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The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm

¹ Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia
² Department of Molecular Genetics, MD Anderson Cancer Center, University of Texas, Houston TX 77030, USA
³ Laboratory of Developmental Biology, Institute of Molecular and Cell Biology, Osaka University, 565 Osaka, Japan
⁴ Samuel Lunenfeld Research Institute, Mount Sinae Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada

View larger version (24K): [in a new window]   Fig. 1. The expression pattern of (A) Hnf3ß, (B) Chrd, (C) GsclacZ, (D) Cer1 and (E) Otx2 in the ES, MS, LS and the EB stage embryos. Gene expression was revealed by whole-mount mRNA in situ hybridization (A,B,D,E); by immunostaining (A, asterisk); and by X-gal staining for ß-galactosidase activity (C). In (B), ES stage embryo, which displays no Chrd activity, is not shown and combined in situ hybridization of T and Chrd are for EB stage embryo. Scale bar: 300 µm.

View larger version (57K): [in a new window]   Fig. 2. Axis induction by the MGO. (A) The scheme of tissue transplantation: a tissue fragment dissected from the anterior end of the primitive streak (Region V, VI; Fig. 5C) of mid-streak (MS) stage embryo is transplanted to the lateral region of the late-streak/no bud (LS/0B) stage host embryo. (B) After 24 hours of in vitro development, the host embryo contains an elongated structure (white arrowheads) of EGFP-expressing graft-derived tissues on its left flank. Sox2 activity, which is expressed in the neural tube of the host embryo, is also expressed in the ectoderm overlying the graft-derived tissue (black arrowheads, one out of two embryos containing grafts showed Sox2 induction). (C) EGFP-expressing graft tissue (white arrowhead) located at hindbrain level has induced Otx2 expression (black arrowhead, six of seven embryos containing grafts showed Otx2 induction) which is normally expressed in the fore- and midbrain regions. hf, head folds; nt, neural tube; som, somites. Scale bar: 200 µm.

View larger version (29K): [in a new window]   Fig. 3. The contribution of the organizer of the ES (EGO), MS (MGO) and the LS (node) stage embryo to the axial mesoderm and the neuroectoderm (ventral tissue of the forebrain and the floor plate of the neural tube) of the early-somite stage embryo. Both the EGO and the MGO contribute to the axial mesoderm and the neuroectoderm from the most anterior region to the level of the presomitic mesoderm. The node contributes to the notochord and floor plate spanning from the second to third somite to the anterior primitive streak (PS). Unbroken line, major contribution; broken line, lesser contribution. The early-somite stage embryo is shown from lateral (left) and the dorsal (right) views.

View larger version (98K): [in a new window]   Fig. 4. Contribution of the EGO and MGO to the anterior mesoderm and foregut endoderm. (A) GsclacZ-expressing tissue is found in the prechordal mesoderm and the foregut of the early-somite stage embryo. (B) EGFP-expressing cells derived from the transplanted EGO cells are distributed throughout the anteroposterior axis of the early somite stage host embryo, but only those that are localized (C-E) in the prechordal mesoderm (D), the foregut endoderm and the head processes (E) express the GsclacZ transgene (arrowheads). (F) MGO-derived cells that are found in the cranial tissues of the early-somite stage host embryo also express GsclacZ transgene when they are localized in the prechordal mesoderm (G) and the foregut (H, arrowhead), but not in the notochord of the hindbrain (box). Broken lines in C,F indicate the planes of section shown in D,E,G,H. Abbreviations: A-P, anterior-posterior axis; crm, cranial mesenchyme; hf, head-fold; som, somite; ne, neuroectoderm. Scale bars: 200 µm in A,B,C,F; 50 µm in D,H; 20 µm in E,G.

View larger version (89K): [in a new window]   Fig. 5. Fine mapping of the developmental fate of cells within and adjacent to the EGO and MGO. (A) Regions I-IV in the posterior midline epiblast of the ES embryo, where cells are tested for contribution to axial mesoderm. Regions I to IV are located anterior to the recently formed primitive streak, with region III and IV located in the domain of the EGO delineated by Hnf3ß and GsclacZ activity (Fig. 1). (B) The contribution to axial mesoderm by cells in Regions I to IV, expressed as the percentage of embryos showing graft-derived cells in this tissue. (C) Regions V-VII in the posterior midline epiblast of the MS embryo, where cells are tested for contribution to axial mesoderm. Regions V and VI are located in the anterior end of the elongating primitive streak within the domain of the MGO, which is defined by the expression of Hnf3ß, Chrd and GsclacZ. Region VII is located in the anterior segment of the primitive streak. (D) The contribution to axial mesoderm by cells in Regions V to VII, expressed as the percentage of embryos showing graft-derived cells in this tissue. (E) Cells that were transplanted to Region III of the early-streak embryo (t=0) colonized the axial tissues of the host embryo after 36 hours of culture (t=36). Histological sections at the level of the forebrain (fb),hindbrain (hb) and trunk (tr) of an embryo showing graft-derived cells in the prechordal mesoderm and the notochord (inset). (F) Cells that were transplanted to Region VI of the MS embryo (t=0) colonized the axial tissues of the host embryo after 36 hours of culture (t=36). Histological sections at the level of the forebrain (fb), hindbrain (hb) and trunk (tr) of an embryo showing graft-derived cells in the prechordal mesoderm (inset a), the notochord of the midbrain (inset b) and the trunk. (G) Cells transplanted to epiblast lateral to Region III of the ES embryo (t=0) colonized the cranial mesenchyme and the heart of the host embryo after 48 hours of culture. (H) Cells transplanted to Region VII of the MS embryo (t=0) colonized the notochord in the trunk of the host embryo after 36 hours of culture (t=36). EGFP-expressing cells show green fluorescence and lacZ-expressing cells are stained blue by X-gal reagent. Scale bars: in E, 50 µm; in G (t=0) and H (t=0),100 µm; in F, 50 µm; in H (t=36), 50 µm.

View larger version (59K): [in a new window]   Fig. 6. Movement of cells derived from the EGO and the MGO. (A) Serial images taken at 6 hour intervals showing the localization of the EGFP-expressing cells derived from the EGO during 30 hours of in vitro development from the ES to the early neural plate stage. At 0-6 hours, the EGO-derived cells are displaced anteriorly to the site of the MGO, followed by the dispersal of the EGO-derived cells out of the MGO (lateral and posterior view at 12 hours) laterally to the mesodermal layer and along the midline of the embryo (arrowheads at 12 hours). The EGO-derived cells are spreading anteriorly in both the paraxial and axial mesoderm under the head folds (18-30 hours). Embryos are viewed from the left side at 0, 6, 12 and 18 hours, posterior side at 12 hours, and anterior side at 24 and 30 hours. (B) The EGO-derived cells in the mesodermal (yellow fluorescent DiI-labeled cells) and axial (green fluorescent EGFP-expressing cells, arrowhead) position in the MS stage embryo colonize, respectively, the paraxial (orange-red DiI-labeled cells) and axial mesoderm (green fluorescent cells, arrowheads in C) of the early-somite stage host embryo (C,D). (B) Lateral view, anterior towards the left; (C,D) dorsal view, anterior towards the top. (E) Serial images showing the localization of the EGFP-expressing MGO-derived cells during development from the MS to the early neural plate stage. The MGO-derived cells migrate anteriorly in a tight column along the midline at 6-15 hours. After 24 hours of culture, some MGO-derived cells are in the axial mesoderm as well as underneath the anterior of the neural plate. 0-10 hours: lateral view, anterior towards the left; 15-24 hours: anterior view. Broken lines mark the distance of the MGO cells from the anterior end of the body axis. Scale bars: in E, 150 µm in A,B,E; in D, 50 µm in C,D.

View larger version (77K): [in a new window]   Fig. 7. Contribution of node-derived cells to the notochord. (A) EGFP-expressing cells transplanted to the node of the early-bud stage embryo (t=0 hour). (B) Posterior displacement of the grafted cells and the formation of a trail of notochordal as the node is drawn posteriorly as the primitive streak regresses (t=12 hours after grafting). (C) lacZ-expressing graft-derived cells in the notochord of the early-somite stage host embryo (24 hours after grafting). (D) A histological section of the embryo at the plane indicated by the broken line in C showing the colonization of the notochord (nc) and the ventral neural tube (nt) by the graft-derived cells. (A,B) Lateral view, anterior towards the left; (C) dorsal view, anteriorwards to the top. Scale bar in A, 150 µm in A,B and 50 µm in C,D.

View larger version (29K): [in a new window]   Fig. 8. The movement of cells derived from the gastrula organizer relative to the morphogenetic movement of the mesoderm during gastrulation from the ES (early-streak) to the EB (early-bud) stage. Left column: lateral view of the embryo with anterior towards the left; right column: flattened dorsal view, anterior towards the top. The early gastrula organizer (EGO, gray) cells are first found in the posterior epiblast at a distance of about 50 µm anterior to the newly formed primitive streak (black) of the ES embryo. These cells initially remain static in a tight cluster before the elongating primitive streak reaches them. Cells in the posterior region (Region IV, Fig. 5A) of the EGO are incorporated first into the advancing primitive streak and they ingress (grey arrows) to join the mesoderm (pink) lateral to the anterior end of the primitive streak. The cells in the anterior region of the EGO (the bulk of the progenitor of the prechordal mesoderm) stay in the midline and are carried anteriorly to meet the progenitors of the prechordal mesoderm and the head process in the epiblast of Regions I and II. These cells collectively constitute the mid-gastrula organizer (MGO). Cells leaving the MGO move in a tight column anteriorly along the midline of the embryo (black arrow, pointing anteriorly) and form the prechordal mesoderm and the head process. These progenitors of the axial mesoderm are moving in concert with the paraxial stream of EGO-derived cells in the mesoderm. The movement of these two streams of EGO-derived cells is part of the global movement of the mesoderm and the definitive (gut) endoderm towards the anterior region of the gastrulating embryo (Tam et al., 2001). At the MS stage, the bulk of the precursors of the trunk notochord is not found in the MGO and is localized in the primitive streak immediately posterior to the MGO. These cells are recruited into the node after the departure of the anterior axial mesoderm and they are laid down as the notochord as the node is displaced posteriorly (black arrow, pointing posteriorly) with the regression of the primitive streak.