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.

Ahn, BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. 2001, Pubmed

Ahn, BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. 2001, Pubmed
Alexander, Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton. 2011, Pubmed , Xenbase
Blitz, Finding partners: how BMPs select their targets. 2009, Pubmed
Bosman, Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels. 2006, Pubmed
Cai, Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2. 2011, Pubmed
Chesnutt, Coordinate regulation of neural tube patterning and proliferation by TGFbeta and WNT activity. 2004, Pubmed
Correia, Bmp2 is required for migration but not for induction of neural crest cells in the mouse. 2007, Pubmed
Coucouvanis, BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. 1999, Pubmed
Danesh, BMP and BMP receptor expression during murine organogenesis. 2009, Pubmed
Downs, Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. 1993, Pubmed
Dunn, Haploinsufficient phenotypes in Bmp4 heterozygous null mice and modification by mutations in Gli3 and Alx4. 1997, Pubmed , Xenbase
Feng, Specificity and versatility in tgf-beta signaling through Smads. 2005, Pubmed
Fernandes, Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly. 2007, Pubmed
Finley, BMP-4 inhibits neural differentiation of murine embryonic stem cells. 1999, Pubmed
Fujiwara, Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse. 2001, Pubmed
Furuta, Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. 1997, Pubmed
Gao, Dpp-responsive silencers are bound by a trimeric Mad-Medea complex. 2005, Pubmed
Guha, In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. 2002, Pubmed
Hata, Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. 1998, Pubmed , Xenbase
Hawley, Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction. 1995, Pubmed , Xenbase
Hayashi, SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast. 2002, Pubmed
Hayashi, The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. 1997, Pubmed
Hens, Key stages of mammary gland development: molecular mechanisms involved in the formation of the embryonic mammary gland. 2005, Pubmed
Holzschuh, Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish. 2005, Pubmed
Ille, Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord. 2007, Pubmed
Imamura, Smad6 inhibits signalling by the TGF-beta superfamily. 1997, Pubmed
Johnson, Interaction of Smad complexes with tripartite DNA-binding sites. 1999, Pubmed
Kaartinen, Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells. 2004, Pubmed
Kawakami, BMP signaling during bone pattern determination in the developing limb. 1996, Pubmed
Kim, Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. 1997, Pubmed
Klaus, Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. 2007, Pubmed
Korchynskyi, Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. 2002, Pubmed
Kühn, Generation and analysis of interleukin-4 deficient mice. 1991, Pubmed
Larraín, Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling. 2001, Pubmed , Xenbase
Lawson, Bmp4 is required for the generation of primordial germ cells in the mouse embryo. 1999, Pubmed
Liem, A role for the roof plate and its resident TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord. 1997, Pubmed , Xenbase
Lorda-Diez, Expression of Id2 in the developing limb is associated with zones of active BMP signaling and marks the regions of growth and differentiation of the developing digits. 2009, Pubmed
Luo, BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. 1995, Pubmed
Lyons, Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. 1995, Pubmed
Lyons, Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). 1990, Pubmed
Macias, Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development. 1997, Pubmed
Madabhushi, Anterior visceral endoderm directs ventral morphogenesis and placement of head and heart via BMP2 expression. 2011, Pubmed
Mailleux, Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. 2002, Pubmed
Maretto, Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. 2003, Pubmed
Mikkola, Genetic basis of skin appendage development. 2007, Pubmed
Miyazono, BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. 2005, Pubmed
Monteiro, Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins. 2004, Pubmed
Moustakas, Non-Smad TGF-beta signals. 2005, Pubmed
Nakahiro, Identification of BMP-responsive elements in the mouse Id2 gene. 2010, Pubmed
Nakao, Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. 1997, Pubmed , Xenbase
Nakayama, Smad6 functions as an intracellular antagonist of some TGF-beta family members during Xenopus embryogenesis. 1998, Pubmed , Xenbase
Oelgeschläger, Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos. 2003, Pubmed , Xenbase
Ohuchi, Fibroblast growth factor 10 is required for proper development of the mouse whiskers. 2003, Pubmed
Piccolo, Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. 1996, Pubmed , Xenbase
Pizette, BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes. 2000, Pubmed
Pyrowolakis, A simple molecular complex mediates widespread BMP-induced repression during Drosophila development. 2004, Pubmed
Roelink, Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. 1994, Pubmed , Xenbase
Roelink, Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. 1995, Pubmed
Schultheiss, A role for bone morphogenetic proteins in the induction of cardiac myogenesis. 1997, Pubmed
Selever, Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development. 2004, Pubmed
Shi, Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. 1998, Pubmed
Tomás, FLRT3 as a key player on chick limb development. 2011, Pubmed
Tremblay, Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. 2001, Pubmed , Xenbase
von Bubnoff, Intracellular BMP signaling regulation in vertebrates: pathway or network? 2001, Pubmed , Xenbase
von Bubnoff, Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes. 2005, Pubmed , Xenbase
Watson, Mammary development in the embryo and adult: a journey of morphogenesis and commitment. 2008, Pubmed
Winnier, Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. 1995, Pubmed
Xu, Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm. 1998, Pubmed
Yao, Schnurri transcription factors from Drosophila and vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism. 2006, Pubmed , Xenbase
Ybot-Gonzalez, Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling. 2007, Pubmed
Ying, BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. 2003, Pubmed
Ying, Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse. 2001, Pubmed
Zawel, Human Smad3 and Smad4 are sequence-specific transcription activators. 1998, Pubmed
Zhang, Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. 1996, Pubmed
Zhao, FGF mediated Sulf1 regulation. 2007, Pubmed
Zimmerman, The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. 1996, Pubmed , Xenbase
Zou, Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage. 1997, Pubmed