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 Kudoh, T. Articles by Dawid, I. B. Search for Related Content PubMed PubMed Citation Articles by Kudoh, T. Articles by Dawid, I. B.

Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm

¹ Department of Anatomy and Developmental Biology, University College London, Gower St, London WC1E 6BT, UK
² Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

View larger version (79K): [in a new window]   Fig. 1. Expression of cyp26 at gastrula to early somitogenesis stages. Views of whole embryos (orientation indicated to the left of each row) at the stages indicated on top of each column. cyp26 was stained by in situ hybridization (purple), while ntl (red) was used to mark the blastoderm margin (A, A', open arrowhead) and developing notochord (C,F, asterisk). cyp26 is expressed in the presumptive anterior neural ectoderm (A,A' filled arrowhead) and at the blastoderm margin (A,A' open arrowhead) throughout gastrulation (B-D). Subsequently, expression in the anterior neural ectoderm decreases rapidly (E'', F'' filled arrowhead), while expression continues in the tail bud.

View larger version (91K): [in a new window]   Fig. 2. cyp26 and hoxb1b expression domains define an early AP boundary within the prospective neuroectoderm. Dorsal views of whole embryos at the stages indicated on top of. Genes analyzed are indicated at the left of the column or in the figure, with text color matching the in situ stain. cyp26 expression starts at the 30 to 40% epiboly stage (A) in presumptive anterior neural ectoderm (filled arrowhead) and at the blastoderm margin (open arrowhead), leaving a narrow gap (asterisk). As gastrulation proceeds, hoxb1b expression is initiated within the gap at about the 60% epiboly stage (C,G,K). From this time onwards, cyp26 and hoxb1b are expressed in a complementary manner in the neural ectoderm throughout gastrulation; a narrow domain of reduced expression is observed at the interface between the expression domains of these two genes (K-M, arrow). (N-Q) cyp26 expression (red) in relation to expression domains of other AP-specific genes (purple), raldh2 (N,O), iro1 (P) and krox20 (Q). There is a narrow gap between cyp26 and raldh2 expression domains at early gastrula (N, arrow), and this gap becomes wider by late gastrula (O, arrow). At the end of gastrulation, cyp26 and the anterior domain of iro1 expression share a common posterior boundary (P, arrow). There is a gap between cyp26 and rhombomere3 staining of krox20 (Q, arrow); thus the posterior boundary of cyp26 at this stage appears to be positioned around rhombomere 1.

View larger version (110K): [in a new window]   Fig. 3. Fgf signaling alters the expression of early AP-specific genes. Dorsal views of whole embryos at late gastrula stage. One- to two-cell embryos were injected with mRNAs for fgf3 (B,F,J) or RNA encoding the dominant negative Fgf receptor, XFD (D,H,L). Embryos were stained either with cyp26 (A-D), hoxb1b (E-H) or otx2 (I-L). The anterior expression of cyp26 and otx2 was suppressed by Fgf3 (B,J) and expanded in a posterior direction by XFD (D,L). Expression of the posterior gene hoxb1b was expanded by Fgf3 (F) and suppressed by XFD (H, arrowhead). The entire embryo is affected by injection at the one-cell stage (B), while in some cases only half the embryo is affected when one of two cells is injected (H).

View larger version (73K): [in a new window]   Fig. 4. RA alters the expression of early AP-specific genes. Dorsal views of whole embryos at late gastrula stage. Embryos at the 40% epiboly stage were treated with 10–6 M RA for 80 minutes. The embryos were fixed at the 80 to 90% epiboly stage, and stained with cyp26 (A,B), hoxb1b (C,D), meis3 (E,F) or otx2 (G,H). The posterior genes hoxb1b and meis3 were ectopically induced in the anterior region by RA (B,D), while the anterior gene otx2 was suppressed (H). Although cyp26 is expressed in the anterior region, its expression was activated by RA(B).

View larger version (99K): [in a new window]   Fig. 5. Cyp26 can suppress posterior genes but does not induce anterior genes in the posterior region. Dorsal views of whole embryos at late gastrula stage. cyp26 mRNA was injected at the two-cell stage into one blastomere. Some of the embryos were treated with RA (K,L). Embryos were stained for hoxb1b (A,B), meis3 (C,D), iro1 (E,F), otx2 (G,H,K,L) and otx2/hoxb1b together (I,J). hoxb1b, meis3 and the posterior domain of iro1 were suppressed by cyp26 injection (B,D,F,J, arrowhead), but otx2 and the anterior domain of iro1 expression were neither suppressed nor expanded (F,H,J). The suppression of otx2 by RA was partially rescued by cyp26 (L, arrowhead).

View larger version (99K): [in a new window]   Fig. 6. Epistatic analysis of the function of Fgf and RA in patterning the neural ectoderm. Dorsal views of whole embryos at late gastrula stage. RNA for fgf3, alone or together with cyp26 RNA, was injected at the one-cell stage. The embryos were stained for hoxb1b (A-C) or otx2 (D-F). XFD mRNA was injected at the one-to-two cell stage, and half of the embryos were treated with RA. Embryos were stained for hoxb1b (G-I) or otx2 (J-L).

View larger version (87K): [in a new window]   Fig. 7. Epistatic analysis of the function of Wnt and Fgf in patterning the neural ectoderm. Dorsal views of whole embryos at late gastrula stage. Gain and loss of function of Wnt activity was achieved by LiCl treatment (B,G,L) and dkk1 injection (C,H,M), respectively. Epistasis with Fgf was examined by XFD injection followed by LiCl treatment (D,I,N) and by fgf3 +dkk1 co-injection (E,J,O). Embryos were fixed at late gastrula and stained with cyp26 (A-E), otx2 (F-J) and hoxb1b (K-O). Arrowheads in C and M indicate the limit of caudal expansion of cyp26 expression (C) and remaining hoxb1b expression (M). In some cases, only half the embryo is affected when one of two cells is injected (N, arrowhead).

View larger version (87K): [in a new window]   Fig. 8. Epistatic analysis of the function of Wnt and RA in patterning the neural ectoderm. Dorsal views of whole embryos at late gastrula stage (A-F,I,J), and animal views of 55% epiboly stage embryos (G,H). cyp26-injected embryos were treated with LiCl at the 50% epiboly stage (B,E), and dkk1-injected embryos were treated with RA at 40% epiboly stage (C,F). Embryos were fixed at late gastrula and stained with otx2 (A-C) and hoxb1b (D-F). Regulation of the expression of raldh2 was examined in dkk1-injected embryos (H,J). At 55% epiboly stage, raldh2 expression is not suppressed at the blastoderm margin by dkk1 injection though the raldh2 negative area in the dorsal-most margin is slightly expanded (H). At late gastrula stage, raldh2 expression remains restricted to the blastoderm margin in dkk1-injected embryos (J, arrowhead).

View larger version (57K): [in a new window]   Fig. 9. Abrogation of Cyp26 activity causes moderate posteriorization. Dorsal views of whole embryos at late gastrula stage (A-H) and lateral views of late blastula stage embryos (I,J). A control morpholino or mCYP1, which is complementary to cyp26 mRNA sequence, was injected at 5 ng per embryo. Embryos were stained with otx2 (A,B), hoxb1b (C,D), meis3 (E,F) and iro1 (G,H). In addition, cyp26-GFP fusion construct was injected with either morpholino, and fluorescence was examined. (I,J). (A,C,E,G,I) Control morpholino; (B,D,F,H,J) mCYP1-injected embryos.

View larger version (17K): [in a new window]   Fig. 10. Summary of experiments. The experimental results are summarized as a cartoon. otx2 and hob1b are used as representative markers of anterior and posterior neural ectoderm, respectively, as these genes were examined in all of experiments. The top picture is a dorsal view of a late gastrula embryo divided into anterior (purple) and posterior (red) neural ectodermal regions and undifferentiated mesendoderm at the blastoderm margin (green). One half of the dorsal ectoderm is surrounded by yellow line and experimental results are summarized in this area as marker genes for mesoderm and axial area were not analyzed in any detail in this study.

View larger version (23K): [in a new window]   Fig. 11. A model for interactions between Fgf, Wnt and RA signaling in the neural ectoderm during gastrulation. (A) Sequence of posteriorization signals. Fgf and/or Wnt signals initiate the first step of posteriorization by suppressing expression of anterior genes, represented here by cyp26 and otx2. This process is not mediated by RA. In the posterior domain, where cyp26 expression is suppressed by Fgfs/Wnts, RA accumulates at least in part due to the activity of Raldh2, and activates posterior genes such as hoxb1b and meis3. (B) At the late blastula stage, Fgfs/Wnts are expressed in the mesoderm at the blastoderm margin. Fgf/Wnt signals from the margin block the expression of cyp26 in the adjacent ectoderm, which will give rise to the posterior neural plate. cyp26 is expressed in the anterior domain, at a distance from the source of the Fgf/Wnt signals; as a consequence, RA is degraded and the expression of posterior genes is prevented. After the beginning of gastrulation, convergence-extension movements lead to a widening of the cyp26-negative area, allowing RA to accumulate to a level where it can activate posterior genes such as hoxb1b. Subsequent cell movements expand the domain that will give rise to the posterior neural ectoderm.