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First published online 16 October 2008
doi: 10.1242/dev.029025

Development 135, 3719-3729 (2008)
Published by The Company of Biologists 2008
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Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation

Sang-Wook Cha*, Emmanuel Tadjuidje*, Qinghua Tao, Christopher Wylie and Janet Heasman{dagger}

Division of Developmental Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.


Figure 1
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  		Fig. 1. Maternal Dkk1 is ubiquitously expressed. (A) Control uninjected (uninj.) and sibling antisense oligo (AS1 5 ng)-injected oocytes were frozen as mature oocytes, mid- (stage 8.5), early (stage 10.5) and late gastrula (stage 12) stage oocytes, and assayed by real-time RT-PCR for the relative expression of Dkk1 mRNA. (B) Batches of two whole and four animal- or four vegetal-half uninjected stage 6 oocytes were assayed by real-time RT-PCR for the relative expression of VegT and Dkk1 mRNA. (C) Batches of two whole and four right-, left-, dorsal- or ventral-half four-cell stage embryos were assayed by real-time RT-PCR for the relative expression of Dkk1 mRNA. (D) Control uninjected (uninj.) and sibling antisense oligo (AS1 5 ng, 10 ng)-injected oocytes were frozen after 48 hours in culture as mature oocytes and assayed for maternal Dkk1 mRNA expression.

 

Figure 2
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  		Fig. 2. Maternal Dkk1 inhibits canonical Wnt signaling. (A) Embryos derived from sibling control (uninj.) and Dkk1-depleted oocytes (5, 7.5 and 10 ng oligo injected) at the early tailbud stage. This phenotype was seen in seven experiments in a total of 85% of cases (124/165). (B) The phenotype of Dkk1-depleted (Dkk-) embryos was partially rescued by the reintroduction of 20 pg human Dkk1 mRNA (Dkk-+mRNA) before fertilization. Here, Dkk1-depleted embryos (14/17) had the elongated phenotype shown compared with 4/14 for Dkk1-+mRNA and 0/24 uninjected; 20 pg human Dkk1 mRNA alone (Dkk mRNA) caused enlargement of head structures (16/18). The experiment was repeated with a similar result. (C) The relative expression levels of Xnr3 and Xnr5 in control (uninj.), in Dkk1 depleted (Dkk-), in 20 pg human Dkk1 mRNA (Dkk mRNA) and in Dkk1 depleted+20 pg human Dkk1 mRNA injected (Dkk-+mRNA) embryos assayed by real-time RT-PCR at the late blastula stage; siblings of those shown in B. (D) TOPflash reporter activation after injection into two dorsal cells of four-cell stage control embryos compared with sibling Dkk1-depleted embryos frozen at the eight-cell, mid-(stage 8), late blastula (stages 9, 9.5) and early gastrula stages (stages 10, 10.5). (E) Western blot of total β-catenin protein in control and Dkk1-depleted sibling early blastulae (stage 7), using {alpha}-tubulin as a loading control. Quantitation is shown on the right. (F,G) In situ hybridization of sibling control and Dkk1-depleted early gastrulae (Xnr5, F; chordin, G). (H) Western blot of phospho-Smad2 and -Smad1 proteins in control and Dkk1-depleted sibling at late blastulae (stage 9.5) and early gastrulae (stage 10.5). Before freezing, embryos were hemisected into batches of four dorsal (dor) and four ventral (ven) halves.

 

Figure 3
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  		Fig. 3. Maternal Dkk1 inhibits non-canonical Wnt signaling. (A) Embryos derived from sibling control, β-catenin-depleted (β-catMO), Dkk1-depleted and Dkk1/β-catenin-depleted oocytes at the early gastrula stage. The Dkk1-depleted abnormal shape change was seen in 22/26 cases, and was present in 0/25 β-catenin-depleted, 18/21 Dkk/β-catenin-depleted cases and 2/24 controls. The experiment was repeated with similar results. (B) Embryos derived from sibling control, Dkk1-depleted, β-catenin-depleted and Dkk/β-catenin-depleted oocytes at the early tailbud stage. 18/20 Dkk1-depleted embryos had the phenotype shown, whereas β-catenin-depleted embryos were ventralized (20/20, 10 ng MO; 18/18, 20 ng MO). Dkk/β-catenin-depleted embryos were also ventralized (19/20 Dkk-/10ng MO; 17/17 Dkk-/20ng MO) and 24/24 controls were normal. (C) The relative expression levels of Xnr5 in control, Dkk1-depleted, β-catenin-depleted and Dkk1/β-catenin-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (D) Equatorial zones from control, Dkk1-depleted, β-catenin-depleted and Dkk1/β-catenin-depleted dissected at the mid-blastula stage and cultured until the late neurula stage. Dkk1-depleted (9/9) and Dkk1/β-catenin-depleted explants (10/11 Dkk-/10 ng MO; 11/11 Dkk-/20 ng MO) were hyper-elongated compared with control, whereas β-catenin-depleted embryos were round (8/8 with 10 ng MO; 9/9 with 20 ng MO). The experiment was repeated with similar results. (E) Western blot of p-JNK-1 protein in control and Dkk1-depleted sibling at late blastulae (stage 9.5) and early gastrulae (stage 10.5), hemisected into dorsal (dor) and ventral (ven) halves before freezing. A total JNK antibody was used as loading control. (F) Western blot of p-JNK-1 protein in control and Dkk1-depleted vegetal masses compared with equatorial explants. Ten vegetal masses and five equators were compared. Quantitation is shown on the right. P<0.01, Student's t-test. (G) Western blot of p-JNK-1 protein in control, β-catenin-depleted, Dkk1-depleted and Dkk1/β-catenin-depleted (Dkk-/β-catMO) late blastulae. Quantitation is shown on the right. P<0.03, Student's t-test. (H) Equatorial zones from control, Dkk-depleted and JNK inhibitor SP600125-treated equatorial zones dissected at the mid-blastula stage and cultured until the late neurula stage. 0/10 uninjected, 10/10 Dkk- and 1/10 Dkk-/SP600125-treated explants had the Dkk1-depleted shape. The experiment was repeated with similar results.

 

Figure 4
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  		Fig. 4. Maternal Wnt5a is essential for both canonical and non-canonical Wnt signaling. (A) Control uninjected and sibling, Wnt5a antisense oligo-injected oocytes were assayed by real-time RT-PCR for the relative expression of Wnt5a, Wnt11, β-catenin and Dishevelled 2 (Dvl2) mRNA. (B) Real-time RT-PCR assay of the relative expression of Wnt5a in uninjected and Wnt5a-depleted oocytes at blastulae (stage 9) and gastrulae (stages 10, 12). (C) Embryos derived from sibling control and Wnt5a-depleted oocytes at the early tailbud stage. This phenotype was seen in four experiments in a total of 99% of cases (102/104); controls were normal in 99% of cases (91/92). (D) The phenotype of Wnt5a depleted (Wnt5a-) embryos was partially rescued by the reintroduction of 25 pg Xenopus Wnt5a mRNA (Wnt5a-+Wnt5a mRNA) before fertilization. Wnt5a-depleted early tailbud stage embryos (5/6) had the ventralized phenotype shown compared with 0/6 Wnt5a-+25 pg Wnt5a mRNA embryos and 0/10 Wnt5a-+100 pg Wnt5a mRNA embryos; 11/11 controls were normal. The experiment was repeated with a similar result. (E) The relative expression levels of Xnr3 and siamois in control, Wnt5a-depleted and Wnt5a-depleted+100 pg Xenopus Wnt5a mRNA-injected embryos assayed by real-time RT-PCR at the early gastrula stage. (F) TOPflash reporter activation in control and Wnt5a-depleted embryos frozen at the late blastula stage. (G) TOPflash reporter activation in Wnt5a mRNA overexpressing embryos (100 pg), β-catenin-depleted embryos and sibling embryos injected with both Wnt5a mRNA and β-catenin MO. (H) Western blot of p-JNK-1 protein in control, Dkk1-depleted, Wnt11-depleted, Wnt5a-depleted, Dkk1/Wnt11- and Dkk1/Wnt5a-depleted late blastulae. Quantitation is shown below. P<0.01, Student's t-test. (I) The relative expression levels of Xnr5 and chordin in control, Dkk1-depleted, Wnt11-depleted and Dkk1/Wnt11-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (J) The relative expression levels of Xnr5 and chordin in control, Dkk1-depleted, Wnt5a-deleted and Dkk1/Wnt5a-depleted embryos assayed by real-time RT-PCR at the early gastrula stage. (K,L) Batches of two whole and four dorsal- or ventral-half, 32-cell stage control and Wnt5a-depleted embryos assayed by real-time RT-PCR for the relative expression of Wnt11 mRNA (K) and Wnt5a mRNA (L).

 

Figure 5
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  		Fig. 5. Interactions between Wnt11 and Wnt5a. (A) Wnt11-HA or Xnr2-HA mRNA (1 ng) was injected either alone or together with Wnt5a-Myc mRNA (1 ng) in the same cells at the two-cell stage. Immunoprecipitation was performed with anti-Myc antibody. Co-immunoprecipitation shows Xnr2-HA present in the lysate (lanes 1-2) but absent in the anti-Myc IP-complex (lanes 5-6); Wnt11-HA was present in the whole lysates (lanes 3-4) and precipitated with Wnt5a-Myc (lanes 7-8). Arrowhead, IgG band; asterisk, Wnt11-HA band. (B) Schematic description of same cells (SC) and different cells (DC) injected with tagged Wnt11 and Wnt5a mRNAs at the four-cell stage. (C) Co-immunoprecipitation shows Wnt11-HA present in the whole lysates (lanes 1-2) and precipitated with anti-Myc from both SC and DC injections (lanes 3-4). Wnt5a-HA is detected in whole lysates (lanes 5-6) and after anti-GFP co-IP from SC and from DC injections (lanes 7-8). (D) Schematic description of the TOPflash reporter assay after Wnt mRNA overexpression in the oocyte. (E) TOPflash Luciferase activity measure in blastulae derived from control (uninj), Wnt5a-injected (Wn5a 5 pg), Wnt11 injected (Wnt11 5 and 10 pg) or co-injected (Wnt5a 2.5 pg+Wnt11 5 pg) oocytes.

 

Figure 6
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  		Fig. 6. Wnt proteins form homodimers. (A) Western blot analysis of gastrulae injected at the two-cell stage with 0.5 ng or 1 ng of Wnt11-HA mRNA. Lanes 1 and 2, no reducing agent; bands of Wnt11-HA are present at 50 kDa (monomer) and 100 kDa (dimer). Lanes 3-5, β-ME added (1, 5, 10%); only the 50 kDa band is visible. (B) Western blot of 125, 250 or 500 pg Wnt11-HA mRNA injected embryos+iodoacetamide (IAA, 10 mM). There is a 100 kDa band (*) in the 250 and 500 pg lanes both in the absence (lanes 2, 3) and presence (lanes 5, 6) of IAA. β-ME converts to monomers (lanes 7, 8). (C) Western blot of 0.25, 0.5, 1 ng Wnt11-HA, Wnt8-HA or Wnt5a-Myc mRNA-injected embryos with IAA-containing buffer and non-reducing electrophoresis. Wnt11 (lanes 1-3), Wnt8 (lanes 4-6) and Wnt5a (lanes 7-9) form dimers. (D) Anti-GFP western blot analysis of embryos injected with 100 pg of Wnt11-GFP mRNA alone or +50, 100, 200, 500 pg of Wnt5a-Myc mRNA; non-reducing conditions. Wnt11-GFP alone (lane 1) produces a 75 kDa band (monomer) and a 150 kDa band (dimer). Co-injection with increasing doses of Wnt5a mRNA (lanes 2-5) showed no band of the expected 125 kDa (*) of Wnt5a-Myc/Wnt11-GFP heterodimer. Lanes 6 and 7 show that the signal in lanes 1-5 is specific to Wnt11-GFP. Arrowhead indicates a non-specific band. (E) Anti-HA WB in non-reducing conditions where 0.5 ng of Wnt11-HA mRNA alone (lane 1) produced 50 and 100 kDa bands. Co-injection with 0.5 ng of Wnt11{Delta}C-Flag mRNA (30 kDa) (lane 2) did not produce the 80 kDa band (*) expected for Wnt11{Delta}C-Flag/Wnt11-HA heterodimer. Similarly, the pattern of Wnt5a-Myc bands (lane 6) was not changed by the co-injection of Wnt11{Delta}C-Flag (lane 4); *expected positions for a Wnt5a-Myc/Wnt11{Delta}C-Flag heterodimer (80 kDa). **Wnt11{Delta}C-Flag homodimer. No heterodimer bands were detected in co-injection samples (lanes 7, 12, asterisks) compared with Wnt11{Delta}C-Flag alone (lane 8). Arrowheads indicate non-specific bands. (F) Analysis of Wnt11-HA and Wnt5a-Myc Co-IP under non-reducing conditions (NR) shows that only dimers (100 kDa) and oligomers (>100 kDa) of Wnt11-HA were precipitated by Wnt5a-Myc (lane 3); under reducing conditions (R), the IP product was seen a single monomer band (lane 4). (G) Western blot under non-reducing conditions of control and Dkk1-depleted oocytes injected with 1 ng Wnt11-HA or Wnt5a-Myc mRNAs. Dimer forms of Wnt proteins are marked with asterisks. Repeated experiments showed similar results.

 

Figure 7
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  		Fig. 7. Models of Wnt11/5a signaling activity in dorsal axis formation. (A) Model 1. The relative distribution of Wnt11 and Wnt5a protein in eight-cell stage and Wnt signaling activity at late blastula stage embryos as described in the text. (B) Model 2. Possible composition of the Wnt11/5a signaling complexes, as described in the text.

 

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