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

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 HighWire Citing Articles via Google Scholar Google Scholar Articles by David, N. B. Articles by Rosa, F. M. Search for Related Content PubMed PubMed Citation Articles by David, N. B. Articles by Rosa, F. M.

Cell autonomous commitment to an endodermal fate and behaviour by activation of Nodal signalling

Groupe Danio, U 368 INSERM, Ecole Normale Supérieure, 46, rue d’ Ulm, F-75230 Paris Cedex 05, France

View larger version (57K): [in a new window]   Fig. 1. Endoderm commitment occurs at the onset of gastrulation. (A) Diagram of the experimental procedure. DMNB-caged fluorescein was injected at the 1-cell stage. At the late blastula stage, fluorescein was uncaged in a few cells at the animal pole or at the very margin of the donor embryo. At 50% epiboly-germ ring, labelled cells were transplanted to the animal pole of a late blastula (dome) host. (B,C) Anterior to the left, dorsal to the top. (B) Cells transplanted from the animal pole of the donor populated the eye and the forebrain and differentiated accordingly. (C) In contrast, marginalmost cells taken just before or at the onset of gastrulation colonised the endoderm-derived territories and expressed the endodermal marker fkd7 (section in D). Some of these cells also populated the hatching gland (not shown). The white dots indicate the limit of the YSL. e, eye; no, notochord; nt, neural tube; AP, Animal Pole; M, Margin.

View larger version (74K): [in a new window]   Fig. 2. Endoderm fate is induced by transplantation of the cells to the margin and by activation of Nodal signalling. (A) Diagram of the experimental procedure. (B,G) In late blastula (sphere stage, lateral view), a few cells (arrowhead) expressing GFP (green) alone or combined with Tar* were transplanted to the margin (B) or the animal pole (G) of a host embryo. (C-F,H-I) Anterior to the left, dorsal to the top. At 24 hpf, GFP-expressing cells transplanted to the margin mainly become endodermal (C) and mesodermal derivatives (D) (see Table 1). By contrast, activated (Tar* + GFP) cells almost exclusively become endodermal derivatives, such as pharynx (E) and gut (F), and mesendodermal hatching gland. (H) GFP-expressing cells transplanted to the animal pole populate the eye and the forebrain, whereas (I) Tar*-expressing cells contribute progeny only to the endodermal pharynx (white arrow) and to mesendodermal derivatives (hatching gland, white arrowhead). (J-L) Transverse sections showing the expression of the endodermal markers nkx2.3 (J), insulin (K) and fkd7 (L, arrowhead) by grafted Tar*-expressing cells. In all the experiments, nls-lacZ, which encodes nuclear ß-galactosidase was coexpressed with GFP as a lineage tracer for immunohistochemistry (brown nuclei) to identify grafted cells. Dorsal to the top. no, notochord; nt, nerve tube.

View larger version (41K): [in a new window]   Fig. 3. Endoderm-committed blastomeres and activated cells adopt an endodermal identity at the onset of gastrulation. During gastrulation (60% epiboly), endoderm-committed blastomeres (ECB) (A) or activated cells (C,D) transplanted to the animal pole (arrowheads) already express the endodermal genes sox17 and her5. Control cells do not (B). (B-D) Insets show close-up views of the region containing grafted cells.

View larger version (76K): [in a new window]   Fig. 4. Transient Nodal signalling autonomously commits cells to an endodermal fate. One single activated cell (white arrowhead) transplanted to the animal pole of a blastula host (A) expresses sox17 during gastrulation (arrowhead in B) and become endodermal derivatives at 24 hpf (arrow in C). Co-injection of Tar* and FASTSID at the 2-cell stage (D) results in the cells that were transplanted to the animal pole being incorporated into the eye and the brain (E). By contrast, delayed injection of FASTSID (F) results in cells grafted to the animal pole adopting an endodermal fate (G).

View larger version (76K): [in a new window]   Fig. 5. Endoderm-committed blastomeres and activated cells translocate directly to the YSL during gastrulation and develop an endodermal phenotype. Cells expressing GFP alone (A-D) or in combination with Tar* (E-H) or ECB (I-J) were transplanted to the animal pole of a host blastula. The group of grafted cells was monitored during development (lateral views). Until the beginning of gastrulation, grafted cells remain epiblastic (B,F,I), but during gastrulation, in contrast to control cells (C), Tar*-activated cells and ECB leave the epiblast to reach the surface of the YSL, without going through the margin (G,J). (D,H) Morphology of a single control and an activated grafted cell during gastrulation (70% epiboly). Note the filopodia characteristic of migrating endodermal cells in H (arrowheads).

View larger version (43K): [in a new window]   Fig. 6. The YSL is not required to maintain the endodermal identity of ECB and activated cells during gastrulation. (A) Diagram of the experimental procedure. At the blastula stage, activated cells (Tar* + GFP) or ECB were transplanted to the animal pole of host embryos. Animal caps containing the grafted cells (B) were immediately removed, cultured and fixed at the end of gastrulation (90% epiboly). (C,D) sox17 expression was revealed by in situ hybridisation (blue), and activated cells were identified by detection of nuclear ß-galactosidase (brown, D). (D) Inset shows a close-up view of the region containing transplanted cells.

View larger version (68K): [in a new window]   Fig. 7. Wild-type activated cells can form endoderm in cas mutants. (A) At the blastula stage, a few wild-type cells expressing Tar* were transplanted to a cas host. (B) Flat-mount of a young gastrula (60% epiboly) showing the expression of sox17 (blue) by activated cells (brown nuclear staining). Notice the absence of endogenous sox17 expression in cas embryos. The inset shows a close-up view of the region containing the grafted cells. (C-K) Expression of nkx2.3, insulin and fkd7 in 24 hpf wild-type (C,F,I), cas (D,G,J) and grafted cas (E,H,K) embryos. (C-E) The pharyngeal expression of nkx2.3 (C) is absent in cas embryos (D). This expression can be restored by the graft of wild-type activated cells (E). (F-H) Pancreatic expression of insulin (arrow in F), which is absent in cas embryos (G), can be restored by the graft (arrow in H). The expression of nkx2.5 (arrowheads) identifies the cardia bifida of cas embryos and was used to genotype them. (I-K) The expression of fkd7 in the gut (I) is lost in cas embryos (J). This expression can be restored by wild-type activated cells (arrows in K). (L,M) Transverse sections through the pancreatic and gut regions of grafted cas embryos showing that the expression of the insulin and fkd7 (blue, arrowhead in M) is restricted to grafted cells (brown nuclear staining). (N-P) Live grafted cas embryos. At 96 hpf, grafted cells (green) differentiate into endodermal derivatives such as pharyngeal epithelium or segments of gut (arrowheads). In O, the yolk (y) appears green because of its autofluorescence. (C-H) Dorsal views, anterior to the left. (I-K,N-P) Lateral views, anterior to the left, dorsal to the top. no, notochord; nt, nerve tube.

View larger version (75K): [in a new window]   Fig. 8. Restoring wild-type endoderm in cas mutants allows heart fusion. (A-C) nkx2.5 and fkd7 expression were detected by in situ hybridisation in 24 hpf embryos. (A) Wild-type embryo, (B) control cas embryo, (C) and cas embryo grafted with wild-type activated cells. Arrowheads indicate the heart in wild-type embryos, hemicardia in cas and fused hemicardia in grafted cas embryos. (D-F) Transverse sections through the heart region of 24 hpf embryos stained for nkx2.5 expression. (D) Wild-type embryo with a single heart. (E) Control cas embryo with two hemicardia in lateral position. (F) cas embryo grafted with wild-type activated cells. The two hemicardia have fused at the midline. The inset is a close-up view of the heart region showing that grafted cells (brown nuclei) are not incorporated into the heart but are endodermal derivatives overlaying it. (G-H) cas embryos grafted with wild-type activated cells exhibiting an axis duplication. Arrowheads point to the unfused hemicardia in the primary axis (H) and the arrow indicates the heart in the secondary axis (G, H). (G) Lateral view of a live embryo, anterior left. (H) Ventral view, following staining for nkx2.5 expression. nt, nerve tube.