Fig 1.
Phenotypes of in vivo micro-dissected cell types at D18.
(A) Schematic view of the extra-embryonic tissues (EETs) that are in the vicinity of the embryonic tissues: ExEctoderm (or Trophoblast, in magenta), ExMesoderm (red), ExEndoderm (green). (B) Co-immuno-fluorescence and confocal microscopy (including z scans: Z) on whole D18 EETs with antibodies against reported in vivo markers for the Trophoblast (FURIN or PCSK3 [27]), ExMesoderm (VIMENTIN [66]), and ExEndoderm (AFP [67]). (C) Gene profiling using the bovine 10K array (GPL7417, [26]). All expressed genes were considered. Scale bar: 10 μm.
Fig 2.
Phenotypes of in vitro cultured cell types at D18.
(A) Schematic view of whole EET surrounding the embryonic disc (ED). Chorion (ch) is composed of ectoderm (or Trophoblast; magenta line) and ExMesoderm (red), yolk sac (ys) of ExMesoderm and ExEndoderm (green). (B) bTCs, bXMCs, and bXECs were primarily cultured on collagen (colIV) or plastic (where plastic means a tissue-culture-treated surface). Co-immunofluorescence is shown at 16h, 72h, or a week of culture with the antibodies used in vivo (Fig 1: Trophoblast—FURIN, ExMesoderm—VIM, or ExEndoderm—AFP) as well as phase-contrast images of each cell type after 72h of culture. (C) Immunodetection of cytoskeleton organization and cell proliferation with pan-Keratins (KRTs) and Ki67 respectively, after 1 week of culture. Scale bar: 10 μm.
Fig 3.
Dynamic monitoring for cell adhesion and proliferation.
The adhesion and proliferation of bTCs, bXECs, and bXMCs on seven ECM components were individually monitored using the xCELLigence System; measurements were taken every 5 minutes during the first 16h and every 30 minutes during the last 24h of the assay. (A) Adhesion profiles are illustrated at the indicated time intervals (12–16 hours for bTCs and bXECs, 7–12 hours for bXMCs) on the left panels. For each cell type and ECM component, the adhesion index is indicated as the slope (1/hr; right panel). (B) Proliferation profiles are illustrated at the indicated time intervals (21–27 hours for bTCs and bXECs, 6–10 hours for bXMCs) on the left panels. For each cell type and ECM component, the proliferation index is indicated as the doubling time (right panel). The slope and doubling time of each growth curve were calculated using RTCA 1.2 software.
Fig 4.
bXMC spread on four fibronectin patterns (disc, crossbow, I, or Y) of three sizes each [68]. (A) Number of cells that spread on each pattern. Cells were labeled with antibodies against (B) α and γ tubulins (green, magenta respectively), DAPI (blue), and (C) F-actin (red). (D) Over ten F-actin labeling images were averaged and color coded with the rainbow look-up table to highlight intensity (i.e tension) variations. Scale bar: 10 μm.
Fig 5.
ECM composition affects gene expression in bTCs.
(A) Phase-contrast images of bTCs after 16h, 48h, 72h, and a week of culture on collagen IV (colIV) or Matrigel (mat). Note the formation of a vesicle (white arrow) after a week of culture on Matrigel. (B) RT-PCR on IFN-tau, PAG1, PRL, and c93/SOLD1/SSLP1 in bTCs cultured for 16h, 48h, 72h, and a week on collagen IV, as compared to week-old Matrigel cultures and to samples from in vivo D18 EETs. (C) Secretion of IFN-tau and PRL into the bTC culture media: 16h, 48h, 72h, and one week on collagen IV or Matrigel. (D) Immunofluorescent detection of DLX3 in bTCs after a week of culture on collagen IV or Matrigel, as compared to whole in vivo D18 EET. Note the strong expression of DLX3 in nuclei of the bi-nucleated cells (white arrows). (E) bTC vesicles (developed after 1 week on Matrigel) were co-cultured with bXEC. By 24h of co-culture, they had spread on top of the bXEC layer. (F) In vivo bTC vesicles, recovered from uterine flushes at D18.
Fig 6.
Differential gene expression among extra-embryonic cell types.
(A) Hierarchical clustering and functions of 191 differentially expressed genes (DEGs). DEGs are clustered into three distinct cell types (Trophoblast, ExEndoderm, ExMesoderm) and four groups of genes (“core trophoblast”, “core epithelium”, “core ExEndoderm”, and “core ExMesoderm”). Samples are displayed in the vertical axis, genes on horizontal axis. Log2-transformed signal intensities are depicted with color: high expression levels in red, intermediate expression levels in black, and low expression levels in green. (B) Top biological functions of the four gene clusters using Ingenuity Pathway Analysis (IPA).
Fig 7.
(A) Microarray analysis (upper panel) and RT-PCR (lower panel) demonstrate gene expression changes typical of a developmental EMT (from an embryonic disc at D18 to XMCs). (B-D) Microarray data reveal signatures associated with mesenchymal stem cells, angiogenesis, and hematopoietic niche in bXMCs and in vivo D18-D25 EETs (GSE13013). (E) Western Blot analysis shows TGFB2/3 precursor secretions by bXMCs cultured for 16h, 48h, 72h, and a week on plastic. (F) Consistent with in silico data, we found evidence of blood vessels in the allantois (as revealed by the expression of the HOXA9 pro-angiogenic transcription factor [69, 70]) and blood islands in the yolk sac (as evidenced by a vitelline and a mesenchymal layer (VIM+) that surrounds erythrocyte precursors, i.e. round nucleated erythroblasts expressing more CD47 than CD44 [71–73]).
Fig 8.
Nascent mesoderm and crinoline formation.
(A, D, E) Whole mount in situ hybridization (WISH) with Brachyury, HAND1, or BMP4 DIG-labeled riboprobes. (B) Brachyury WISH with VIM, CD44, DAPI co-immunofluorescence. (C, F) Cross-sections from tissues in B and E, respectively. (F) SDF1 (CXCL12) immunostaining after a BMP4 WISH. (G-I) VIM, CD44, DAPI co-immunofluorescence. A to F: Dorsal views, G to I: ventral views. T: trophoblast, ExEn: extra-embryonic endoderm, ExM: extra-embryonic mesoderm.