.Z.2 mRNA, respectively. Statistical test was Fisher’s Exact Test (FET). FET p-value < 0.05 = ∗, FET p-value < 0.005 =∗∗, FET p-value < 0.00005 = ∗∗∗∗. n=2 independent experiments. (J) Western blot of cellular extract from dissected X. laevis at neurula stage, with WT, H2A.Z.1 MO (5.0 μM), H2A.Z.1 MO (5.0 μM) with HA-tagged H2A.Z.1 mRNA overexpression, H2A.Z.2 MO (2.5 μM), H2A.Z.2 MO (2.5 μM) with HA-tagged H2A.Z.2 mRNA overexpression, and H2A.Z.1 MO (5.0 μM) with H2A.Z.2 MO (2.5 μM) samples. All mRNA injected at 2.5ng each. Antibodies against HA-tag (overexpressed HA-tagged H2A.Z.1 or H2A.Z.2, protein bands indicated by red arrows) (mouse HA) and alpha tubulin (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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