Fig. 1.

Identification of a fungal filtrate with activity consistentwith inhibition of BMP signaling. (A) Untreated control, not treated with fungal filtrate. WT, wild type. (B) An example of a phenotype induced by the filtrate of A. aquilegiae mixed in 1:1 ratio with E3 medium, with treatment at 6-48 hpf. (C) Phenotype induced by 750 nM DMH-1. The arrows in B and C indicate loss of the ventral fin. (D) Schematic overview of purification of active component. The filtrate is extracted with 3×1/3 volume ethyl acetate. The ethyl acetate fractions are than combined and dried. The residue is dissolved in DMSO and subsequently loaded onto a preparative HPLC column. Fractions are collected every 63 s. (E,F) Phenotypes induced by purified fraction, at high (E) and low (F) concentrations. In A-C,E,F, ten embryos were incubated per condition; representative pictures are shown. (G) Dose-dependent inhibitory effects of purified fraction of fungal filtrate on BMP2-induced Smad1/5-dependent BRE-luc transcriptional reporter activity. Lack of significant effect of vehicle control DMSO and potent antagonizing effect of BMPR type I kinase inhibitor LDN-193189 are shown. Results are expressed as mean±s.d., *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test, two-tailed, unpaired).