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.

Fibrodysplasia ossificans progressiva mutant ACVR1 signals by multiple modalities in the developing zebrafish

“…While wild-type embryos formed a gradient of pSmad1/5 expression that peaks ventrally and decreases dorsally ( Figure 2a ; Mucha et al, 2018 ), Acvr1l-KD embryos lacked detectable pSmad1/5 signal ( Figure 2a’ ). The mean nuclear pSmad1/5 fluorescence of Acvr1l - KD embryos was significantly decreased compared to WT embryos ( Figure 2h ).…”

“…The zebrafish embryo is an excellent genetically tractable in vivo vertebrate model for investigating the signaling mechanism of the ACVR1-FOP receptor ( Shen et al, 2009 ; Mucha et al, 2018 ). In the early zebrafish embryo, BMP acts as a morphogen to pattern the dorsoventral (DV) axis in a process that is conserved throughout the animal kingdom ( Zinski et al, 2018 ).…”

“…Perturbations to this BMP signaling gradient in the developing embryo result in distinct, dose-dependent patterning phenotypes ( Figure 1b ). Over-activation of the BMP signaling pathway by FOP-ACVR1 causes ventralization, an excess of ventral cell fate specification at the expense of dorsal fates ( Shen et al, 2009 ; Mucha et al, 2018 ), while loss of endogenous acvr1l expression leads to an opposite dorsalization. Loss of acvr1l in the zebrafish can be rescued by human ACVR1 , demonstrating their conserved activity ( Shen et al, 2009 ).…”

The Horizon of a Therapy for Rare Genetic Diseases: A “Druggable” Future for Fibrodysplasia Ossificans Progressiva

“…Indeed, ligand-independent signaling, increased SMAD1/5 activation, enhanced chondrogenic and osteogenic properties in different cellular models, and a reduction in FKBP12 binding were also observed for the L196P, Q207E (GS domain) and G356D (kinase domain) substitutions [ 18 , 19 , 20 , 21 ]. Moreover, in vivo studies in zebrafish embryos demonstrated that G356D/W/R substitutions also enhanced the SMAD1/5 signaling and induced embryo ventralization with loss of dorsal cell fate markers, as observed for the R206H mutation [ 22 ]. The activation state of the different mutants is not exactly the same but they are still BMP-responsive and maintain the ability to be down-regulated by known BMP inhibitors such as regulatory SMAD6 and 7, over-expression of FKBP12 or pharmacological treatment with known inhibitors of the signaling such as dorsomorphin and LDN193189 [ 18 , 20 , 21 ].…”

Zebrafish Models of Rare Hereditary Pediatric Diseases

“…Another excellent example for the use of phenologs in modeling pediatric disorders is fibrodysplasia ossificans progressiva (FOP). In-depth knowledge about the molecular mechanism of early dorso-ventral (DV) patterning in zebrafish development, including the phenotype of ventralized embryos, has been helpful both in the identification of the causative mutations of FOP [ 63 , 64 ] and that of putative drugs [ 142 ].…”