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First published online 1 October 2003
doi: 10.1242/dev.00685

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Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

¹ Program in Neurobiology and Behavior, Department of Biological Structure, Box 357420, University of Washington, Seattle, WA 98195, USA
² Program in MCB, Department of Biological Structure, Box 357420, University of Washington, Seattle, WA 98195, USA

View larger version (100K): [in a new window]   Fig. 3. Wnt3a induces Irx3 and Gbx2 in prospective dT explants. (A) The experimental procedure. Prospective vT and dT explants from HH stage 8 embryos were embedded in collagen, cultured in the presence or absence of soluble Wnt3a, fixed and assayed for Irx3, Gbx2 and Dlx2 expression by in situ hybridization. (B) The data was quantified and analyzed statistically using the -square significance test. (C-N) Representative explants in the culture condition indicated following in situ hybridization for Irx3 (C-F), Gbx2 (G-J) and Dlx2 (K-N). Some panels show more than one explant. The total number of explants in each experimental condition are indicated above each bar in B. *** indicates P<0.0001. Scale bar: 250 µm in C-F, 100 µm in G-N.

View larger version (92K): [in a new window]   Fig. 5. Wnt is required in ovo for proper induction of Gbx2 and repression of Dlx2 in the diencephalon. (A-D) Adjacent sections were processed by in situ hybridization to visualize the expression of Gbx2 (A,C) and Dlx2 (B,D). Electroporation was used to misexpress Dkk1 in the developing diencephalon of two HH stage-9 embryos (C,D). Ectopic expression of Dkk1 resulted in ectopic expression of Dlx2 (arrow in D) in the normally Gbx2-positive dT (A). Arrowheads indicate the position of the zli. Scale bar: 100 µm.

View larger version (42K): [in a new window]   Fig. 1. Six3, Irx3, Gbx2, Dlx2 and Wnt3 expression in the developing forebrain. (A-J) In situ hybridization of Six3 (A,B), Irx3 (C,D), Dlx2 (E,F), Gbx2 (G,H) and Wnt3 (I,J) at HH stage 7-8 (upper panels) and HH stage 19 (lower panels). At HH stage 7-8, Six3 (A) and Irx3 (C) are anterior and posterior to the prospective zli, respectively. At this stage, Gbx2 (G) expression is confined to the prospective hindbrain, and Dlx2 (E) and Wnt3 (I) expression is negligible. At HH stage 19, the expression domains of Irx3 (D), Gbx2 (H) and Wnt3 (J) are posterior to the zli, whereas Six3 (B) and Dlx2 (F) are expressed anterior to this structure. Arrowheads indicate the position of the zli. Scale bar: 100 µm.

View larger version (60K): [in a new window]   Fig. 2. Wnt signaling is sufficient to inhibit Six3 expression and is required for Irx3 expression in headfold explants. (A-H) Headfold explants were cultured in the presence or absence of Wnt3a, Dkk1 and cki7, and Six3 or Irx3 expression assayed by in situ hybridization. Representative headfold explants are shown following culture and in situ hybridization for Six3 (A-D) or Irx3 (E-H). n=the number of explants tested per condition. Scale bar: 100 µm.

View larger version (83K): [in a new window]   Fig. 4. Wnt signaling is required for dT-specific differentiation in response to zli-derived signals. (A) Diagram of experimental procedure. HH stage 17 zli explants were co-cultured with either prospective vT or dT explants from HH stage 8 embryos in the presence or absence of cki7 for 36-48 hours, fixed, and then processed by in situ hybridization. The horizontal line indicates the limit of the notochord and predicts the future location of the zli. (B) Co-culture induction data was quantified and analyzed statistically using a 2 significance test. (C-J) Representative in situ hybridization images for Dlx2 (D,F,H,J) and Gbx2 (C,E,G,I) in the indicated co-culture conditions. Dotted lines represent the border between prospective dT or vT explants and zli explants, as determined by using CellTracker. The total number of explants in each experimental condition are indicated above each bar in B. *, P<0.05. Scale bar: 100 µm.

View larger version (90K): [in a new window]   Fig. 6. In ovo misexpression of Six3 suppressed diencephalic Wnt3 and Gbx2 expression and upregulated Dlx2 in the dT. (A-F) Serial sections of embryos 48 hours after electroporation with Six3-IRES-GFP at HH stage 9-10. In sections in which GFP was visualized in the dT (A), Gbx2 expression was suppressed (B, n=8). In 25% of these embryos Dlx2 was induced ectopically in the dT (arrowheads in C). Misexpression of Six3-IRES-GFP in the dT (D) also results the down-regulation of Wnt3 (E,F, n=2). Scale bar: 100 µm.

View larger version (16K): [in a new window]   Fig. 7. Wnt signaling and diencephalic patterning. A widespread Wnt signal induces the expression of Irx3 in the developing forebrain, which specifies a posterior, dT-committed fate. Irx3 expression allows the induction of Gbx2 by signals released from the zli, which might include Wnt3 and Wnt3a. Neural tissue that overlies the prechordal plate is exposed to Wnt antagonists, which results in the expression of Six3. In turn, Six3 allows the induction of Dlx2 in response to zli-derived signals and represses the expression of Irx3, Wnt3 and Gbx2. This model predicts that the zli will form at the interface between the domains of Six3 and Irx3 expression, above the transition between the notochord and the prechordal plate. Wnt3 is expressed throughout the prospective dT, just posterior to the zli (Fig. 1B) (Roelink and Nusse, 1991). The diencephalic phenotype of the Wnt3a-knockout mouse is unknown because it has a lethal gastrulation defect that prevents analysis of the role of Wnt3 in forebrain development (Lee et al., 1997; Liu et al., 1999). Because of their largely overlapping expression patterns and nearly identical protein sequences, it is likely that Wnt3 and Wnt3a have partially redundant functions in brain development, which would explain the relatively mild phenotype observed in Wnt3a–/– mice. Given the expression patterns of Wnt3 and Wnt3a in the diencephalon, these molecules are good candidates for inductive signals that either confer or maintain posterior identity on prospective dT tissue.