Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation

Abstract

Signaling by bone morphogenetic proteins (Bmps) plays a pivotal role in developmental and pathological processes, and is regulated by a complex interplay with secreted Bmp binding factors, including Crossveinless 2 (Cvl2). Although structurally related to the Bmp antagonist Chordin, Crossveinless 2 has been described to be both a Bmp agonist and antagonist. Here, we present the first loss-of-function study of a vertebrate cvl2 homologue, showing that zebrafish cvl2 is required in a positive feedback loop to promote Bmp signaling during embryonic dorsoventral patterning. In vivo, Cvl2 protein undergoes proteolytic cleavage and this cleavage converts Cvl2 from an anti-to a pro-Bmp factor. Embryonic epistasis analyses and protein interaction assays indicate that the pro-Bmp function of Cvl2 is partly accomplished by competing with Chordin for binding to Bmps. Studies in cell culture and embryos further suggest that the anti-Bmp effect of uncleaved Cvl2 is due to its association with the extracellular matrix, which is not found for cleaved Cvl2. Our data identify Cvl2 as an essential pro-Bmp factor during zebrafish embryogenesis, emphasizing the functional diversity of Bmp binding CR-domain proteins. Differential proteolytic processing as a mode of regulation might account for anti-Bmp effects in other contexts.

von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

“…CV2-VWC1 bound to BMP-2/-7 and GDF-5 with affinities similar to those of full-length CV2 (Fig. 1B) (24). The VWC domains 2, 3, 4, and 5 and the fragment VWC2-5 had no binding affinity for these ligands (data not shown and Fig.…”

“…These results are consistent with our observation that excessive doses of CV2 inhibited BMP-2-induced ALP activity. However, in vivo studies showed that CV2 is an essential pro-BMP regulator of BMP signaling during zebrafish gastrulation (24) and in mouse organogenesis (26). Although the mechanism of the in vivo pro-BMP activity of CV2 remains uncertain, a model for positive feedback is intriguing (45).…”

“…Studies showed that chordin and CHL2 inhibit BMP signaling (20,21). Tsg and CV2 are BMP-modulating proteins for which both anti-and pro-BMP activities were reported (22)(23)(24). Tsg forms a ternary complex with chordin and BMP, making chordin a better BMP inhibitor.…”

Secreted bone morphogenetic protein antagonists of the Chordin family

“…Kielin/Chordin-like protein (Kcp) with 18 Chordin-like CR domains, one thrombospondin domain (Tsp), and one von Willebrand factor D (vWF-D) domain enhances BMP signaling (13). Crossveinless-2 with five Chordin-like CR domains and one vWF-D domain is a BMP agonist/antagonist (14,15). Connectivetissue growth factor, which has four structural domains including the Chordin-like CR domain, insulin-like growth factor domain, Tsp domain, and C-terminal knot, modulates BMP signaling and TGFb signaling negatively and positively, respectively (16).…”

Crystallization and preliminary X-ray analysis of the complex of the first von Willebrand type C domain bound to bone morphogenetic protein 2

“…Modulator proteins of the chordin family present a unique exception to this inhibitory-only function in that they exhibit two contrary activities (Larrain et al, 2001). Depending on the cellular context, chordin and another family member, crossveinless 2 (CV2), can either down-regulate BMP signalling (anti-BMP activity) or enhance BMP signalling (pro-BMP activity) (Binnerts et al, 2004;Ikeya et al, 2006;Rentzsch et al, 2006). The molecular mechanism of this switch in the regulatory mechanism is so far unknown.…”