Figure 2. Expression of bone morphogenic protein response element: destabilized enhanced green fluorescent protein (BRE:d2GFP) protein and mRNA, and phospho-Smad1/5/8 immunoreactivty in 13- to 17-somite embryos. A–F: Whole embryos were imaged for both d2GFP protein (A,C,E) and mRNA (B,D,F; green) and phospho-Smad1/5/8 immunoreactivity (magenta) (A2–F2). Note the enriched and overlapping d2GFP and phospho-Smad1/5/8 immunoreactivity in the tail and lower trunk regions. Higher magnification images of the tail bud region showed colocalization of d2GFP protein and mRNA and phospho-Smad1/5/8 immunoreactivity in some cells (arrowheads), although others expressed only d2GFP protein or mRNA, or phospho-Smad1/5/8. G–J: Expression of transcripts for known BMP-regulated targets id1, hey1, and msxE (H–J) were found in regions associated with the BRE promoter (G).

Figure 3. Expression of enhanced green fluorescent protein (eGFP), destabilized eGFP (d2GFP), or Kusabira-Orange 2 (KO2)-PEST driven by the bone morphogenic protein response element (BRE) promoter lines during larval development. A,C,E,G: The BRE:eGFP was expressed in the dorsal developing eye (e), somites (s), tail (t), heart (h) and pineal (p) during the first 5 days post fertilization. B,D,F,H: Similarly, d2GFP was found in the same areas, but at lower levels. I–K: Annotated expression of enlarged view of 3 dpf BRE:eGFP transgenic zebrafish larvae (I), BRE:d2GFP transgenic zebrafish larvae (J), and BRE:KO2-PEST transgenic zebrafish larvae (K). Montaging artifacts are visible in the trunk region at the border of adjacent image frames.

Figure 4. Diverse tissue expression of enhanced green fluorescent protein (eGFP) or destabilized eGFP (d2GFP) in bone morphogenic protein response element (BRE) transgenic lines. A: Expression of eGFP in cranial vasculature of 2 day postfertilization (dpf) BRE:eGFP larvae. B: Similar expression of d2GFP in 2 dpf cranial vasculature of BRE:d2GFP larvae. C: Expression of d2GFP in intersomitic vasculature of 2 dpf BRE:d2GFP larvae. D: Expression of eGFP in 2 dpf somite muscle cells and underlying notochord cells of BRE:eGFP larvae. E–H: Higher magnification of boxed regions in A–D. Arrows indicate the vascular structures in E, F, and G, and the somite muscle cells in H. I: Expression of eGFP in pineal cells of 5 dpf BRE:eGFP larvae (arrow). J: Expression of eGFP in somite muscle (s) and dorsal spinal neurons (arrows) of 5 dpf BRE:eGFP larvae. K: Expression of eGFP in medial longitudinal fasciculus axons (arrow) of 5 dpf BRE:eGFP larvae. L: Expression of eGFP in midbrain–hindbrain neurons (arrow) of 5 dpf BRE:eGFP larvae. Note the ependymal cells that are also weakly eGFP-positive (asterisks). M: Expression of d2GFP in pineal cells of 5 dpf BRE:d2GFP larvae (arrow). N: Expression of d2GFP in somite muscle (s) and dorsal spinal neurons (arrows) of 5 dpf BRE:eGFP larvae. O: Expression of d2GFP in medial longitudinal fasciculus axons (arrow) of 5 dpf BRE:d2GFP larvae. P: Expression of d2GFP in hindbrain neural progenitor cells (arrow) of 5 dpf BRE:d2GFP larvae.

Figure 5. Bone morphogenic protein response element:enhanced green fluorescent protein (BRE:eGFP) in adult fish. A: eGFP expression viewed in whole fish. B: Dissected kidney showing eGFP expression. C,D: Higher magnification of proximal(C) and distal (D) regions (white brackets)

Figure 6. Expression of enhanced green fluorescent protein (eGFP) in regenerating fins of bone morphogenic protein response element:eGFP (BRE:eGFP) adult fish. A–F: BRE:eGFP expression in adult caudal fins before and during regeneration. Times post-amputation are indicated above each image. Note that BRE:eGFP expression is increased in the regenerating fin tissue following amputation. G: Magnified view of 7 day post-amputation blastemal cells. H–N: Corresponding transmitted light images are shown below.

Figure 7. Adult ocular bone morphogenic protein response element:enhanced green fluorescent protein (BRE:eGFP) expression revealed in cryosections. A: BRE:eGFP expression was obvious in the lens and dorsal ciliary margin, but not in the central retina (r). B: Transmitted light image of A. C: BRE:eGFP expression in the ciliary marginal zone (cmz) and anterior stromal and vascular cells (as, arrows). D: Transmitted light image of C. E: BRE:eGFP expression in dorsal retina showing Müller glia (mg) and scleral cells (s). F: Transmitted light image of E. G: BRE:eGFP expression in central retina showing choroidal rete vascular cells (cv). Note the lack of glial expression in the central retina. H: Transmitted light image of G. Scale bars = 200 μm in A,B, 50 μm in C–H

Figure 8. BRE:eGFP expression in adult dorsal Müller glia. A: Central transverse cryosection of an adult bone morphogenic protein response element:enhanced green fluorescent protein (BRE:eGFP) eye. Note that the lens was not imaged as part of the montage. Müller glia (mg); optic nerve head (onh). Region showing eGFP-positive Müller glia is indicated with a white bracket. B: Higher magnification of BRE:eGFP in dorsal Müller glia. C: Expression of glutamine synthetase immunoreactivity (anti-GS), a Müller glia marker, in the same section as B. D: Colocalization of B and C.

Figure 9. Bone morphogenic protein response element:enhanced green fluorescent protein (BRE:eGFP) expression in adult tissues revealed through cryosections. A: BRE:eGFP expression in the atrium and ventricle of the heart (dotted line) and in the gill filaments. B: BRE:eGFP expression in the vasculature of the brain. C,D: Higher magnification images of the (C) cardiac atrium and (D) gill filaments.

Figure 10. Destabilized enhanced green fluorescent protein (d2GFP) levels are altered when BMP activity is manipulated. A–C: Overexpression of Bmp2b (B) driven by the inducible hsp70 heat-shock promoter increases d2GFP expression levels relative to endogenous BRE:d2GFP in control larvae (A) at 24 hpf when imaged 6 hours after a 30 minute heat shock at 37°C. Note the loss of the dorsal high-ventral low gradient pattern in the eye which is replaced by a uniformly high level of d2GFP expression. Conversely, inhibition of BMP signaling by hsp70 overexpression of Noggin3 downregulates d2GFP expression (C). The embryo position is indicated by dashed lines. D–I: At 3 dpf, overexpression of Bmp2b increases both d2GFP and pSmad1/5/8 immunoreactivity relative to normal levels, particularly in the jaw (arrow) and pectoral fin (arrowhead). J–O: In the 3 dpf eye, Bmp2b overexpression increased d2GFP expression and pSmad1/5/8 immunoreactivity.

Figure 11. Destabilized enhanced green fluorescent protein (d2GFP) expression driven by the bone morphogenic protein response element (BRE) promoter is down-regulated by dorsomorphin. A–D: Larvae expressing BRE:d2GFP were treated with dimethyl sulfoxide (DMSO) as a control (B,D) or with 50 μM dorsomorphin (A,C) at 24 hpf for 3 hr. The d2GFP expression was reduced, particularly in the head and eye following treatment. Original magnification: ×10 in A,B; ×40 in C,D.

Supp. Fig. S2. A–F: Treatment of 13- to 17-somite embryos with 50 μM dorsomorphin, or dimethyl sulfoxide (DMSO) control, for 3 hr down-regulates mRNA levels of bone morphogenic protein (BMP) -regulated genes by varying degrees: strongly, id1 (A,D); moderately, msxE (B,E); no observed change, hey1 (C,F).