Journal: Nature Communications

Article Title: Organizer-derived Bmp2 is required for the formation of a correct Bmp activity gradient during embryonic development

doi: 10.1038/ncomms4766

Figure Lengend Snippet: ( a – c ) Chd protein distribution detected by confocal microscopy at different stages. Embryos injected with Tg(−2.067gsc:tBr-gfp) or Tg(−2.272bmp2b:tBr-gfp) DNA were harvested along with uninjected embryos at indicated stages for immunostaining using an anti-Chordin antibody and DAPI staining. Representative z-stack images were shown in animal-pole view with dorsal to the right. ( d – g ) The average ratios ( e – g ) of Chd to DAPI signals at the ventralmost or dorsalmost areas on optical equatorial sections at the blastodermal margin as illustrated in d . The strategy for estimating relative intensity was the same as described in the legend of . For each group, the average relative immunostaining intensity was calculated from 3 to 5 embryos and three optical sections at and nearby the blastodermal margin per embryo. The error bars represented s.d. The Chd/DAPI ratio in the ventralmost region at each stage was set to 1.0. Statistical significance (Student’s t -test): NS, nonsignificant ( P >0.05); * P <0.05; ** P <0.01. ( h – k ) chd transcripts distribution detected by in situ hybridization at indicated stages. Representative embryos were animal-pole views with dorsal to the right. The ratio of embryos with representative pattern was indicated for those shown in h – j ( l ) The ratio of embryos showing an expansion of chd expression at the shield stage (as shown in k ) n , number of observed embryos.

Article Snippet: The used antibodies for immunofluorescence were as follows: rabbit anti-zebrafish Bmp2b(NT) antibody (Anaspec, 1:50 diluted), rabbit anti-zebrafish Chordin (GeneTex, 1:50 diluted), mouse anti-Smad1 antibody (Santa Cruz), rabbit anti-phospho-Smad1/5/8 (Cell Signaling, 1:100 diluted), mouse anti-β-catenin antibody (Cell signaling, 1:100 diluted), sheep anti-Digoxigenin antibody (Roche, 1:4,000 diluted), DyLight 488-conjugated AffiniPure Goat anti-mouse IgG and DyLight 549-conjugated AffiniPure goat anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch, 1:100 diluted).

Techniques: Confocal Microscopy, Injection, Immunostaining, Staining, In Situ Hybridization, Expressing

Journal: Current biology : CB

Article Title: The Drosophila pioneer factor Zelda modulates the nuclear microenvironment of a Dorsal target enhancer to potentiate transcriptional output

doi: 10.1016/j.cub.2019.03.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-DL antibody (7A4) , Developmental Studies Hybridoma Bank , Cat# anti-Dorsal 7A4, RRID:AB_528204.

Techniques: Recombinant, Labeling, Hybridization, Plasmid Preparation, Software, Microscopy

Journal: REPRODUCTION

Article Title: Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

doi: 10.1530/rep-11-0150

Figure Lengend Snippet: Figure 3 Immunohistochemical staining of bovine ovary sections showing the distribution of chordin, gremlin, noggin and follistatin immunoreactivity (brown) in the granulosa and theca layer of small antral follicles (w3 to 5 mm diameter). In eachpanel the left-hand image shows an antibody-treated section while the right-hand image shows an adjacent section treated with the appropriate non-immune serum or purified IgG. The scale bar in each right panel represents 50 mm.

Article Snippet: The following primary antibodies (and appropriately matched control serum/purified IgG for adjacent tissue section) were diluted in blocking buffer and incubated with sections overnight at 4 8C: goat anti-mouse gremlin (R&D Systems) at 5 mg/ml, goat anti-mouse chordin (R&D Systems) at 5 mg/ml, rabbit anti-human noggin (Abcam Ltd, Cambridge, UK) at 5 mg/ml and rabbit anti-human follistatin (FP22; supplied by NIDDK, Baltimore, MD, USA) at 1:500 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural crest origin of olfactory ensheathing glia

doi: 10.1073/pnas.1012248107

Figure Lengend Snippet: Chick neural crest cells form OECs along the length of the olfactory nerve from the lamina propria to the olfactory bulb. Isotopic grafts of GFPchick midbrain-level neural fold at the 5 ss label migratory NCCs. At E10.25, p75NTR-positive OECs are associated with p75NTR-positive olfactory axons/neurons in the lamina propria (A and B). These p75NTR-positive OECs, like the rest of the lamina propria and the cribriform plate, are GFPchick NCC-derived (arrowheads highlight examples) (A1, A2, B1, and B2). Similarly, the p75NTR-positive OEC processes ensheathing olfactory axon bundles in the olfactory nerve (C and D) are from GFPchick NCC-derived cells (C1, C2, D1, and D2), whose cell bodies can be seen as brighter patches of immunofluorescence between the axon bundles (the p75NTR immunoreactivity is confined to their cell processes). P0-positive OEC processes (E) are also from GFPchick NCC-derived cells (E1 and E2). (F) Low-power composite view showing that GFPchick NCC-derived cells not only surround the olfactory bulb but also are found throughout the ONL. [The scattered GFPchick NCC-derived cells in the olfactory bulb are smooth muscle actin-positive pericytes associated with the forebrain vasculature (also see Fig. S3).] (G–G2) Higher-power view showing GFPchick NCC-derived cells throughout the ONL, with cells in the outer layer expressing both p75NTR (blue) and P0 (red), and cells in the inner layer expressing P0 but not p75NTR. (H–H2) In a different E10.25 embryo, the olfactory bulb is surrounded by GFPchick NCC-derived cells, and almost all of the P0-positive OECs inside the olfactory bulb are GFPchick NCC-derived (arrowheads highlight examples). Arrows highlight some P0-positive OECs that are not GFPchick graft-derived; these are likely to be derived from host NCCs. CP, cribriform plate; LP, lamina propria; nβ3-tub, neuronal β-III tubulin; OE, olfactory epithelium; ON, olfactory nerve; ONL, olfactory nerve layer.

Article Snippet: Primary antibodies used were the following: anti-GFP (rabbit; Invitrogen), FITC-conjugated anti-GFP (goat; Abcam), anti-neuronal β-III tubulin (TuJ1, mouse IgG2a; Invitrogen), anti-P0 [1E8, mouse IgG1, Developmental Studies Hybridoma Bank (DSHB), developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242], anti-chick p75 NTR and anti-mouse p75 NTR (both rabbit; gifts of L. Reichardt, University of California at San Francisco), anti-quail (QCPN, mouse IgG1; gift of M. Bronner-Fraser, Caltech, Pasadena, CA; also available from the DSHB), and anti-smooth muscle actin (mouse IgG2a; Sigma).

Techniques: Derivative Assay, Immunofluorescence, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neural crest origin of olfactory ensheathing glia

doi: 10.1073/pnas.1012248107

Figure Lengend Snippet: Avian neural crest cells form OECs on the olfactory nerve. Isotopic grafts of GFPchick (A–B2 and E–F2) or quail (C–D) midbrain-level neural fold at the 5-ss label migratory NCCs that colonize the olfactory nerve as well as the surrounding frontonasal mesenchyme. At E4.5 (A–B2), nonneuronal NCCs are associated with the olfactory nerve. (Faint green staining in the apical olfactory epithelium is background.) At E5.5 (C–D), many Sox10-positive cells are seen on the olfactory nerve on the grafted side of the embryo. Quail NCC-derived cells (green nuclei) on the olfactory nerve express Sox10 (D). At E6.5 (E–F2), a significant proportion of olfactory nerve-associated GFPchick NCCs and their processes express the OEC marker P0 (arrowheads, F–F2) (22). nβ3-tub, neuronal β-III tubulin; OB, olfactory bulb; OE, olfactory epithelium; ON, olfactory nerve.

Article Snippet: Primary antibodies used were the following: anti-GFP (rabbit; Invitrogen), FITC-conjugated anti-GFP (goat; Abcam), anti-neuronal β-III tubulin (TuJ1, mouse IgG2a; Invitrogen), anti-P0 [1E8, mouse IgG1, Developmental Studies Hybridoma Bank (DSHB), developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242], anti-chick p75 NTR and anti-mouse p75 NTR (both rabbit; gifts of L. Reichardt, University of California at San Francisco), anti-quail (QCPN, mouse IgG1; gift of M. Bronner-Fraser, Caltech, Pasadena, CA; also available from the DSHB), and anti-smooth muscle actin (mouse IgG2a; Sigma).

Techniques: Staining, Derivative Assay, Marker

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: Representative confocal images of an E8.5 embryo labelled by immunofluorescence for Sox2 and Brachyury (n=3 embryos). (a) Wholemount E8.5 and (b-d) are transverse sections at the level of the CLE domain (demarcated by the white dotted line in (a)). Sox2 labels the neuroepithelium along the A/P axis and the CLE whereas Brachyrury labels the PSM and tailbud mesoderm. (e) is a magnification of (d) and shows the location of the NMPs (Sox2/Brachyury co-expressing cells) in the CLE epithelium. Representative in situ hybridisation images of E8.5 embryos stained for mRNA expression of (a) cMyc (n= 10 embryos) and (b) MycN (n=4 embryos) (c) Fgf8 (n=3 embryos), (d) Wnt3a (n=3 embryos) and (e) LFringe (n=5 embryos). (a’-e’) are transverse sections of the CLE and underlying cPSM domain indicated by white dotted lines on (a-e). (a’) cMyc , (b’) Fgf8 and (c’) Wnt3a show high levels of expression in the CLE. (b’) MycN and (e’) LFringe show high levels of expression in the cPSM. Representative confocal images of immunofluorescence labelling of E8.5 embryos for cMyc and Sox2 (a-g; n=3 embryos) and cMyc and Brachyury (h-n; n= 3 embryos). Sox2/cMyc co-expressing cells are evident in the transverse sections of the CLE (e’-g’) panels correspond to sections at the level of the domain demarcated by the white dotted line in (a). Brachyury/cMyc co-expressing cells are evident both in CLE and underlying cPSM (l’-m’ panels, correspond to white dotted line in (e)). Scale bars are 100 μm

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: Immunofluorescence, Expressing, In Situ, Hybridization, Staining

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: Representative in situ hybridisation of (a) wholemount E9.5 embryo labelled for cMyc at E9.5 (n=12 embryos).(b-b’) high levels of cMyc are present in the caudal most neuroepithelium and adjacent PSM (black arrowheads) (c) side view of an E10.5 tail labelled for cMyc mRNA. (d) Three different expression profiles for cMyc in the PSM of E10.5 embryos, reminiscent of the three phases of the segmentation clock gene expression (n=10 embryos). Representative confocal images of immunofluorescence labelling for cMyc in E9.5 and E10.5 embryos. (a-f) show cMyc and Brachyury staining in wholemount embryos. (a’-f’) are higher magnification images of (a-f) showing cMyc/Brachyury co-expressing cells in the tailbud (n=5 embryos). (g-l) show cMyc and Sox2 labelling in wholemount embryos and (g’-l’) are higher magnification images of (g-l) showing cMyc/Sox2 co-expressing cells in the tailbud. (n=5 embryos). Scale bars are 100 μm.

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: In Situ, Hybridization, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: Representative in situ hybridisation images on CLE/cPSM explants treated with DMSO (a-f) or 10 μΜ JQ1 (a’-f’) for 6h. Fgf8 (a-a’; n=8/8 embryos), Wnt3a (b-b’; 5/5 embryos) and Sox2 (f-f’; 3/3 embryos) expression is suppressed upon JQ1 treatment. In contrast expression of Axin2 (c-c’; n=4/4 embryos), Sprouty2 (d-d’; n=4/4 embryos) and Brachyury (e-e’; n= 6/6 embryos) is not affected. Scale bar is 100 μm. Representative in situ hybridisation images of half tail explants from E8.5 embryos (micro-dissected below the level of the last somite pair) treated either with DMSO or 10 μΜ JQ1 for 6h show no effect on expression of the NOTCH target gene LFringe (n=4/4 4mbryos). RT-qPCR analysis of CLE/cPSM explants for Hes5 expression show no differences upon treatment with 10 μΜ JQ1 or 75 μΜ 10074G5 for 6h. Scale bar is 100 μm Characterisation of gene expression changes in CLE/cPSM explants upon 10 μΜ JQ1 or 75 μΜ 10074G5 for 6h. Relative gene expression, normalised to actin levels. Data from 3 independent experiments, presented as Mean ± SEM.

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: In Situ, Hybridization, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: Representative confocal images of in vitro generated hNMPs from H9 pluripotent stem cells, show co-localisation of Sox2/Brachyury positive cells on Day3 of the differentiation protocol. Scale bar is 100 μm. qRTPCR expression analysis of Sox2 and Brachyury between Day3 and Day4 of the differentiation protocol shows that gene expression levels for these two genes remain unaltered in the 24h timeframe (data from two independent experiments, expression normalised to levels of PRT2 gene)

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: In Vitro, Generated, Expressing

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: Representative confocal images of CLE/cPSM explants cultured for 10h, either in DMSO or in 10 μΜ JQ1 and subsequently stained for pH3 by immunofluorescence(n=3 embryos). The Tunel assay was employed to analyse cell death. Apoptotic nuclei were detected in peripheral edges of both DMSO and JQ1 treated explants (n=3 embryos). No significant differences were found between DMSO and JQ1 treated explants in number of pH3 positive loci (explants micro-dissected from 3 different embryos, quantified in 18 optical sections per condition). Data presented as Mean ± SEM. CLE/cPSM explants treated in DMSO (a-d) or with 10 μΜ JQ1 (a’-d’) for 10h analysed by in situ hybridisation. At this time frame, both Sox2 (n=3/3) and Brachyury (n=7/10) expression is downregulated, however differentiation markers such as Pax6 (n=0/5 embryos) and Paraxis (n=0/3 embryos) are not expressed in either control DMSO or JQ1 treated explants. CLE/cPSM explants after 10h of culture in DMSO (a-d) or in 10 μΜ JQ1 (a’-b’). Representative in situ hybridisation images of explants treated with CT99021 (a,b,a’,b’) or 100 nM RA (c,d,c’,d’) for 14h or 20h post the 10h culture in DMSO or 10 μΜ JQ1. RT-qPCR analysis of gene expression changes in control CLE/cPSM explants cultured in DMSO or in 10 μΜ JQ1 for 10h. Relative gene expression, normalised to actin levels. Data from 3 independent experiments, presented as Mean ± SEM. Scale bars are 100 μm.

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: Cell Culture, Staining, Immunofluorescence, TUNEL Assay, In Situ, Hybridization, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: MYC activity is required for maintenance of the Neuromesodermal Progenitor signalling network and for correct timing of segmentation clock gene oscillations

doi: 10.1101/212035

Figure Lengend Snippet: (a-f) Representative images of E10.5 Control embryos (n=3 embryos) labelled by in situ hybridization for Fgf8 expression show high expression levels in the hind limbs and in the tailbud. (c) is a transverse section in the limb bud region demarcated by the white dotted line in (b). (d-f) are transverse sections of the CNH and tailbud demarcated by white dotted box in (b). (g-r) Are confocal images on transverse sections of the CNH and tailbud labelled by immunofluorescence for Sox2 and Brachyury and show large number of co-expressing cells in the tailbud mesenchyme (data from 10 confocal sections from 2 embryos). Representative images of E10.5 cMyc conditional inducible mutant embryos (n=3 embryos) labelled by in situ hybridization for Fgf8 expression (a’-f’) show high levels in the hind limbs, whereas very low levels are detected in the tailbud. (c’) is a transverse section in the limb bud region demarcated by the white dotted line in (b’). (d’-f’) are transverse sections of the CNH and tailbud demarcated by white dotted box in (b’). (g’-z’) Confocal images on transverse sections of the CNH and tailbud labelled by immunofluorescence for Sox2, Brachyury and GFP show large number of co-expressing cells in the tailbud mesenchyme. GFP cells are mostly absent from the domain of the CNH (s’), which coincides with the domain where Fgf8 expression is still detected (f’) (data from 10 confocal sections from 2 embryos). Scale bars are 100 μm.

Article Snippet: E8.5 wholemount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 (neural marker; raised in goat, Immune Systems), Brachyury (mesoderm marker; raised in rabbit, Santa Cruz biotechnology or raised in goat, R&D Systems) and Phospo Histone 3 (pH3; Upstate). cMyc positive cells were labelled using the polyclonal anti c-Myc antibody (raised in rabbit, Abcam).

Techniques: In Situ Hybridization, Expressing, Immunofluorescence, Mutagenesis