QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 August 2003
doi: 10.1242/dev.00712

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chapman, S. C. Articles by Lumsden, A. Search for Related Content PubMed PubMed Citation Articles by Chapman, S. C. Articles by Lumsden, A.

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm

¹ MRC Centre for Developmental Neurobiology, Kings College London, New Hunts House, Guy's Hospital, London SE1 1UL, UK
² University of Utah School of Medicine, Department of Neurobiology and Anatomy, and Children's Health Research Center, Room 401 MREB, 20 North 1900 East, Salt Lake City, UT 84132-3401 USA

View larger version (50K): [in a new window]   Fig. 1. Early chick embryonic stages and formation of the lower layer. At stage X, detached cells originating from epiblast lie in the subgerminal cavity (green circles). Koller's sickle (black crescent) and the posterior marginal zone produce a sheet of lower layer cells extending rostrally, forming the primary hypoblast (green sheet) by stage XIV. Endoblast (dark green) formation from the caudal part of the embryo then begins. Stage 2, a broad, short triangular streak forms in the caudal part of the embryo. The streak begins to elongate at stage 3a/b, but only reaches the centre of the area pellucida at stage 3c (equivalent to HH stage 3+) with definitive endoderm ingressing through the rostral tip of the streak (blue). Stage 3a and 3b are difficult to separate in practice (stage 3a, a short and broad linear streak with no groove yet visible; stage 3b, a longer narrower linear streak with visible primitive groove). The defining feature of stage 3a/b is that the streak has not yet reached the centre of the area pellucida, the widest zone across the left/right axis of the embryo. Stage 3c, the elongated and grooved streak extends to the centre, with stage 3d characterised by the rostral extension of the streak beyond the centre point. Maximum streak extension is at stage 4, with a noticeable change in the morphological character of the ectoderm, with the onset of neural specification at stage 3d. By stage 4, definitive endoderm has almost completely ingressed and both primary hypoblast and endoblast have been displaced towards the rostral and caudal poles of the embryo, respectively. Definitive endoderm spreads by a polonaise movement; after ingressing through the tip of the streak, the cells spread rostrally and then laterally. A noticeable swelling at the rostralmost part of the streak indicates the formation of Hensen's node. Stage 4+ heralds the beginning of ingression of axial mesoderm, recognised by a triangular-shaped ingression rostral to Hensen's node.

View larger version (67K): [in a new window]   Fig. 2. Transections. At all stages shown, transections were either Type B (broken blue line) or Type C (broken green line). Type B transections were made at the rostral extent of the ingressing primitive streak, excluding the extending streak and later ingressing axial mesoderm from the rostral blastoderm isolates (RBIs). Type C transections were made by measuring 125 µm rostral to the tip of the primitive streak (approximately one node diameter) and then transecting through all layers. The centre of the area pellucida is marked by a red line and assists in staging before transecting the embryo. The yellow circle represents the central epiblast (CE) population of cells. This group of cells is known to have organising properties and is rostral to the extending streak at stage 3a/b. At stages 3c, the CE is becoming incorporated into the extending streak. By stage 3d and 4, CE is incorporated into the rostral streak and forms part of Hensen's node. At each of the stages note the relative positions of the CE, the rostral extent of the primitive streak, the centre of the area pellucida, and the level of type B and type C transections.

View larger version (66K): [in a new window]   Fig. 3. Sox2 expression enlarges progressively with neural specification. A panel of whole-mount embryos probed for Sox2 transcripts by in situ hybridisation. Anterior is towards the top. (A) At stage 3d, neural specification of the prospective neural ectoderm occurs. The first cells with detectable expression of Sox2 lie in close proximity rostral and lateral to the definitive streak. (B) As neuralisation proceeds and Hensen's node forms, expression of Sox2 expands outward from the streak (stages 4/4+). The caudal boundary of expression remains constant at this stage. (C) By stage 5, the neural plate has pan-neural expression of Sox2 over the whole of the still flat neuroectoderm. (D) At stage 5+, the node regresses, drawing the posterior boundary of expression caudally. The ventral neural plate looks lighter in colour as the neural tube begins to form, becoming narrower as the neural folds move medially. Some variation in transcript levels occurs with stronger expression of Sox2 in the anteriormost neural plate.

View larger version (106K): [in a new window]   Fig. 4. Whole-mount in situ hybridisation of Ganf. Embryos stained for Ganf (blue) and Chordin (red). (A,C,E) Whole-mount, dorsal view with anterior towards the top of the page. (B,D,F) Sagittal sections (40-50 µm) with anterior towards the left. (A,B) At stage 4 (18 hours) Ganf is expressed for the first time. Arrows indicate the gap between neuroectodermal Ganf expression and Chordin in Hensen's node and ingressing axial mesoderm. (C,D) At stage 5, axial mesoderm underlies Ganf expressing tissue and there is some suggestion that mesodermal cells may also express Ganf at this point. (E,F) The headfold begins to form at stage 6 (24 hours) and the Ganf-positive zone narrows as convergent extension takes place in advance of neural tube formation.

View larger version (72K): [in a new window]   Fig. 5. Ganf is colocalised within the Sox2 expression domain. Anterior is towards the top. Sox2 alone results in a pink colour, and where Ganf and Sox2 overlap a brown colour results. In all the embryos tested, Ganf is always colocalised within the Sox2 expression domain. (A) Stage 4/4+: a broken line marks the rostral extent of Sox2 expression. Ganf is strongest directly rostral to Hensen's node. (B) Sox2 expression extends rostrally as neural specification occurs throughout the neural plate at stages 4+/5. The Ganf expression domain has also enlarged, but remains within the Sox2 expression region. (C) By stage 5+, the streak has begun to regress (note gap between Ganf expression and Hensen's node) and Sox2 expression extends more caudally (pink).

View larger version (70K): [in a new window]   Fig. 6. Sox2 expression in transected embryos. (A-C) RBIs to the top and CBI below, processed after 24 hours in agar/albumen culture. (A,C) Stage 3a/b and 3d RBIs have expression of Sox2 in most cases (see Table 1), whereas (B) stage 3c RBIs do not. All caudal isolates have expression of the pan-neural marker.

View larger version (90K): [in a new window]   Fig. 7. Ganf expression in intact and lower layer deficient transected embryos. RBIs of type B transections. Isolates are cultured in collagen gel to maintain tissue integrity following lower layer excision. All CBIs were positive for Ganf expression (not shown). (A,B) Stage 3a/b. Intact RBI has Ganf expression (A), whereas RBI with lower layer removed (B) is negative for transcripts. CE is present in RBIs and is required for neural specification to occur, whereas the hypoblast is required for Ganf expression. (C,D) Stage 3c. Both control (C) and experimental (D) RBIs are negative for transcripts. (E,F) Stage 3d. As for stage 3a/b, except that the RBI does not include CE cells. Ganf expression occurs because the rostral tissue is already neuralised. Loss of the lower layer indicates the requirement for hypoblast and ADE.

View larger version (141K): [in a new window]   Fig. 8. Mesoderm is excluded from RBIs. (A-D) Transected isolates at the level of the node at stage 3a/b-4 tested for the presence of mesoderm in RBIs (top) and CBIs (below) immediately after transecting (asterisks indicate the nodes). In all cases, the CBIs were positive for Wnt8c transcripts, as expected, whereas the RBIs were negative.

View larger version (92K): [in a new window]   Fig. 9. The lower layer is sufficient for Ganf induction in rostral epiblast. RBIs (top) and CBIs (below) cultured in collagen gel. (A) After transection, rostral ectoderm recombined with rostral hypoblast is sufficient to induce Ganf expression (top arrow). The CBI is also positive for Ganf expression (bottom arrow). (B) Caudal endoderm taken from a position lateral to the streak is unable to induce Ganf expression (0/6) when recombined with rostral ectoderm. CBI is positive for Ganf expression (arrows).

View larger version (26K): [in a new window]   Fig. 10. Schematic representation of tissue positions in revised early patterning model. Anterior is towards the left, epiblast/neuroectoderm layer in yellow, with the rostralmost level of the primitive streak (hatched yellow) marked by vertical arrows. At stage 3a/b, note that CE is rostral to the streak, but becomes incorporated into the extending streak until it forms part of Hensen's node at stage 4. Definitive endoderm is represented by bar in blue. Only at stage 4+ does axial mesoderm begin ingressing (red). Hypoblast is responsible for establishing anterior identity in overlying epiblast at stage 3a/b (red arrowheads). Only at stage 3d and 4 is the prechordal plate endoderm fully specified and has a maintenance role in the overlying neuroectoderm layer (dark blue arrows). The CE population signals in the plane of the ectoderm and becomes incorporated into the rostral primitive streak, and together with Hensen's node forms the head organiser (horizontal arrows). After ingression of the axial mesoderm at stage 4+, a trunk/tail organiser function for Hensen's node is revealed. am, axial mesoderm; ce, central epiblast; de, definitive endoderm; ds, definitive streak; en, endoblast; ep, epiblast; gc, germ cell crescent; hy, hypoblast; hn, Hensen's node; ne, neuroectoderm; pcpe, prechordal plate endoderm; ps, primitive streak; se, stomodeal ectoderm.