Dkk2 promotes neural crest specification by activating Wnt/β-catenin signaling in a GSK3β independent manner

  1. Arun Devotta
  2. Chang-Soo Hong
  3. Jean-Pierre Saint-Jeannet  Is a corresponding author
  1. New York University, United States
  2. Daegu University, Republic of Korea
11 figures and 1 additional file

Figures

Figure 1 with 2 supplements
Dkk2 knockdown blocks neural crest formation in vivo.

(a) The translation blocking MO (Dkk2MO) targets the initiation codon. (b) Western blot using lysates from embryos injected with Dkk2-Flag mRNA (10 ng) alone or in combination with increasing …

https://doi.org/10.7554/eLife.34404.002
Figure 1—source data 1

Quantification of Dkk2 knockdown phenotype.

https://doi.org/10.7554/eLife.34404.005
Figure 1—figure supplement 1
Quantification of unspliced vs. spliced transcripts in MO-injected embryos.

(a) Schematic representation of the dkk2 locus. The position of the splice blocking MO (Dkk2SMO) is shown (red line). The PCR primers used to analyze the spliced (E1/E2) and unspliced (I2/I3) …

https://doi.org/10.7554/eLife.34404.003
Figure 1—figure supplement 2
Dkk2 plasmid DNA injection rescues snai2 expression in morphant embryos.

Injection of Xenopus Dkk2 plasmid DNA (10 pg) restores snai2 expression in Dkk2SMO-injected (30 ng) embryos. Results from two independent experiments. For the phenotype of Dkk2 gain of function see F…

https://doi.org/10.7554/eLife.34404.004
Dkk2 knockdown does not affect the expression of neural plate border specifiers and mesoderm formation.

(a). Unilateral injection of Dkk2SMO (20 ng) did not affect the expression levels of pax3, sox8 and snai1, although their expression was shifted laterally. (b) The neural crest specifier twist1 was …

https://doi.org/10.7554/eLife.34404.006
Figure 2—source data 1

Quantification of Dkk2 knockdown phenotype.

https://doi.org/10.7554/eLife.34404.007
Dkk2 knockdown blocks neural crest induction by Wnt8 in neuralized animal cap explants.

(a) At the 2 cell stage, mRNAs encoding noggin (50 pg) and wnt8 (100 pg) were injected in the animal pole region alone or in combination with Dkk2SMO (30 ng) or CoMO (30 ng). At the blastula stage …

https://doi.org/10.7554/eLife.34404.008
Developmental expression of dkk2.

(a) Temporal expression of dkk2.L and dkk2.S by qRT-PCR. (b) By in situ hybridization, at all stages examined (NF stage 11-33/34) dkk2 does not appear to be spatially restricted. Sense probe is …

https://doi.org/10.7554/eLife.34404.009
Expression of Lrp6 and β-catenin rescue neural crest formation in Dkk2-depleted embryos.

(a) Unilateral injection of Dkk2SMO (30 ng) reduced snai2 expression. This phenotype was efficiently rescued by injection of plasmid DNA encoding lrp6 (50 pg) or β-catenin (50 pg), and to a lesser …

https://doi.org/10.7554/eLife.34404.010
Figure 5—source data 1

Quantification of Dkk2 knockdown phenotype upon β-catenin, lrp6 or wnt8 coexpression.

https://doi.org/10.7554/eLife.34404.011
Dkk2 overexpression induces snai2 expression in vivo but cannot substitute for Wnt8 activity in neuralized animal cap explants.

(a) Unilateral injection of dkk2 mRNA (500 pg) or dkk2 plasmid DNA (25 pg) expanded snai2 expression domain laterally. (b) Quantification of the Dkk2 overexpression phenotype. The number of embryos …

https://doi.org/10.7554/eLife.34404.012
Figure 6—source data 1

Quantification of Dkk2 gain-of-function phenotype.

https://doi.org/10.7554/eLife.34404.013
Dkk2 neural crest-inducing activity requires active Wnt/β-catenin signaling.

(a) Unilateral injection of dkk2 plasmid DNA (50 pg) expanded snai2 expression domain laterally (arrowhead). This activity was blocked in the context of β-catenin- (β-catMO; 20 ng), Lrp6- (Lrp6MO; …

https://doi.org/10.7554/eLife.34404.014
Figure 7—source data 1

Quantification of β-catenin, Lrp6 and Wnt8 knockdown phenotypes upon Dkk2 coexpression.

https://doi.org/10.7554/eLife.34404.015
Dkk2 mediates its activity independently of GSK3.

(a) BIO treatment (10 μM) expanded sox10 expression domain anteriorly. Unilateral injection of Dkk2SMO (30 ng) reduced sox10 expression a phenotype that cannot be rescued by BIO treatment. In …

https://doi.org/10.7554/eLife.34404.016
Figure 8—source data 1

Quantification of Dkk2 knockdown and Dkk1 overexpression phenotypes upon BIO treatment.

https://doi.org/10.7554/eLife.34404.017
Figure 9 with 1 supplement
Model for neural crest induction by Dkk2 and Wnt8.

During neural crest induction, Lrp6 mediates two independent signaling events triggered by Wnt8 and Dkk2 converging on β-catenin to promote neural crest formation (snai2 induction). The Lrp6/Wnt/Fzd …

https://doi.org/10.7554/eLife.34404.018
Figure 9—figure supplement 1
Dkk2 function is required for dorsal axis duplication by Wnt8.

(a) Ventral injection of wnt8 mRNA (50 pg) in the equatorial region of 4 cell stage embryos resulted in secondary dorsal axis formation at stage 30. Injection of Dkk2SMO (30 ng) significantly …

https://doi.org/10.7554/eLife.34404.019

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