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First published online 18 February 2004
doi: 10.1242/dev.01007

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Reiterated Wnt signaling during zebrafish neural crest development

Jessica L. Lewis¹, Jennifer Bonner², Melinda Modrell^3,*, Jared W. Ragland¹, Randall T. Moon^1,4,5, Richard I. Dorsky² and David W. Raible^1,3,5,

¹ Molecular and Cellular Biology Program, University of Washington School of Medicine, Seattle, WA 98195, USA
² Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA
³ Department of Biological Structure, University of Washington School of Medicine, Seattle, WA 98195, USA
⁴ Howard Hughes Medical Institute and Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA
⁵ Center for Developmental Biology, University of Washington School of Medicine, Seattle, WA 98195, USA

View larger version (122K): [in a new window]   Fig. 1. Induction of hs-Tcf expression blocks zygotic Wnt signaling in vivo. (A) In control (non-transgenic) embryos at shield stage following 2 hours of heat shock at 37°C, gsc expression is normally limited to the axial mesoderm (animal pole view, dorsal to the right). (B) In transgenic Tcf-expressing sibling embryos the mesoderm is dorsalized, as indicated by radial gsc expression 2 hours after heat shock at 37°C. (C-F) TOPdGFP in-situ hybridization of heat shocked embryos expressing the Tcf transgene (D,F) compared with non-transgenic siblings (C,E). Embryos were fixed following 2 hour (C,D) and 6 hour (E,F) recovery periods. TOPdGFP is down regulated throughout the embryo, notably in the brain and spinal cord.

View larger version (87K): [in a new window]   Fig. 2. Temporal limit for the Wnt/ß-catenin signaling requirement. Foxd3-positive neural crest cells, identified by anti-Foxd3 primary antibody (red), flank the neural plate after heat activation at bud (A), 3-somite (B), and 6-somite stages (C). Transgenic embryos show significant loss of Foxd3 expression upon heat activation of hs-Tcf (green) (D,E), while Foxd3 expression is comparable to that of wild-type siblings after induction at 6-somites (F). All images are dorsal views, anterior to top, of stacked 10X confocal series after fixation at 3-somites (A,D) or 10-somites (B,C,E,F). Expression of the transgene is shown in the insets (D,E,F).

View larger version (72K): [in a new window]   Fig. 3. Loss of canonical Wnt signaling does not alter Hu expression. (A,B) Foxd3-positive neural crest cells (outline empty arrowhead), identified by anti-Foxd3 primary antibody (red), flank the neural plate after heat activation at the 3-somite stage. As shown in (B), transgenic embryos show significant loss of Foxd3 expression upon heat activation of hs-Tcf (green) as compared to with wild-type siblings (A). (C,D,E) Anti-Hu antibody, which recognizes the pan-neuronal marker Hu, was were used to identify Hu-positive cells in heat-activation treated embryos (red). Normal expression is detected in the trigeminal ganglia (white arrowhead), Rohon-Beard sensory neurons (white arrow), and primary motorneurons in the spinal column (asterisk) in Tcf embryos (D) as compared to with their wild-type siblings (C). Overlap between Hu and transgene expression is seen at higher power (E). All images are dorsal views of flat-mounted embryos fixed at the 10-somite stage, anterior to top, of stacked confocal series obtained at 10X (A-D) or 20X (E).

View larger version (101K): [in a new window]   Fig. 4. Canonical Wnt/ß-catenin signaling is required in neural crest precursors cell-autonomously. (A) As a control, rhodamine dextran-labeled wild-type neural crest precursors (red) were transplanted into a wild-type host embryo. The transplanted cells express Foxd3, detected with anti-Foxd3 antibody (green) when transplanted into a wild-type host embryo. Inset is a single confocal slice to indicate co-localization in single transplanted cell (white arrowhead). (B,C) Transgenic crest precursors fail to express Foxd3 when transplanted into a wild-type environment. Embryo shown is a dorsal view with anterior to the top. Inset in (B) is magnified in (C). (D-F) Wild-type cells express Foxd3 when transplanted into a transgenic environment. (D) Foxd3-positive nuclei, detected with anti-Foxd3 primary antibody (red), are observed in transplanted cells from wild-type donor embryos. (E) GFP-positive nuclei (green) are observed in the Tcf background after heat-activation of the transgene. (F) Stacked confocal images have been merged to show of failure of Foxd3-positive nuclei to co-localize with background GFP expression.

View larger version (26K): [in a new window]   Fig. 5. Wnt8 and pax3 show overlapping expression in the presumptive neural crest domain. (A) Whole-mount in-situ hybridization at the 80% epiboly stage reveals expression of wnt8 transcripts at the margin and along the dorsal side of the embryo. (B) Expression of pax3 in the presumptive neural crest (arrow). (C) The wnt8 expression domain is in proximity to, and partially overlapping with, expression of pax3 in the presumptive neural crest region (black arrow). All images are lateral views, with dorsal to the right, of 80% epiboly stage embryos after hybridization.

View larger version (100K): [in a new window]   Fig. 6. Wnt8 is required for neural crest induction in vivo. Whole-mount in-situ hybridization at the 6-somite stage reveals loss of early neural crest markers foxd3 (A-C), pax3 (D-F), and sox10 (G-I) upon injection of 20 mg/ml wnt8.1 Morpholino oligonucleotides (B,E,H). Such loss was not detected upon injection of 20 mg/ml wnt8.2 Morpholino (C,F,I), suggesting it does not have a role in neural crest induction. Uninjected controls (A,D,G) show normal expression of markers in the neural crest; foxd3 is also expressed in the notochord. All images are dorsal views, anterior to top, of 3-somite embryos.

View larger version (80K): [in a new window]   Fig. 7. Functional Wnt8 is not required for late expression of neural crest markers. Whole-mount in-situ hybridization for neural crest marker, sox10, on 10-somite (A,B) and 24hpf (C,D) stage embryos. (E,F) Whole-mount in-situ hybridization for melanophore-specific marker, mitfa. (G,H) Whole-mount in-situ hybridization for cranial neural crest marker, dlx2. Injection of 20 mg/ml wnt8.1 antisense Morpholino oligonucleotides does not significantly reduce or eliminate the expression of any of these neural crest markers. All images of uninjected control siblings (A,C,E,G) and wnt8.1 MO-injected embryos (B,D,F,H) are lateral views (B-F) or dorsal views (A-B,G-H) with anterior to the left. Scale bar: 100 µm in (A,B); 200 µm in (C-F); 50 µm in (G,H).

View larger version (89K): [in a new window]   Fig. 8. Wnt/ß-catenin signaling is required for mitfa expression and branchial arch expression of dlx2 upon crest migration. (A,B) Whole-mount in-situ hybridization for melanophore-specific marker, mitfa. (C,D) Whole-mount in-situ hybridization for neural crest marker, sox10. (E-H) Whole-mount in-situ hybridization for cranial neural crest marker, dlx2. Black arrowhead indicates few remaining mitfa-expressing cells. Open arrowhead indicates normal pattern of sox10-expressing neural crest. While telencephalon and diencephalon expression is unchanged in TCF transgenic embryos, dlx2 expression is observed in the developing branchial arches. All images are lateral views, anterior to the left, of wild-type siblings (A,C,E,G) and hsTcf-GFP transgenic embryos (D,E,F,H) after heat shock at the 18-somite stage and fixation at the 26-somite stage, except (G,H) which are dorsal views of flat-mounted embryos, anterior to the left.