Fig 1.
Quantification of morphological traits relative to embryonic age or epiblast size.
A. Embryo length in mm (triangles; crosses geometric mean) and epiblast length in μm (dots; solid squares geo-mean) are plotted on a log scale against embryo age; n = 126. B-G. Morphological traits plotted against the maximal diameter of the epiblast (length) in μm; n = 32. In E., the shape of the epiblast in transverse section has been categorised as “Lens” for concave lens shaped epiblast without cavities, as “Cav” for concave epiblasts with intra-epiblast cavities and as “Dome” for embryonic ectoderm (EmE) discs protruding above the surface of the embryo, predominantly 2-cell layers thick. F. Rauber’s Layer (RL)-stage has been assigned as per Table 1. G. “Structure” refers to the additional embryonic structure that becomes visible at the indicated epiblast size: H, undifferentiated hypoblast; VH, both VH and PH are visible; AVH; differentiation of VH into AVH is visible; Mes, in addition to the AVH, endomesodermal cells are seen.
Fig 2.
Intra-epiblast cavitation and RL disappearance as marked by CRIPTO and FURIN expression respectively.
A. CRIPTO expression in a stage 3-AVH embryo with a 160 μm epiblast before sectioning. B-E. Progressive cross-sections of embryo shown in panel A. Section numbers are indicated, each section was 8 μm thick. The progressive merging and eventual rupturing of large cavities within the epiblast can be seen in this series. No RL remained in this embryo as seen by CRIPTO expression throughout the disc. The inner region of the epiblast has already assumed an EmE-like 2-cell layer appearance in panels B-D. Star, intra-epiblast cavity. F, G. FURIN expression in F., stage 2-VH and G., stage 4-EmE embryos, showing expression in RL as well as the mural trophoblast (mTB). Bars represent 100 μm.
Fig 3.
Cattle embryo staging system from post-hatching to the start of gastrulation.
Criteria for, and diagrammatic representation and typical sections (H&E or CER1 stained) of, cattle embryos at the five stages of development between hatching and the start of gastrulation, based on data from sectioning 32 embryos. All bars are 100 μm. AVH, anterior VH; EmE, embryonic ectoderm; Endo, endoderm; Epi, epiblast; H, undifferentiated hypoblast; Meso, mesoderm; mTB, mural trophoblast; PH, parietal (mural) hypoblast; PS, primitive streak; RL, Rauber’s Layer (polar TB); VH, (embryonic) visceral hypoblast.
Fig 4.
Gene expression via real time PCR.
A. FURIN, B. CER1 and C. EOMES expression relative to the geomean of three housekeeping genes (see Methods and S3 Fig) in 3-AVH to 5-EG stage micro-dissected embryos. Solid circles represent embryonic discs (with remaining RL material at stage 3-AVH) whereas squares are mural TB. Error bars are s.e.m.. Sample sizes as follows: for discs, stages 3, 4, 5; n = 7, 9, 15; for mTB, stages 3, 4, 5; n = 4, 8, 7; where some of the n samples were themselves pools of two embryos.
Fig 5.
All embryos are orientated with anterior to the left, with dorsal (top) views (A, B, E, F) and representative cross-sections as indicated by stippled lines. A. Asymmetric onset of CER1 at stage 3-AVH. B. Slightly later stage 3-AVH embryo. C. H&E stained section of embryo B showing distinct AVH cells of increased height and with extensions toward epiblast (yellow arrow). Intra-epiblast vacuoles can be seen (arrowheads). D. Section of embryo B adjacent to section C (7 μm) showing confinement of CER1 staining to AVH cells. E., G. Stage 4-EmE embryo with CER1 marking the AVH, which still exhibits extensions toward EmE (arrows). The entire visceral hypoblast contains vacuoles (arrowheads) covered by a membrane on the EmE side. F, H. Stage 5-EG embryo without anterior hypoblast CER1 staining but showing CER1 expression in the anterior part of the primitive streak as well as in delaminating cells which are presumptive mesendoderm cells (MesEn). Bars, 100 μm.
Fig 6.
A, B. Adjacent sections of stage 1-RL embryo after WMISH for NODAL, stained with H&E (A) or not (B). NODAL is restricted to the epiblast and not expressed in the hypoblast underlying the epiblast, which is indistinguishable from that underlying the mural TB. C, D. Adjacent H&E and Nodal sections of stage 2-VH embryo. Only the visceral hypoblast (VH) and inner (ventral) epiblast are NODAL-positive. Rauber’s layer (RL) is starting to disintegrate. E, F, G. By stage 3-AVH, NODAL expression levels are weaker. NODAL is seen in the AVH (section F) and in the epiblast is confined to posterior region (section G). H, I. By stage 4-EmE, NODAL is still restricted to the AVH in the hypoblast tissue and is expressed only in the posterior EmE. J-M. Overstained stage 4-EmE embryo indicating expression throughout VH but only presumptive posterior EmE expression (arrow in M., anterior EmE). Bar, 100 μm.
Fig 7.
EOMES WMISH of stage 2-VH (A, sideview; B, H&E stained section; C, adjacent section) and advanced stage 4-EmE (D-F) embryos. At both stages the visceral hypoblast (VH) is stained. D-F. At stage 4-EmE, the posterior EmE, which will form the primitive streak, is EOMES-positive. E and F are adjacent mid-saggital sections, anterior to the left. Bar, 100 μm.
Fig 8.
BMP4 expression is excluded from the AVH.
A. Stage 3-AVH embryo showing BMP4 staining extending beyond the embryonic disc which is shown by a stippled circle. B-G. Pairs of adjacent sections as indicated in panel A. The # numbers refer to section, each being 7 μm thick. The AVH is shown via arrows and can be seen to be excluded from BMP4 stain, which extends into the parietal hypoblast (PH) as well as labelling all the epiblast. Bar, 100 μm.
Fig 9.
BRACHYURY expression commences at stage 4-EmE.
A, B. Stage 4-EmE and 5-EmE whole mount embryos stained for BRACHYURY. C, D. Saggital sections of embryos A and B, respectively, anterior to the right. D. BRACHYURY is strongly expressed in the primitive streak (PrS) but only weakly in the nascent Mesendoderm cells (Meso). Extraembryonic mesoderm (ExM) can be seen separating the mTB and PH posterior to the embryo. Bars, 100 μm.
Table 1.
Staging system used for classifying disappearance of Rauber’s layer (RL).
Fig 10.
Comparison across several species of establishment of hypoblast signalling centre.
In mice, negative signals (black arrows; BMP) from the trophoblast (in blue) are believed to inhibit the establishment of a distal hypoblast signalling centre until the epiblast (in red) has proliferated sufficiently to remove some of the visceral hypoblast (in yellow; in mice termed the visceral primitive endoderm, or VE) from this influence allowing the DVE (orange) to form at distances greater than 60 μm. A conceptual flattening out of the mouse egg cylinder is shown. In rabbits, CER1, marking the initially centrally and symmetrically located DVE equivalent, termed the AMC, again forms at a distance from the trophoblast margin. In cattle, persistence of the polar trophoblast (Rauber’s layer) and a more lens-like transverse shape of the epiblast infer that the VH remains under the influence of putative inhibitory signals, precluding a DVE-like precursor population of the AVH.
Fig 11.
Summary of gene expression patterns between stages 1-RL and 5-EG, potential interactions and comparison to mouse embryos.
A. Expression of genes are shown at the stages analysed and colour coded: FURIN dark blue, NODAL light blue, EOMES orange, CRIPTO grey, BMP4 yellow, CER1 red and BRACHYURY green. B. Gene/protein regulatory interactions as described in mouse embryos are depicted in shaded boxes representing trophoblast (dark), epiblast/EmE (mid) and hypoblast (light). References are listed in the discussion. C. Comparison of gene expression domains in mouse (M) and cattle (C) embryos at cattle stages 1–5. The mouse equivalent stages are described in Table 2. aPS, anterior primitive streak; “Epi” refers to expression in all of the epiblast; prPH, proximal parietal hypoblast/endoderm; po, posterior.
Table 2.
Morphological comparison of stage-matched cattle and mouse embryos.