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First published online 6 December 2006
doi: 10.1242/dev.02724

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Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

Patrick P. L. Tam^1,2,*, Poh-Lynn Khoo¹, Samara L. Lewis¹, Heidi Bildsoe¹, Nicole Wong¹, Tania E. Tsang¹, Jacqueline M. Gad^1, and Lorraine Robb³

¹ Embryology Unit, Children's Medical Research Institute, University of Sydney, Locked bag 23, Wentworthville, New South Wales 2145, Australia.
² Faculty of Medicine, University of Sydney, Locked bag 23, Wentworthville, New South Wales 2145, Australia.
³ The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Victoria 3050, Australia.

View larger version (63K): [in this window] [in a new window]   Fig. 1. Sites of electroporation and domains in the endoderm for fate mapping experiments. (A) Regions of the endoderm for testing cell fates by electroporation: anterior-proximal (AP), anterior-distal (AD), distal (Dist), lateral (Lat), posterior-distal (PD), posterior-middle (PM) and posterior-proximal (PP). (B) Regions of the endoderm for scoring the distribution of GFP-expressing cells in early-head-fold-stage embryos. (C-C'') Early-somite-stage embryo. The sub-division of the endoderm is shown as a schematic lateral view (C), an oblique view (C') and a ventral view (C''). In the early-somite-stage embryo, although the domain of embryonic foregut may be approximately delineated by the foregut portal, the precise sub-division of gut segments is not morphologically evident, and a substantial length of the mid- and hind-gut is yet to be formed. Embryonic endoderm of the anterior (A, rostral segment of the foregut), middle (M, posterior segment of the foregut and endoderm at the somitic level) and posterior (P, endoderm associated with the presomitic mesoderm) regions. AYS, anterior yolk sac; LYS, lateral yolk sac; PYS, posterior yolk sac.

View larger version (89K): [in this window] [in a new window]   Fig. 2. Localization of EGFP and lacZ-expressing cells in the endoderm following electroporation of expression vectors. Distribution of EGFP-expressing cells at the mid-streak (MS) stage (3 hours), at the early-head-fold-stage (24 hours) and in the early-somite-stage embryo (46-48 hours) after electroporation of the endoderm of mid-streak-stage (E7.0) embryos at seven sites: (A) lateral, (B) anterior-proximal, (C) anterior-distal, (D) posterior-proximal, (E) posterior-middle, (F) posterior-distal and (G) distal. Examples of the pattern of distribution of EGFP-fluorescent cells after electroporation and in vitro culture are shown as a set of three (at 3 hours, 24 hours and 46-48 hours) for each site. All figures are lateral views with anterior to the left, except for anterior views of anterior-proximal (24 hours and 46-48 hours) and anterior-distal (46-48 hours), and posterior views of posterior-proximal (24 hours and 46-48 hours) and posterior-middle (46-48 hours) sites. (H-L) Localization of the electroporated lacZ-expressing cells in (H,J) whole-mount and (I,K,L) histological sections of the embryo. (H,I) Endoderm of an E7.0 MS-stage embryo after 3 hours of in vitro culture. (J-L) endoderm of the dorsal foregut (K) and the middle region (L) of an early-somite-stage embryo (J) after 24 hours of in vitro culture. The faint staining in I at the basal region of the epilast is probably due to the spillage of the product of the X-gal-staining reaction, as opposed to genuine staining, which would be found in the whole cell.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Fate maps of the precursors of embryonic and extraembryonic endoderm and the pattern of cell movement in the endoderm of the mid-streak stage embryo. (A) The expression domain of Cer1 and Sox17 in the endoderm of a mid-streak embryo (anterior to the left). Cer1 expression overlaps in the distal, posterior-distal and posterior-middle regions, which contribute to the gut endoderm, but is also expressed in the endoderm immediately distal and lateral to the primitive streak, and in the anterior proximal endoderm. Sox17 expression overlaps in the posterior-middle region and is also expressed in the extraembryonic visceral endoderm. Both genes are expressed in the anterior visceral endoderm (asterisk, anterior proximal region). (B) Fate maps of the endoderm of mid-streak-stage gastrula (left figures in B' and B'') showing the localization of the precursors of embryonic (gut) endoderm (B') and extraembryonic (yolk sac) endoderm (B'') of the early-somite-stage embryo (right figures in B' and B''). (C) Color coding of the seven sites in the mid-streak-stage embryo for testing endodermal cell fates (see Fig. 1A). (D-G') The trajectories of the progenitors of the yolk sac endoderm (D-E') and embryonic endoderm (F-G') during development from the mid-streak to the mid- to late-streak stage (D,F) and then to the early-head-fold stage (E,E',G,G'), showing the distribution of cells arising from (E,G) anterior and distal sites and (E',G') posterior sites separately. Cells originated from each of the seven sites are color-coded. In D and F, the origin of the arrow shows the location of the cells at the mid-streak stage and the head of the arrow marks the predicted position of these cells at the mid- to late-streak stage, based on the cell-fate data. Double-headed arrows indicate the expansion of the distal and posterior-distal endoderm. Red line in the posterior region of the embryo (B,C) marks the primitive streak.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Impaired cell movement in the endoderm of Mixl1GFP/GFP mutant embryos. (A,B) The displacement of DiI-labeled endoderm (yellow because of overlaying of red dye color on the green fluorescence) in Mixl1+/GFP (A) and Mixl1GFP/GFP (B) mutant embryos after 1-, 6-, 12- and 24-hours of in vitro culture. (C,C') Summary of the distribution of DiI-labeled endodermal cells along the anterior-posterior axis of the embryos and in the posterior yolk sac 24 hours after labeling. Each bar represents the pattern of distribution of labeled cells in a single representative embryo after the endoderm overlying the anterior (C) or the posterior (C') region of the primitive streak was painted.

View larger version (78K): [in this window] [in a new window]   Fig. 5. Allocation of the endoderm to the dorsal and ventral cell population of the embryonic foregut. (A,B) DiI labeling of the endoderm overlying the anterior end of the primitive streak in mid-streak (A) and mid- to late-streak (B) embryos (as staged by the distal extension of the GFP-expression domain), followed by visualization of the labeled cells at 0 hour (merged GFP and DiI, left images), 12 hours (DiI only, middle images) and 24 hours (top right images, DiI only; bottom right images, merged GFP and DiI) of in vitro development. (C) The allocation and the path of displacement of lateral (green) and medial (dark blue and light blue) populations of the anterior endoderm at the mid-streak and mid- to late-streak stages of gastrulation. The diagrams show the trajectories of these two populations of anterior endoderm in the normal (left panels) and the flattened-disc (right panels) configurations of the embryo (Tam and Gad, 2004). (D,D') The spatial correlation of medial and lateral divisions of the anterior endoderm of the late-bud-stage embryo (D, lateral view showing Sox17 expression in the anterior endoderm) and the resultant dorsal (roof) and prospective ventral (floor) regions of the foregut portal of the early-somite-stage embryo (D', transverse histological section).

View larger version (115K): [in this window] [in a new window]   Fig. 6. Allocation of the embryonic gut endoderm during gastrulation. (A) Five separate sites in the endoderm were painted with DiI (red) or DiO (green) and the movement of these cells tracked at 3 hours and 28 hours post-labelling. (B) Cells from the distal site (Dist, red) were found mostly in the prechordal region, whereas those from the posterior-distal site (PD, green) were distributed to the middle (somitic) region of the embryo. Dotted circle (red in A, blue in B-E) marks the anterior region of the primitive streak (APS) (C) Cells from the PD to posterior-middle (PM) region (red) were distributed to the medial regions from the forebrain to the posterior (presomitic mesoderm) level (dashed line), whereas the APS cells (green cells from the yellow region) were found mainly in the middle endoderm. (D) Cells from the painted (green) region covering the whole primitive streak from APS to posterior-proximal (PP) region were distributed mostly to the lateral and intermediate regions of the anterior endoderm and to the most posterior endoderm and posterior yolk sac. Some labelled cells were present in the middle medial endoderm. (E) From the posterior endoderm, Dist, PD and APS cells (red) were distributed to the medial endoderm spanning from the forebrain to the somitic level. APS, PM and PD cells (green) were distributed to the lateral endoderm at the forebrain to hindbrain level and to the posterior endoderm. Overlapping contribution from the APS cells is observed in the medial middle endoderm (yellow domain). Few labelled cells are found in the lateral posterior endoderm. (F-I) Histological examination of (F) E7.0 MS embryo (inset: whole embryo) showing DiIlabelled cells in the endoderm and (G) labelled embryo after 24 hours of in vitro culture containing labelled cells in the foregut portal (H) and in the endoderm (I) at the somite level.