Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.

SummaryMorphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm.

Dorsoventral (DV) patterning of vertebrate embryos requires the concerted action of the Bone MorphogeneticProtein (BMP) and Wnt signaling pathways. In contrast to our understanding of the role of BMP in establishing ventral fates, our understanding of the role of Wnts in ventralizing embryos is less complete. Wnt8 is required for ventral patterning in both Xenopus and zebrafish; however, its mechanism of action remains unclear. We have used the zebrafish to address the requirement for Wnt8 in restricting the size of the dorsal organizer. Epistasis experiments suggest that Wnt8 achieves this restriction by regulating the early expression of the transcriptional repressors Vent and Vox. Our data show that vent and vox are direct transcriptional targets of Wnt8/β-catenin. Additionally, we show that Wnt8 and Bmp2b co-regulate vent and vox in a dynamic fashion. Thus, whereas both Wnt8 and zygotic BMP are ventralizing agents that regulate common target genes, their temporally different modes of action are necessary to pattern the embryo harmoniously along its DV axis.

Edited by Joan Massagué and Wilhelm JustKeywords: BMP Gradient Morphogen Signalling Smad TGF-b superfamily a b s t r a c tThe bone morphogenetic protein (BMP) signalling pathway is critical for embryonic development and tissue homeostasis, and impaired BMP signalling has been implicated in multiple diseases. Molecular tools have been developed to visualise BMP activity in vivo and these have allowed a better understanding of the intricate ways in which BMP activity is regulated spatially. In particular, generation and interpretation of BMP activity gradients during development result from the complex interplay between core BMP signalling components and specific regulators. In this essay we discuss the mechanisms by which spatial regulation of BMP activity is achieved and its functional consequences.