Rabadán MA et al. (2016), Delamination of neural crest cells requires tra...

XB-ART-52045

Development 2016 Jun 15;14312:2194-205. doi: 10.1242/dev.134981.

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Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2.

Rabadán MA , Herrera A , Fanlo L , Usieto S , Carmona-Fontaine C , Barriga EH , Mayor R , Pons S , Martí E .

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Delamination of neural crest (NC) cells is a bona fide physiological model of epithelial-to-mesenchymal transition (EMT), a process that is influenced by Wnt/β-catenin signalling. Using two in vivo models, we show that Wnt/β-catenin signalling is transiently inhibited at the time of NC delamination. In attempting to define the mechanism underlying this inhibition, we found that the scaffold proteins Dact1 and Dact2, which are expressed in pre-migratory NC cells, are required for NC delamination in Xenopus and chick embryos, whereas they do not affect the motile properties of migratory NC cells. Dact1/2 inhibit Wnt/β-catenin signalling upstream of the transcriptional activity of T cell factor (TCF), which is required for EMT to proceed. Dact1/2 regulate the subcellular distribution of β-catenin, preventing β-catenin from acting as a transcriptional co-activator to TCF, yet without affecting its stability. Together, these data identify a novel yet important regulatory element that inhibits β-catenin signalling, which then affects NC delamination.

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Species referenced: Xenopus laevis
Genes referenced: ctnnb1 dact1 itk nts sox2 twist1 wnt8a
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	Fig. 2. Inhibition of the Wnt canonical pathway is required for NC delamination. (A) Scheme showing the components of the canonical Wnt pathway. (B) Scheme representing the TOP-Flash electroporation of chick embryos at HH10 for in vivo luciferase assays. Quantification of Luc/Renilla activity 24 hpe with the indicated DNAs. Lrp6DN inhibits and β-catenin-CA activates the TOP-Flash reporter (mean±s.e.m., n=6-10 embryos/condition; *P<0.001, one-way ANOVA). (C) Scheme showing the location of the transfected cells in chick embryos electroporated at HH10 (reporter GFP expression). (D-F) Selected images of embryos electroporated with DNA at HH10 in which GFP-expressing cell migration was analysed 24 hpe in whole-mount preparations. Lateral view shows the NC streams over 3 somites of the embryos. (D′, E′,F′) Selected transverse sections of electroporated NTs showing the migratory NC cells (green) in the three conditions costained with DAPI (blue). (G) Quantification of the EGFP-expressing cells (mean total±s.e.m. after electroporation, n=10-20 sections of ≥4 embryos/condition; *P<0.001, one-way ANOVA). (H,I) Xenopus laevis embryos injected with RNA encoding Wnt8 (Wnt-ON) or dnTcf3 (Wnt-OFF) were fixed at stage 22. In situ hybridisation for Xenopus twist1 shows that the cephalic NC streams have migrated less on the Wnt-ON injected side of the embryos whereas they have progressed further in the Wnt-OFF injected side of the embryos (n≥10/15 embryos/condition). (J) Schematic representation of pre-migratory NC cells in which activation of the Wnt pathway prevents delamination from the dorsal NT.
	Fig. 4. Dact1/2 activity regulates NC delamination. (A) Scheme of chick embryos electroporated at HH10 in which the location of the transfected cells was analysed (reporter GFP expression). (B) Quantification of the ratio (±s.e.m.) of EGFP-expressing cells in control, shDact2 (D2-LOF) and Dact2 (D2-GOF) electroporated embryos (n=10-20 sections from ≥4 embryos/condition; P<0.01, *P<0.001, one-way ANOVA). (C-E) Selected images of embryos electroporated at HH10 with the DNAs indicated in which the migration of the GFP-expressing cells was analysed 24 hpe in a whole-mount preparation. Lateral view shows NC streams over 3 somites of the embryos. (C′,D′,E′) Selected transverse sections of electroporated NTs showing migratory NC cells (green) in the three conditions. DAPI (blue) shows nuclear staining. (F) Scheme representing Xenopus embryos injected at the eight cell stage and allowed to develop until the initiation of cephalic NC migration. (G-K) Xenopus laevis embryos injected with control, dact1 MO (LOF) or dact1 RNA (GOF) were fixed at stage 22. In situ hybridisation for Xenopus twist1 highlights the migratory cephalic NC streams that are shorter in (75%) dact1 LOF embryos (brackets in I,I′) and longer in (60%) dact1 GOF embryos (brackets in K,K′). (H,J) Alcian Blue staining performed at a later stage (St46) showing that cartilage derivatives are smaller. (L) Scheme summarising the effects on the NC migratory streams (n≥10/15 embryos/condition).
	Fig. 5. DACT activity is dispensable for NC cell motility. (A) Scheme representing the explant culture and time-lapse assay for chick embryo NTs electroporated at HH10 in which the cell distance from the source (explanted NT) was analysed at 25, 50, 75 and 100 μm. (B) Selected images showing Dact2 GOFexplants at the beginning (t0) and end (t256) of the culture. (C) The plot shows the percentages of cells that cross defined points at 25 μm from the NT. (D) NC cells overexpressing Dact2 migrate slightly faster. (E) The persistence of both cell types was identical [mean±s.e.m., n≥100 cells of ≥3 explants/condition; *P<0.05, unpaired t-test (two-sided); see also Movie 1]. (F) Scheme representing the explant culture and time-lapse assay for Xenopus laevis embryos injected at the eight cell stage. (G-I) Selected images of control, dact1 MO (LOF) or dact1 mRNA expressing (GOF) explants at the beginning (t0) and end (t370) of the culture. (G′,H′,I′) Delaunay triangulations of NC explants show the more restricted dispersion in dact1 LOF compared with the control MO explants, reflected by the reduced area between the neighbours. (J,K) The speed and persistence of both cell types is identical. (L) Dispersion is impaired in Dact1 LOF explants (mean±s.e.m.; n≥100 cells from ≥3 explants/condition; see also Movie 2).
	Fig. S3 (related to Figure 4). Loss of DACT1 function in Xenopus embryos in vivo. (A) The DACT1–MO used was as published previously. We used the same morpholinos and reproduced the controls of DACT1-MO by injecting Xenopus laevis embryos with control-MO or DACT1-MO (two concentrations). The embryos were fixed at stage 19 and Sox2 expression was examined by in situ hybridization to show the effects on the NT. (B-C) Specificity of the DACT1-MO was tested by the rescued of NC migration in embryos co-injected with DACT1 (resistant to MO). Xenopus laevis were injected at the 8 cell stage and allowed to develop until the initiation of cephalic NC migration. In situ hybridization for XTwist highlights the reduced migratory cephalic NC streams after DACT1-MO injection and the rescue of NC migration after co-injection with DACT1 mRNA. (C) Quantification showing the percentage NC cell migration in embryos of each experimental condition (n ≥ 10/15 embryos/condition).
	Fig. S6 (related to Figure 7). The -catenin sequence contains three putative SUMO acceptor residues. (A) -catenin contains three putative SUMOylation sites. Schematic representation of the b-catenin protein in which the arrowheads indicate the position of the three putative SUMO acceptor lysines: K19, K312 and K666. (B) Multiple sequence alignment of full length -catenin using Multalin version 5.4.1. Grey squares highlight the conserved lysines (K) located in inverted SUMOylation consensus motifs E/DxKψ. (C) Forced SUMOylation of -catenin was insufficient for the nuclear translocation of -catenin (selected images of HEK-293 cells transfected with the DNAs indicated). In the presence of SUMO-1 (green), -catenin (red) was localized throughout the cell (cytoplasm + nucleus). (D) Forced SUMOylation of b-catenin was insufficient to inhibit the canonical Wnt/-catenin activity. Quantification of Luc/Renilla activity 24 h after transfection of the DNAs indicated into HEK cells. The activation the Top-Flash reporter by -catenin was resistant to the SUMOylation induced by co-transfection of SUMO-1, while -catenin activation of the Top-Flash reporter was inhibited by co-transfection of DACT2. DACT2 mediated inhibition is resistant to SUMO-1 activity. (E) Quantification of Luc/Renilla activity 24 hpe of HH10 chick embryos with the indicated DNAs. As occurred in HEK cells, -catenin activation of the Top-Flash reporter was resistant to SUMOylation induced by the co-transfection of SUMO-1. The activation of the Top-Flash reporter by -catenin was inhibited by co-transfection of DACT2. This DACT2 mediated inhibition is resistant to SUMO-1 activity (mean ±s.e.m., n = 6-10 embryos/condition: ***p<0.001).

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