BMP signaling (pSmad1/5/9) gradient formation and simulations of five major dorsal-ventral patterning models over relevant zebrafish developmental stages (3 hr).

(A) Two-dimensional Hammer-Aitoff projections (2D maps) of pSmad1/5/9-immunostained individual wild type zebrafish embryos at different developmental stages. Embryos were imaged using light sheet microscopy (see Materials and methods for details). (B) Quantification of ventral-to-dorsal average pSmad1/5/9 distributions in one-dimensional projections of 2D maps generated for embryos at different developmental stages (n = 3 for each stage) as in (A). Error bars denote standard error. (C–G) Gradient formation kinetics simulated for Models 1–5 at relevant zebrafish developmental stages.

Theoretical predictions for the influence of the inhibitor Chordin on the BMP signaling gradient and experimental test.

(A–E) Simulations of BMP distributions in five major models of dorsal-ventral patterning in the presence (black) or absence (red) of Chordin. The BMP and Chordin sources are indicated below each graph in green and blue, respectively. Note that the spatial production rates in Models 3 and 4 are modulated over time by feedback. (F–G) Quantification of average pSmad1/5/9 distributions in wild type (black) and chordin^−/− (red) embryos using one-dimensional projections of 2D maps. Wild type n = 7, chordin^−/− mutants n = 10. Error bars denote standard error. (H) p-values (unpaired two-tailed t-test assuming equal variance) calculated as a function of space between pSmad1/5/9 distributions in wild type and chordin^−/− embryos shown in (F) indicate no significant difference of pSmad1/5/9 on the ventral side but a dramatic expansion into dorsal-lateral domains.

Gradient formation kinetics of fluorescently tagged BMP and Chordin.

(A) Schematic of BMP2b-sfGFP and -Dendra2 fusion constructs. (B) Fluorescent BMP2b fusion constructs can induce ventralization, a BMP-overexpression phenotype (Kishimoto et al., 1997). mRNA amounts equimolar to 2 pg of BMP2b mRNA were injected at the one-cell stage, and images were taken 30 hr post-fertilization (hpf). (C) Rescue of a BMP2b mutant (swr^−/−) with BMP2b-Dendra2. 2.74 pg of BMP2b-Dendra2-encoding mRNA were injected at the one-cell stage, and images were taken at 30 hpf. In a separate experiment with 1 pg of BMP2b-sfGFP-encoding mRNA, 20% (9/44) of all injected swr^−/− mutants were rescued, 16% (7/44) were ventralized, and 64% (28/44) were dorsalized. (D) Schematic of Chordin-sfGFP and -Dendra2 fusion constructs. (E) Fluorescent Chordin constructs can induce dorsalization, a Chordin-overexpression phenotype. mRNA amounts equimolar to 30 pg of Chordin mRNA were injected into wild type embryos at the one-cell stage, and images were taken at 30 hpf. F + G) Light sheet microscopy images of BMP- and Chordin-sfGFP gradients forming from a local source in live zebrafish embryos. Approximately 50–75 cells expressing BMP2b-sfGFP (F) or Chordin-sfGFP (G) were transplanted into host embryos at sphere stage (see Materials and methods for details). The images show gradient formation in single optical slices approximately 20 min after transplantation. H + I) Quantification of BMP2b-sfGFP (H) and Chordin-sfGFP (I) gradient formation kinetics from a local source (BMP2b-sfGFP: n = 8; Chordin-sfGFP: n = 5). Dashed lines indicate the distance at which the protein distributions drop to 50% of their maximal concentration 60 min post-transplantation.

Detailed characterization of fluorescently tagged BMP2b and Chordin.

(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.