(
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.