Javier AL , Doan LT , Luong M , Reyes de Mochel NS , Sun A , Monuki ES , Cho KW .

R01HD056219 NICHD NIH HHS , R01 HD056219 NICHD NIH HHS , P30 CA062203 NCI NIH HHS

	Figure 2. The BRE-gal reporter mES cell line can respond to various Bmp ligands.(A) The Xid3 BRE consists of a Smad1 binding site (5′-GACGCC-3′) and a highly-conserved Smad Binding Element (SBE, 5′-GTCTG-3′) for Smad4 binding, separated by a 5-nucleotide spacer. In the diagram, the Smad binding sites are indicated in red and underlined. (B) BRE-gal mES cells were treated with the indicated Bmp ligands, and then stained with X-gal. Column 1 shows reporter response without addition of exogenous Bmp ligand to the culture media. Column 2 shows reporter response after addition of Bmp ligand. Bmp4 and Bmp4/7 were added at 10 ng/ml, and Bmp7 was added at 50 ng/ml. Magnification is at 20×. It should be noted that (C) BRE-gal mES cells were treated with the indicated growth factors and concentrations. There is an increased, dose-dependent response to Bmp2 and Bmp4, compared to other growth factors. (D) BRE-gal mES cells respond more strongly to Bmp4 than Bmp7 at each indicated concentration. Reporter cells were treated with recombinant hBmp4 or hBmp7 for 24 hours at the indicated concentrations. Quantification of lacZ expression was quantified using an enzymatic assay with the colorimetric lactose analog ONPG.
	Figure 3. BRE-gal reporter activity during mid-gestation stage BRE-gal mouse embryos (E8.75–12.5).Wholemount X-gal staining of BRE-gal mouse embryos showed dynamic BRE-dependent Bmp signaling in various regions and tissues throughout development. Embryos are shown in left, lateral views (A1, B1, C1, D1, E1, F1). At E8.75, a dorsal-oblique view of open neural folds (nf) at the site of the future midbrain and hindbrain is shown (A2). Dorsal views of the neural tube are shown (B2, C2, D2, E2, F2). Abbreviations: d, diencephalon; e, eye; er, ear; fb, forebrain; fg, foregut; fl, forelimb bud; h, heart; hb, hindbrain; hl, hindlimb bud; mb, midbrain; nf, neural folds; nt, neural tube; ov, otic vesicle; pa, pharyngeal arch(es); s, somites; t, tailbud; tv, telencephalic vesicles; v4, fourth ventricle. Scalebar 0.5 µm.
	Figure 4. Sections showing BRE-gal reporter activity in various regions of mid-stage BRE-gal mouse embryos.Transverse sections (10–12 µm thickness) of wholemount X-gal stained BRE-gal embryos (E9.5–E12.5) are shown. The dorsal neural tube in the rostral region is shown, with dorsal facing up (A1, B1, C1, D1). The right pharyngeal arches are shown (A2, B2, C2). Abbreviations: md, mandibular component of first branchial arch; mx, maxillary component of first branchial arch; ne, neural ectoderm; pa1, first pharyngeal arch.
	Figure 5. BRE-gal reporter activity during mid-gestation stage BRE-gal mouse embryos (E8.75–12.5).Magnified view of various structures in wholemount X-gal stained BRE-gal embryos are shown. Only the first pharyngeal arch is present at E8.75 (A1), and the second pharyngeal arch follows (A2–A5). By E10.5, the maxillary and mandibular components of the first branchial arch are apparent (A4–A5). At E12.5, the vibrissal follicle placodes (shown within the black, dashed box) appear on the snout, which develops from the maxillary component of the first branchial arch (A6). While the heart tube (B1) forms and loops, the atria and ventricles start to develop as well (B2–B5). At E9.5, the forelimb buds can be seen protruding laterally from the trunk and continue to grow outward (C2–C3, dorsal faces right, indicated with a solid orange line; ventral faces left, indicated with a dashed orange line). BRE-dependent Bmp activity appears primarily on the dorsal side (inset C2–C3, distal edge outlined with solid orange line). By E10.5, the forelimb buds are more prominent (C4–C5, dorsal view). By E12.5, the future digits of the handplate are visible (C6, dorsal view). The apical ectodermal ridge (inset, C4–C6) runs along the dorsal-ventral boundary of the forelimb. Abbreviations: a, atrium; avc, atrioventricular canal; e, eye; lv, left ventricle; nf, neural folds; mx, maxillary component of first branchial arch; nt, neural tube; pa1, first pharyngeal arch; pa2, second pharyngeal arch.
	Figure 6. BRE-gal reporter activity in the mammary buds and vibrissal follicles of an E13.5 mouse embryo.A wholemount X-gal stained BRE-gal mouse embryo at E13.5 is shown in a right, lateral view (A). The upper, right dashed box in (A) indicates the area that is magnified in (B1), and a transverse section of a vibrissal follicle is shown in (B2). The lower, left dashed box in (A) indicates the area that is magnified in (C1), and a transverse section of a mammary bud is shown in (C2). Sections are 12 µm thickness. Abbreviations: cms, condensing mesenchyme; fl, forelimb bud; hl, hindlimb bud; mb, mammary buds; mb2, second mammary bud on right side; mb3, third mammary bud on right side; ms, mesenchyme; s, somites; se, surface ectoderm; vf, vibrissal follicles. Scalebar 1 mm.
	Figure 7. BRE-gal reporter activity in mouse embryos at pre-primitive streak and early primitive streak stages (E5.5–6.5).Wholemount X-gal staining of BRE-gal mouse embryos showed BRE-dependent Bmp signaling in extra-embryonic structures. A pre-primitive streak embryo (E5.5) is shown in (A), with a corresponding sagittal section (B). The plane of this section is slightly oblique. An early primitive streak embryo (E6.5) is shown in (C), with corresponding transverse sections through the (D1) extraembryonic and (D2) embryonic regions. The dashed lines in (C) indicate the plane of the sections shown in (D1, D2). All sections are slightly enlarged with respect to the corresponding image of the whole embryo. Sections are 10 µm thickness. Abbreviations: de, decidua; e, embryonic region; ec, ectoplacental cone; eec, embryonic ectoderm; pc, proamniotic cavity; x, extraembryonic region; xec, extraembryonic ectoderm; xen, extraembryonic visceral endoderm. Scalebar 200 µm.
	Figure 8. BRE-gal reporter activity in mouse embryos at headfold stages (E7.5–8.0).Wholemount X-gal staining of BRE-gal mouse embryos showed BRE-dependent Bmp signaling in embryonic and extra-embryonic structures. Anterior view of early headfold stage embryo (E7.5–E8.0) is shown in (A1), with a corresponding lateral view (A2), with anterior at left. Transverse sections of the early headfold stage embryo are shown in (B1, B2, B3). Anterior view of late headfold stage embryo (E7.5–E8.0) is shown in (C1), with a corresponding lateral view (C2), with anterior at left. Transverse sections of the late headfold stage embryo are shown in (D1, D2, D3). All sections are 10 µm thickness. Abbreviations: ac, amniotic cavity; af, amniotic fold; al, allantois; am, amnion; cd, chorionic dome; ec, ectoplacental cone; eec, embryonic ectoderm; fg, foregut diverticulum; hf, headfolds; ms, mesenchyme; ne, neural ectoderm; ng, neural groove; ps, site of primitive streak; ses, surface ectoderm and somatopleure; xc, exocoelomic cavity; xen, extraembryonic visceral endoderm. Scalebar 200 µm.
	Figure 1. BRE-mediated responsiveness depends on the five nucleotide spacer between Smad binding sites.(A) Luciferase reporter constructs were generated with either the wildtype BRE sequence (BRE-luc WT) containing a five nucleotide (nt) spacer, or a mutant BRE sequence (BRE-luc MT) containing a two nt deletion in the spacer. In the diagram, the Smad binding sites are underlined. (B) Transient luciferase assays showed that Bmp responsiveness in mES cells was abrogated if the length of the spacer is decreased. BRE-luc WT and BRE-luc MT constructs were transfected into wildtype mES cells. Twenty-four hours after transfection, the cells were treated with or without Bmp4 at 10 ng/ml for six hours. BRE-mediated responsiveness in BRE-luc WT mES cells increased after treatment with Bmp4.

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