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Fig. 5

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ZDB-FIG-230505-21

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Wentworth et al., 2022 - Functional testing of BMP pathway variants identified on whole exome sequencing in a patient with delayed-onset fibrodysplasia ossificans progressiva (FOP) using ACVR1^R206H -specific human cellular and zebrafish models

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Fig. 5

Co‐knockdown of BMPR1A and ACVR2A decreases BMP signaling in ACVR1 ^R206H HEK cells after stimulation with BMP4 or AA; knockdown of acvr2aa; acvr2ab; bmpr1aa; bmpr1ab partially rescues the ventralization phenotype in Tg(ACVR1‐R206Ha) zebrafish. (A) pSMAD 1/5/9 levels normalized to GAPDH loading control in ACVR1 ^R206H HEK 293T cells after transient transfection with siRNA targeting both BMPR1A and ACVR2A, and after stimulation with either BMP4, activin A (AA), or nonstimulated. N = 6 biological replicates for each condition except n = 4 for co‐transfected AA. Values of p are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (B) acvr2aa; acvr2ab and bmpr1aa; bmpr1ab knockdown in Tg(ACVR1‐R206Ha) fish partially rescues the ventralization phenotype (Columns 1–4). Percent ventralized heat‐shocked (HS)‐Tg(ACVR1‐R206H) embryos in bmpr1aa; bmpr1ab mutant background after co‐injection with acvr2aa; acvr2ab or control MOs. Kruskal‐Wallis test was performed, statistical significance observed between columns 1 and 2 (p < 0.0001) and columns 2 and 4 (p < 0.0001) (Columns 1–4, n = 24, 46, 73, and 88, respectively). Genotyping was used to confirm that 6/10 V3 ventralized embryos were bmpr1aa ^−/− ; bmpr1ab ^−/− mutants as compared to 1/8 and 0/6 for more severe V4 and V5 ventralized embryos, respectively (see Fig. S7B). Percent dorsalized non‐Tg(ACVR1‐R206H) embryos in bmpr1aa; bmpr1ab mutant background after co‐injection of acvr2aa; acvr2ab or control morpholinos (MOs). Kruskal‐Wallis test was performed and statistical significance was observed between columns 5 and 6 (p < 0.0001) and columns 6 and 8 (p < 0.0001) (Columns 5–8, n = 49, 78, 57, and 71, respectively). (C,D) Ventralized (V3, V4, V5) HS‐Tg(ACVR1‐R206H) embryos in bmpr1aa; bmpr1ab mutant background following acvr2aa; acvr2ab MO co‐injection. (E) Dorsalized non‐Tg(ACVR1‐R206H) embryos in bmpr1aa; bmpr1ab mutant background following acvr2aa; acvr2b MO co‐injection. Parental genotypes are presented above each column in B. Control and acvr2aa; acvr2ab MOs were injected into separate clutches, and uninjected controls were scored from each clutch. Transgenic‐positive and transgenic‐negative embryos were sorted via mCherry fluorescence. MO = morpholino.

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Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ J. Bone Miner. Res.