(A) Ventralized and dorsalized phenotypes at 24 hr post-fertilization (hpf) were categorized using established classification schemes (Mullins et al., 1996; Kishimoto et al., 1997). (B) Embryos were injected with equimolar amounts of mRNA encoding BMP2b (1 pg, n = 102), BMP2b-sfGFP (1.49 pg, n = 101), and BMP2b-Dendra2 (1.47 pg, n = 107) at the one-cell stage. BMP2b-sfGFP induced stronger ventralization, and BMP2b-Dendra2 induced weaker ventralization compared to untagged BMP2b. (C) To determine whether the differences in the degree of ventralization (B) are due to changes in protein activity or protein levels, extracellularly enriched extracts were obtained from zebrafish embryos injected with mRNA amounts equimolar to 444 pg BMP2b-FLAG-encoding mRNA. Levels and processing of FLAG-tagged BMP ligands were assessed using anti-FLAG western blots. Green asterisks to the left of a band indicate properly processed mature BMP2b ligand; blue asterisks indicate unprocessed full-length pro-protein. Similar to FLAG-tagged BMP2b, FLAG-tagged BMP2b-sfGFP and -Dendra2 are properly processed and mostly secreted as mature ligands into the extracellular space. BMP2b-sfGFP-FLAG protein levels are higher compared to FLAG-tagged BMP2b, possibly owing to the rapid folding kinetics of sfGFP (Pédelacq et al., 2006); in contrast, BMP2b-Dendra2-FLAG levels are lower. The correlation between protein levels and activity (B) suggests that the fluorescent BMP2b constructs are equivalent to untagged BMP2b in inducing downstream signaling responses. (D) Phenotype distributions at 24 hpf. Zebrafish embryos were injected at the one-cell stage with equimolar amounts of mRNA encoding Chordin (30 pg, n = 41), Chordin-sfGFP (37 pg, n = 39), Chordin-Dendra2 (37 pg, n = 39), and Chordin-FLAG (30 pg, n = 33) (uninjected: n = 49). (E) Extracellularly enriched fractions were obtained from zebrafish embryos injected with mRNA equimolar to 500 pg of Chordin-FLAG-encoding mRNA. Levels and processing of FLAG-tagged Chordin constructs were assessed using anti-FLAG western blots. Green asterisks indicate properly processed mature Chordin; blue asterisks indicate unprocessed full-length protein. Similar to the correlation between BMP2b construct levels and ventralization activity, the dorsalization activity of Chordin constructs (D) is correlated with protein levels. (F–H) Distribution of BMP2b/Chordin-sfGFP in transplantation donors similar to those used in experiments shown in Figures 3 and 5 . Embryos were injected at the one-cell stage with 500 pg BMP2b-sfGFP- (G) or 1000 pg Chordin-sfGFP- (H) encoding mRNA (compare to uninjected embryo (F)). Embryos were imaged using light sheet microscopy at sphere stage (5–5.5 hpf), when transplantations were carried out in the experiments shown in Figures 3 and 5. Maximum intensity projections are shown.