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First published online 4 July 2007
doi: 10.1242/dev.02877

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Two distinct sources for a population of maturing axial progenitors

Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Schematic representation of grafted and scored regions. (A) Lateral (top) and dorsal (bottom) views of the primitive streak showing the regions dissected. The rostral end of the node (RN) was recognised by its wider notochordal plate, and the border (denoted by B) was recognised by the crown (cr) at the caudal end of the node. (B) Schematic diagrams showing the position of the CNH (red dashed lines) in sagittal sections (upper panel; as shown in Fig. 2 and in Fig. S1 in the supplementary material) and transverse sections (lower panel; as shown in Figs 5 and 6). Black dotted lines on the upper panel show the rostrocaudal position of transverse sections. CNH, chordoneural hinge

View larger version (79K): [in this window] [in a new window]   Fig. 2. Gene expression in the primitive streak and tail bud. Embryos were hybridised with in situ probes for Fgf8 (A-B), Evx1 (C-D), Wnt3a (E-F), T (G-H), Foxa2 (I-J) or Sox1 (M). (A,C,E,G,I) Whole-mount lateral views of E8.5 embryos; (A',C',E',G',I') corresponding sagittal sections. (B,D,F,H,J) E10.5 sagittal sections. Black and white arrows: end of the notochordal plate. Inset in G': transverse section of the caudal half of the node (at the level marked in G' by a white arrowhead), showing a cell in the ectodermal layer strongly expressing T. Black box in B: E10.5 chordoneural hinge (CNH). (K-L',N-O) Confocal images showing Sox1-GFP expression (green) and TO-PRO-3 counterstain (red). (K) Sagittal section of an E8.5 embryo. (L) Sagittal section of the tail tip of an E10.0 Sox1-GFP embryo. (L') Magnified view of the CNH region marked (white box) in L. (N,O) Transverse sections through the tail tip of an E10.0 embryo at the levels marked by vertical white lines in L (N, left line; O, right line). (N') Magnified view of the region marked (white box) in N, showing Sox1-GFP-positive cells in the notochord region (arrowhead). (P) Summary diagram. Blue, ectoderm expressing Sox1 and Foxa2; brown, rostral prospective notochord expressing T and Foxa2; orange, caudal prospective notochord expressing Fgf8, T and Foxa2; dark purple, caudal node and streak ectoderm, and caudal ectoderm in the tail bud; light purple, streak mesoderm and TBM, both areas express Wnt3a, Evx1, Fgf8, T and Sox1 (E10.5). Black boxes in P, E8.5 border and E10.5 CNH. not, notochord; nt, neural tube; hg, hindgut; np, neural plate; ps, primitive streak; tb, tail bud; tbm, tail bud mesoderm.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Decline in gene expression levels as tail elongation arrests. Lateral views of tail buds hybridised with in situ probes for Fgf8 (A-D), Wnt3a (E-H'), Cdx2 (I-L) or T (M-P). Insets in C,G,H,K show dorsal views. (H,H') Two E13.5 tails hybridised with Wnt3a, showing very low (H) or undetectable (H') expression.

View larger version (94K): [in this window] [in a new window]   Fig. 4. Dissection of pieces for grafting. (A) Ventral view of rostral node (RN), border (B) and streak 1 (St1) regions of an E8.5 embryo hybridised with a T (brachyury) in situ probe; (B,C) sagittal sections showing the same region in embryos hybridised with Foxa2 (B) or Fgf8 (C). Arrows delimit the regions dissected. (D-I) In situ hybridisation of dissected pieces. The rostral node expresses Foxa2 (D), but not Fgf8 (G); streak 1 expresses Fgf8 (I), but not Foxa2 (F); however, the border expresses both markers (E,H). (J-M) Transverse sections through mid-streak (St2) of E8.5 embryos hybridised with T (J), Wnt3a (K), Fgf8 (L) and Evx1 (M). Line in J marks the midline, arrows mark the lateral region grafted (Lt).

View larger version (74K): [in this window] [in a new window]   Fig. 5. Homotopic grafts. (A-G) Trunk and tail of embryos showing incorporation of GFP-labelled cells in homotopic grafts, as indicated. Transverse confocal sections through representative embryos are shown in panels labelled with a lowercase letter. Sections Aa...Ga, mid trunk; Ab...Gb, rostral tail bud (chordoneural hinge, CNH); Ac...Gc, the bud caudal to the notochord end. (Ca, arrowhead) Labelling in intermediate mesoderm. (Da, inset) Transverse confocal section through a different embryo showing endothelial contribution of labelled cells. (Fa) Top section, rostral-most unilateral contribution in the somites in lateral 1-3 grafts (arrowhead in F and H); bottom section, caudal unilateral neural tube and bilateral somite contribution (asterisk in F and H). White arrows, notochord; white boxes in Bb and Fb, CNH. (H) Diagram showing contribution of the lateral border and lateral 1-3 regions to the somites and neural tube. The rostral-most somite labelled is represented as average±standard deviation. s, somite; nt, neural tube; TB, tail bud; TBM, tail bud mesoderm.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Heterotopic grafts. (A-F) Trunk and tails of embryos receiving a heterotopic graft as indicated. (G-X) Transverse confocal sections through a representative embryo. Sections through body and tail are at the same levels as in Fig. 5. Insets in F and L, embryos in which donor cells did not incorporate well in the axis and tail bud but formed an ectopic structure. Arrows indicate the notochord. White box in P, chordoneural hinge (CNH). s, somite; nt, neural tube; tbm, tail bud mesoderm.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Cell fate and movements at the caudal end of the E8.5 embryo. Tissues are colour-coded as follows: blue, neurectoderm (flattened so that the dorsal neural tube lies laterally); brown, notochord; pink, paraxial mesoderm; white fill and orange arrow, lateral mesoderm; purple, primitive streak. The checked area contains progenitors of chordoneural hinge (CNH) cells and neural progenitors retained in the tail bud at E10.5. Notice the region overlap. Arrows represent the movement of cells and are colour-coded as follows: blue, neurectoderm; brown, axial mesoderm; red, paraxial mesoderm; orange, lateral mesoderm. Broken arrows, movements of progenitor cells inside the progenitor zone. As well as a net movement rostrally to neural tube from the progenitor zone, lateral progenitors can move towards the midline (blue broken arrows), form mesoderm (purple arrows) and move towards presomitic mesoderm (red arrows). Cells at the midline exit to presomitic mesoderm, and might join a pre-existing stream of presomitic mesoderm. In the border (junction of checked, purple and brown regions), cells exit to ventral neurectoderm, notochord and medial somites (thin red arrow). They might also exit caudally to the primitive streak (purple arrow). Box enclosed by a broken line indicates the most recently formed somite.