13 research outputs found
Robust and non-mosaic BRE-gal expression in dorsal telencephalic midline.
<p>(<b>A</b>) Schematic of the BRE-gal construct showing the 16 bp core of the <i>XId3</i> BRE with Smad1 and Smad4 binding sites separated by 5 bp, which allows for docking by the co-factor Schnurri. Seven concatemerized BREs, all in sense orientation, are upstream of the <i>XId3</i> minimal promoter and nuclear lacZ coding sequence. (<b>B–F</b>) Xgal stains of E8.5-E12.5 whole mount embryos. In the forebrain, BRE-gal expression is initiated in the E8.5 neural folds (nf) (B) and is strongly expressed in the DTM through E12.5 (C–F). Scale bars: 0.5 mm. (<b>G–I</b>) Xgal stains of E10.5–E12.5 coronal cryosections. BRE-gal expression is robust and non-mosaic in the DTM (hem and cpe). Scattered labeling is also seen in the E11.5 and E12.5 marginal zone (H,I). Scale bars: 200 um. (<b>J–L</b>) Xgal or IHC of E12.5 coronal cryosections. BRE-gal expression coincides with Lmx1a, but not with Lhx2, indicating that the BRE-gal expression border approximates the cortex-hem boundary (CHB). Scale bar: 50 um. Abbr: cpe, choroid plexus epithelium; cpl, choroid plaque; cx, cortex; fb, forebrain; ge, ganglionic eminence; hc, hippocampal primordium; hb, hindbrain; mb, midbrain; nc, neocortical primordium; nf, neural folds; rp, roof plate; SBE, Smad binding element; tv, telencephalic vesicle.</p
BRE-gal coexpression with other Bmp signaling readouts in the DTM.
<p>Xgal or IHC of E10.5 (A) and E12.5 (B) coronal sections. BRE-gal is sharply restricted to the DTM, and excluded from cortex and mesenchyme. pSmad is present in the neuroepithelium (graded from high dorsomedial to low ventrolateral) and in the mesenchyme at both stages. Like pSmad, but in contrast to BRE-gal, Msx1-nlacZ is expressed in both the neuroepithelium and mesenchyme. Within the neuroepithelium, the Msx1-nlacZ expression domain is smaller than the BRE-gal domain at both stages. Scale bars: 100 um. Arrows designate mesenchyme; arrowheads designate the cortex-hem boundary. Abbr: cpe, choroid plexus epithelium; cx, cortex; hc, hippocampal primordium; nc, neocortical primordium; rp, roof plate.</p
Absence of BRE-gal or increased pSmad levels in ectopic hem cells.
<p>IHC of coronal sections from E12.5 Lhx2 null chimeras, confocal images. (<b>A</b>) The normal hem (towards bottom of panels) expresses BRE-gal and Lmx1a, but not Lhx2. Lhx2 null patches (white lines) seen in adjacent sections of the medial pallium upregulate Lmx1a, but do not express BRE-gal. Scale bar: 100 um. (<b>B</b>) pSmad levels (red) are not elevated in Lhx2 null patches in the medial pallium (white dashed lines). Scale bar: 50 um. (<b>C</b>) Monochrome pSmad images shown in (B) and boxplots of their relative pSmad intensities (normalized to Hoechst intensity). pSmad intensity is lower in “Lhx2-off” (Lhx2 null) hem cells compared to adjacent “Lhx2-on” medial pallial progenitors (p<0.0001 for both sections). Non-normalized pSmad intensity values were also statistically significant (p = 0.007 for patches away from endogenous hem (top of panel), p = 0.0001 for patches closer to hem (bottom of panel)). Scale bar: 50 um.</p
BRE-gal induction in the postnatal hippocampus.
<p>(<b>A</b>) Xgal stains of P7, P29, and adult (10 months) sagittal sections, with eosin counterstain. Although BRE-gal is absent from the hippocampal anlagen before E17.5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044009#pone-0044009-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044009#pone.0044009.s001" target="_blank">S1A</a>), BRE-gal expression becomes detectable in the dentate gyrus (DG) and cornu ammonis 2 (CA2) field by P7. Expression in these regions then becomes strong and stable in adults. (<b>B</b>) pSmad/BRE-gal IHC of adult sagittal sections. Like BRE-gal, pSmad is expressed in the DG and CA2 regions, but is also expressed in CA3 and CA1. In the DG, virtually all BRE-gal-expressing cells also label for pSmad (right panel). However, some pSmad-positive cells in subgranular and supragranular zones of the DG are BRE-gal-negative (inset; arrows). (<b>C</b>) IHC of adult sagittal sections, confocal images. Nearly all Prox1-expressing DG granule neurons are BRE-gal positive. A few BRE-gal+/Prox1- cells are also observed (arrowheads). BRE-gal is not expressed by Sox2-positive neural stem cells of the subgranular zone (SGZ), although coexpression is seen in some hilar cells (asterisk). (<b>D</b>) IHC of adult sagittal sections, confocal images. Lhx2 is expressed in a subset of Sox2-positive neural stem cells (arrowheads in right panel), but not in BRE-gal-positive granule neurons. Scale bars: 200 um (A,B), 50 um (inset in B,C,D). Abbr: DG, dentate gyrus; CA, cornu ammonis; cpe, choroid plexus epithelium; hcf, hippocampal fissure.</p
Ultrasensitive BRE-gal induction by Bmp4 <i>in vitro</i>.
<p>(<b>A</b>) Xgal stains, dissociated E12.5 cortical cells. BRE-gal expression displays a threshold effect, with strong activation occurring between 16–32 ng/ml. At all concentrations, BRE-gal induction is highly mosaic. Scale bar: 200 um. (<b>B</b>) RT-qPCR, dissociated E12.5 cortical cells. BRE-gal activation is consistent and highly sigmoidal (three independent cultures graphed separately), with an EC50 of 42.23 ng/ml (+/−0.15) and a Hill coefficient of 6.85 (+/−0.08).</p
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 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.</p
Sections showing BRE-gal reporter activity in various regions of mid-stage BRE-gal mouse embryos.
<p>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.</p
BRE-gal reporter activity during mid-gestation stage BRE-gal mouse embryos (E8.75–12.5).
<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 (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.</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 (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 <i>lacZ</i> expression was quantified using an enzymatic assay with the colorimetric lactose analog ONPG.</p
BRE-gal reporter activity in mouse embryos at headfold stages (E7.5–8.0).
<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 (E7.5–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.</p