12 research outputs found
The TATGARAT box of the HSV-1 ICP27 gene is essential for immediate early expression but not critical for efficient replication in vitro or in vivo
Bmp Indicator Mice Reveal Dynamic Regulation of Transcriptional Response
<div><p>Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among <em>Drosophila, Xenopus</em> and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.</p> </div
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Bmp Indicator Mice Reveal Dynamic Regulation of Transcriptional Response
BRE-gal reporter activity in the mammary buds and vibrissal follicles of an E13.5 mouse embryo.
<p>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 Β΅m 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.</p
BRE-gal reporter activity in mouse embryos at pre-primitive streak and early primitive streak stages (E5.5β6.5).
<p>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 Β΅m 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 Β΅m.</p
The BRE-gal reporter mES cell line can respond to various Bmp ligands.
<p>(A) The <i>Xid3</i> BRE consists of a Smad1 binding site (5β²-GACGCC-3β²) and a highly-conserved Smad Binding Element (SBE, 5β²-GTCTG-3β²) 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 20Γ. 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 <i>lacZ</i> expression was quantified using an enzymatic assay with the colorimetric lactose analog ONPG.</p
BRE-gal reporter activity during mid-gestation stage BRE-gal mouse embryos (E8.75β12.5).
<p>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 Β΅m.</p
BRE-gal reporter activity in mouse embryos at headfold stages (E7.5β8.0).
<p>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 (E7.5βE8.0) 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 (E7.5βE8.0) 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 Β΅m 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 Β΅m.</p
BRE-gal reporter activity during mid-gestation stage BRE-gal mouse embryos (E8.75β12.5).
<p>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 (A2βA5). By E10.5, the maxillary and mandibular components of the first branchial arch are apparent (A4βA5). 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 (B2βB5). At E9.5, the forelimb buds can be seen protruding laterally from the trunk and continue to grow outward (C2βC3, 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 C2βC3, distal edge outlined with solid orange line). By E10.5, the forelimb buds are more prominent (C4βC5, dorsal view). By E12.5, the future digits of the handplate are visible (C6, dorsal view). The apical ectodermal ridge (inset, C4βC6) 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.</p