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Axial progenitors with extensive potency are localised to the mouse chordoneural hinge

Centre for Genome Research, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

View larger version (75K): [in a new window]   Fig. 1. Labelling sites, donor origin and graft sites. (A) Schematic showing labelling and grafting experiments. Blue fill denotes sites of DiI label. In 8.5 d.p.c. embryos, the ventral layer of node is exposed as a hiatus in the endoderm and can therefore be labelled separately from the ectoderm layer immediately above it, whereas anterior primitive streak is labelled by inserting a pipette through the endoderm and thus labels all layers. The entire neural ectoderm surface, including the posterior ventral neural plate that overlies the notochord is labelled in 10.5 d.p.c. cultured tail pieces. Broken red lines outline sites dissected for grafting. The broken black line outlines plug of tissue at the node/streak border replaced by graft in host embryo. (B) Dissection of 10.5 d.p.c. tail bud: (left) lateral view of tail bud after removal of paraxial mesoderm, overlaid with position of CNH and TBM (broken red lines); (right, top) the same embryo after removal of dorsal neural tube and hindgut; (right, bottom) the same piece rotated so that the widened end of the notochord is upwards. CNH is outlined in red. (C) Inset shows a dissected clump containing eight GFP-labelled cells amongst 200 unlabelled cells from the CNH of the embryo in Fig. 2C,H,L. Main panel: posterior view of an embryo containing this clump grafted immediately posterior to the node (outlined by a broken white line) at the anterior of the primitive streak, the posterior limit of which is marked by a white arrow.

View larger version (82K): [in a new window]   Fig. 2. Descendants of the anterior streak and node populate different regions of the tail bud. Embryos were DiI labelled or grafted with GFP-expressing cells at 8.5 d.p.c. and cultured for 48 hours. (A-D) Lateral views of the posterior ends of manipulated embryos. (A) Embryo labelled with DiI in the ventral node. Labelled cells populate the notochord (arrowhead) and end short of the tail tip, just anterior to the line showing the plane of section in K. (B) Embryo labelled with DiI in the anterior primitive streak. Labelled descendants colonise somites and are widespread in the tail bud. Arrow, position of somite 20. (C) Embryo grafted with 8.5 d.p.c. node. Label is similarly located to the label in A, but also includes the ventral neurectoderm (asterisk). (D) Embryo grafted with 8.5 d.p.c. anterior primitive streak. Label is similar to that in B. Arrow indicates position of somite 20. (E) Transverse section of the embryo shown in A, showing label in the notochord. (F) Neural tube and notochord, and (G) paraxial mesoderm of dissected embryo shown in B. Labelled cells are present in ventral neural tube, but not notochord (F), and in somites (G). (H) Dissected neural tube and notochord of embryo shown in C, where labelled cells populate notochord and ventral neural tube. (I) Dissected neural tube and notochord, and (J) paraxial mesoderm of the embryo shown in D. Labelling is similar to that in F,G. (K) Transverse section of distal tail bud of embryo in A. No labelling is seen. (L) Dissected tail tip of embryo shown in C. Labelling is confined to notochord and ends short of the tail tip. (M) Dissected neural tube and notochord (transverse view) of embryo in B. Labelled cells are present in posterior neurectoderm and mesoderm (arrow). (N) Dissected distal neural tube and underlying mesoderm of embryo in D. Labelled cells are present in ventral neurectoderm. (O,P) Embryo labelled with DiI in anterior primitive streak (red) and CMFDA in ventral node (green). (O) Fluorescent overlay on brightfield image, and (P) fluorescent image, of dissected neural tube and notochord. Node descendants end sharply under the neural tube. Anterior streak descendants populate the ventral neurectoderm and underlying mesoderm and, posteriorly, encroach on the notochord territory (arrows). Arrowheads indicate notochord; asterisks indicate ventral neural tube.

View larger version (151K): [in a new window]   Fig. 3. The posterior neural plate generates mesoderm after posterior neuropore closure. (A) 10.5 d.p.c. tail piece labelled with DiI in neurectoderm and cultured for 48 hours. Labelled descendants are present in neurectoderm (asterisk) and posterior mesoderm (arrow). (B-D) successively more posterior transverse sections of a second embryo, showing label in neurectoderm (asterisks), mesoderm (arrow) and in the posterior, but not more anterior, notochord (arrowheads in B,C).

View larger version (97K): [in a new window]   Fig. 4. The CNH, but not the TBM, generate labelled descendants in axis, CNH and tail bud. (A,B) Cultured embryos that received a graft of 10.5 d.p.c. (A) and 12.5 d.p.c. (B) TBM. Grafts remain as distinct clumps and do not incorporate in the host. (C,D) Whole embryo (C) and dissected paraxial mesoderm from a second embryo (D) that received a graft of DiI-labelled 10.5 d.p.c. CNH. Labelled cells are present in CNH (arrowhead) and paraxial mesoderm. Arrow indicates position of somite 20. Labelled cells are also present along the dorsal neural tube, possibly because of incomplete incorporation of the graft during posterior neuropore closure. (E-G) Embryos receiving a graft of DiI-labelled 12.5 d.p.c. CNH. Tail (E) and transverse section (F) of a second embryo showing incorporation in notochord (arrowhead). (G) Dissected paraxial mesoderm of a third embryo showing label in somites.

View larger version (79K): [in a new window]   Fig. 5. Grafted cells express markers of differentiation correctly. Embryos are doubly stained with X-gal (light blue; donor cells), and antisense riboprobes as indicated (purple). (A,C,E,G) Whole embryo; (B,D,F,H) transverse section of the adjoining embryo at the level shown by the lines in A,C,E,G. Left hand insets in B,D,F,H show high power images of the regions indicated by black arrowheads. Co-expressing cells are indicated by blue arrowheads. (A-D) Embryos resulting from graft of 8.5 d.p.c. node. (A,B) Embryo hybridised with T riboprobe. Like unlabelled host cells, donor cells in the notochord express T, while those in the ventral neural tube (asterisk and upper right-hand corner of inset) do not. (C,D) Embryo hybridised with Shh riboprobe. Donor cells correctly express Shh in the floorplate (compare floorplate in B with that in D). A comparable high magnification image in a control unlabelled embryo is shown (right-hand inset in D). (E-H) Embryos receiving grafts of 10.5 d.p.c. CNH. (E,F) Embryo hybridised with a Shh riboprobe. Donor cells in the notochord express Shh. (G,H) Embryo hybridised with Dll1 riboprobe. Donor cells in dorsal paraxial mesoderm express Dll1, while those outside the region of host cells expressing Dll1 do not (arrow). Asterisks indicate ventral neural tube.

View larger version (85K): [in a new window]   Fig. 6. Regrafting of GFP-labelled TBM (derived from an anterior streak graft) results in contribution to short stretches of somites, not the tail bud. (A) Whole embryo and (B) dissected paraxial mesoderm (enlarged) of embryo shown in A. Cells have incorporated over a distance of six somites unilaterally. Arrows indicate anterior and posterior borders of labelled cell incorporation.

View larger version (101K): [in a new window]   Fig. 7. Regrafting of GFP-labelled CNH results in contribution to both the axis and tail bud in up to three generations. Diagrams illustrate the history of grafted cells in the cultured embryo shown immediately to the right. (A-C) Whole mount (A) and transverse sections (B,C) of an embryo that received a graft of 10.5 d.p.c. GFP-labelled CNH. In the axis, cells populated the paraxial mesoderm exclusively and either formed small medial graft-derived somites (B), or incorporated into wild-type tissue (C, arrow). (D-F) Whole mount (D) and sections (E,F) of an embryo grafted with CNH cells from the embryo in A-C. Grafted cells populate axial derivatives that are identical to the parent graft. (G-J) Whole mount (G), dissected neural tube/notochord (H) and paraxial mesoderm (I,J) from embryos that had received a graft of 10.5 d.p.c. CNH, derived from an initial 8.5 d.p.c. node graft. Labelled cells populate the posterior end of the notochord and CNH (arrowhead, H), incorporate in paraxial mesoderm (I), but also form small medially located somites that are epithelial posteriorly (arrow in J) and disperse anteriorly, and are located out of register with the endogenous somites (s). (K-O) A third generation graft. Whole mount (K), dissected neural tube and notochord (L,M), and paraxial mesoderm (N,O), showing contribution to notochord (arrowhead in L), posterior neurectoderm (asterisks in L and M), and both ectopic somites located between host somites (s) (N) and interspersed GFP-labelled cells (O) in host somites. Arrows in A,D,G indicate the position of somite 20.

View larger version (27K): [in a new window]   Fig. 8. Location of the progenitor cells during axis elongation. White boxes represent the regions in 8.5 d.p.c. and 10.5 d.p.c. embryos where the stem cell-like population resides. Descendants populate notochord, neural tube and somites (white arrows), and may originate from a common stem cell axial progenitor (broken lines).