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First published online 16 March 2005
doi: 10.1242/dev.01733

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Wnt/ß-catenin regulation of the Sp1-related transcription factor sp5l promotes tail development in zebrafish

Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195, USA

View larger version (122K): [in a new window]   Fig. 1. wnt3a and wnt8 coordinately regulate tail development. (A-C) Wild-type development. (D-L) Embryos from a transgenic TOPdGFP line were injected with wnt3a MOs (D,E,F), wnt8 MOs (G,H,I) or both (J,K,L). Expression of the ß-catenin-responsive reporter was examined at bud stage by in situ hybridization with a probe for GFP (A,D,G,J). Embryos are shown in a dorsal view of the tailbud, with anterior to the top. At the 10-somite stage (B,E,H,K), TOPdGFP expression was again assessed by in situ hybridization, in this case with a Fast Red color reaction. myoD expression, to visualize the defect in tail development, is in blue. Embryos are flat mounted and shown in a dorsal view with anterior to the left. At 24 hpf (C,F,I,L), living embryos of each type are shown in a lateral view, anterior to the left.

View larger version (90K): [in a new window]   Fig. 2. wnt3a and wnt8 are required for the maintenance, not initiation, of tailbud marker expression. (A-L) Embryos injected with the indicated morpholinos were fixed at tailbud stage and stained for ntl (A-D), fgf8 (E-H) and tbx6 (I-L). Embryos are shown in dorsal views, anterior to the top. (M-B') Injected embryos were allowed to develop until the 5-somite stage (M-P) or 10-somite stage (Q-B') and stained for tbx6 (M-T), spt (U-X) or ppt (Y-B'). All embryos are flat mounted and shown in dorsal views with anterior to the top. By the 10-somite stage in the tailbud of wnt3a/wnt8 MO embryos, tbx6 (T) and spt (X) are not expressed, while ppt (B') is still expressed at reduced levels compared with wnt3a MO (Z) or wnt8 MO (A').

View larger version (92K): [in a new window]   Fig. 3. Fgf signaling is not affected in the tailbud of wnt3a/wnt8 morphants. The expression of sprouty4 was examined at the 5-somite (A-D) and 10-somite (D-H) stages in wild-type (A,E), wnt3a MO (B,F), wnt8 MO (C,G), and wnt3a/wnt8 morphants (D,H). All embryos are shown in dorsal views of the tailbud, with anterior to the top.

View larger version (121K): [in a new window]   Fig. 4. wnt3a and wnt3a/wnt8 morphants lack mesodermal fates at the expense of floor plate in the caudal tail. All embryos were examined at approximately 27-28 hpf. More mildly affected wnt3a/wnt8 morphants that made some tail structures were selected for these analyses. (A-D) Tails of living 28 hpf embryos, anterior to the left. Arrows in B and D indicate premature termination of the notochord in wnt3a and wnt3a/wnt8 morphants, respectively, and the arrowhead in D marks enlarged neural tube lumen. (E-P) Embryos were fixed at 26 hpf and stained with the indicated marker. The caudal tip of the tail is outlined in black dashes for clarity where necessary. (E-H) Embryos stained with ntl to visualize the notochord, arrowheads indicate ntl expression in the tailbud; arrows in F and H indicate the tip of the truncated notochord. (I-L) -tropomyosin staining to visualize the somites; arrowheads mark the posterior extent of somite formation. (M-P) Embryos stained for F-spondin to visualize the floor plate; arrow in P indicates the expanded floor plate in wnt3a/wnt8 embryos. (Q-T) Pan-neural ngn1 expression in the neural tube is unaffected by wnt3a and wnt8 inhibition.

View larger version (93K): [in a new window]   Fig. 5. sp5l transcription is induced by ectopic Wnt and repressed by interference with Wnt signaling. (A) Ectopic expression of sp5l at the animal pole of embryos injected with 10 pg wnt8 RNA, 6 hours after injection at shield stage (right panel, dorsal view), compared with wild-type expression in embryos injected with equimolar amounts of GFP RNA (7 pg, left panel). (B) Overexpression of dominant-negative TCF rapidly abolishes sp5l expression. Fish heterozygous for heat-shock-inducible dominant-negative TCF-GFP [Tg (hsp70:TCF-GFP)w26] were outcrossed to wild type, the resulting embryos heat-shocked for 15 minutes or 30 minutes starting at the 1-somite stage and the whole clutch fixed immediately. In situ hybridization for GFP (light brown) in addition to sp5l (blue) was performed to identify transgenic embryos. sp5l expression is severely reduced in 100% (n=21) of transgenics 15 minutes after induction of the transgene (middle panel), and completely abolished in 100% (n=24) of transgenic embryos after 30 minutes (right panel). (C-F) Expression of sp5l in WT, wnt3a, wnt8 and wnt3a/wnt8 morphants at the 3-somite stage.

View larger version (138K): [in a new window]   Fig. 6. sp5l MO enhances wnt3a loss of function. Embryos were injected with 3.5 mg/ml control morpholino (A,B), 2.5 mg/ml sp5l MO + 1 mg/ml control MO (C,D), 1 mg/ml wnt3a MO +2.5 mg/ml control MO (E,F), or 1 mg/ml wnt3a MO + 2.5 mg/ml sp5l MO (G, H). (A,C,E,G) Live embryos are shown at 48 hpf, anterior to the left. (B,D,F,H) Embryos stained for tbx6 expression at the 10-somite stage and shown in a dorsal view of the tailbud region, anterior to the top.

View larger version (57K): [in a new window]   Fig. 7 sp5l RNA overexpression rescues wnt3a morphants. Embryos are shown at 48 hpf, with arrows marking the posterior end of the notochord. (A) wnt3a MO embryos co-injected with 200 pg of a control RNA (renilla luciferase) typically have a truncated notochord (89%, n=104). (B) Most embryos co-injected with wnt3a MO and 200 pg sp5l MO have normal tail development (33% have truncated notochord; n=107). (C) Graph showing penetrance of rescue.

View larger version (25K): [in a new window]   Fig. 8. Model for functions of wnt3a, wnt8 and sp5l in tail development. (A) wnt3a and wnt8 both regulate anterior-posterior (A/P) patterning and dorsal-ventral (D/V) patterning during gastrulation. The thicker arrow from wnt8 represents the larger contribution to early patterning events by wnt8 relative to wnt3a. Specification of the tail organizer is separately listed for clarity, but is probably tightly linked to D/V patterning. During somitogenesis, both genes are required for maintenance of presomitic mesoderm, but later roles for Wnt signaling in promoting mesoderm formation versus floor plate are more wnt3a dependent (represented by the thicker arrow). (B) wnt3a and wnt8 both regulate sp5l expression in the tailbud. Overlap of sp5l expression with Wnt activity (visualized by TOPGFP reporter expression) in the tailbud is represented in green. When wnt3a and wnt8 function is blocked, Wnt activity and sp5l expression in the tailbud is dramatically reduced, resulting in a failure in tail formation.