Variant BMP receptor mutations causing fibrodysplasia ossificans progressiva (FOP) in humans show BMP ligand-independent receptor activation in zebrafish

Abstract

The large majority of cases of the autosomal dominant human disease fibrodysplasia ossificans progressiva (FOP) are caused by gain-of-function Arg206His mutations in the BMP type I receptor ACVR1 (ALK2). The Arg206His mutation is located in the GS domain of the type I receptor. This region is normally phosphorylated by the BMP type II receptor, which activates the type I receptor to phosphorylate its substrate, the signal transducer Smad1/5/8. A small subset of patients with FOP carry variant mutations in ACVR1 altering Gly328 to Trp, Glu or Arg. Since these mutations lie outside the GS domain, the mechanism through which ACVR1 Gly328 mutations cause disease remains unclear. We used a zebrafish embryonic development assay to test the signaling of human ACVR1 Gly328 mutant receptors comparing them to the Arg206His mutant. In this assay increased or decreased BMP pathway activation alters dorsal-ventral axial patterning, providing a sensitive assay for altered BMP signaling levels. We expressed the human ACVR1 Gly328 mutant receptors in zebrafish embryos to investigate their signaling activities. We found that all ACVR1 Gly328 human mutations ventralized wild-type embryos and could partially rescue Bmp7-deficient embryos, indicating that these mutant receptors can activate BMP signaling in a BMP ligand-independent manner. The degree of ventralization or rescue was similar among all three Gly328 mutants. Smad1/5 phosphorylation, a readout of BMP receptor signaling, was mildly increased by ACVR1 Gly328 mutations. Gene expression analyses demonstrate expanded ventral and reciprocal loss of dorsal cell fate markers. This study demonstrates that Gly328 mutants increase receptor activation and BMP ligand-independent signaling through Smad phosphorylation.

Keywords: ACVR1; BMP signaling; Dorsal-ventral embryonic patterning; FOP; Smad1/5; Zebrafish.

Figure 1. Effect of Gly328 mutant receptor expression in acvr1l^tm110b and WT embryos

A. mRNA misexpression in embryos from incrosses of acvr1l^tm110b/+ fish. One quarter are acvr1l^{tm110b/tm110b} mutant embryos. Phenotypes were scored at 24 hpf. B. mRNA misexpression in WT embryos. Phenotypes were scored at 24 hpf. C. Ventralization phenotypes are indistinguishable between WT embryos injected with ACVR1 Gly328 mutant mRNAs and His206 mRNA. hpf – hours post fertilization; C2 – lack of ventral tail fin and vein; V1 – mild ventralization with diminished eye size and broadening of ventral tail fin; V1-2 – loss of notochord, but eye present; V2 – lack of eye, reduced head tissue, loss of notochord; V3 – lack of head tissue; V4 – greatly enlarged yolk extension; V5 – radially ventralized; WT – wild type.

Figure 2. Effect of human Gly328 mutant receptor expression in bmp7^{sb1aub/sb1aub} embryos

A. mRNA misexpression in bmp7^{sb1aub/sb1aub} mutant embryos. Phenotypes were scored at 24 hpf. B. Phenotypes observed in bmp7^{sb1aub/sb1aub} embryos injected with ACVR1 Glu328 mRNA and ACVR1 His206 mRNA. In parantheses, percent embryos with the phenotype. Classifications as in Figure 1 legend; C3 – lack of ventral tail fin and vein, truncated tail, and at least 14 somites present; C4 – preserved head tissue, less than 14 somites evident; C5 – disordered tissue development without discernible anatomical structures; lysis – dead embryos (C5 embryos typically lyse prior to 24 hpf).

Figure 3. Whole-mount immunostaining of P-Smad1/5

WT (A, B, C) and bmp7^{sb1aub/sb1aub} (D, E, F) embryos at mid-gastrulation. Uninjected control embryos in A, D (blue); human ACVR1 Arg328 mRNA injected in B, E (green); ACVR1 His206 mRNA injected in C, F (red). Animal pole views, dorsal (when evident) to the right. P-Smad1/5 quantification in arbitrary units (A.U.) of ACVR1 Arg328 mRNA injected embryos (green graphs) and ACVR1 His206 mRNA injected embryos (red graphs) compared to wild type (G, G′) and bmp7^{sb1aub/sb1aub} (H, H′). The aggregate information is depicted in G and H; measurements for individual embryos are shown in G′ and H′.

Figure 4. Gene expression analysis

Whole-mount in situ hybridization of chordin (A), eve1 (B), and goosecoid and gata2 (C) in WT embryos injected with human ACVR1 mutant mRNAs, compared to uninjected controls. Animal pole views with dorsal to the right. Closed arrows delineate the borders of the ventral markers, open arrows delineate the extent of the dorsal markers.