Walentek P et al. (2013), Wnt11b is involved in cilia-mediated symmetry b...

XB-ART-47881

PLoS One 2013 Jan 01;89:e73646. doi: 10.1371/journal.pone.0073646.

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Wnt11b is involved in cilia-mediated symmetry breakage during Xenopus left-right development.

Walentek P , Schneider I , Schweickert A , Blum M .

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Breakage of bilateral symmetry in amphibian embryos depends on the development of a ciliated epithelium at the gastrocoel roof during early neurulation. Motile cilia at the gastrocoel roof plate (GRP) give rise to leftward flow of extracellular fluids. Flow is required for asymmetric gene expression and organ morphogenesis. Wnt signaling has previously been involved in two steps, Wnt/ß-catenin mediated induction of Foxj1, a regulator of motile cilia, and Wnt/planar cell polarity (PCP) dependent cilia polarization to the posterior pole of cells. We have studied Wnt11b in the context of laterality determination, as this ligand was reported to activate canonical and non-canonical Wnt signaling. Wnt11b was found to be expressed in the so-called superficial mesoderm (SM), from which the GRP derives. Surprisingly, Foxj1 was only marginally affected in loss-of-function experiments, indicating that another ligand acts in this early step of laterality specification. Wnt11b was required, however, for polarization of GRP cilia and GRP morphogenesis, in line with the known function of Wnt/PCP in cilia-driven leftward flow. In addition Xnr1 and Coco expression in the lateral-most GRP cells, which sense flow and generate the first asymmetric signal, was attenuated in morphants, involving Wnt signaling in yet another process related to symmetry breakage in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: actl6a dand5 foxj1 foxj1.2 fzd8 nodal1 nodal3.1 pitx2 wnt11b
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Phenotypes: Xla Wt + fzd8 MO (Fig. S1 F, G) [+]

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	Figure 1: Wnt11b expression in the superficial mesoderm is required for asymmetric Pitx2c expression in the lateral plate mesoderm. (A–D) Wnt11b mRNA expression as determined by whole-mount in situ hybridization of staged embryos at stage (st.) 9.5 (A), st. 10.5 (B), st. 11.5 (C) and st. 13 (D). Specimens are shown in vegetal (A–D) or dorsal (D′) view. Please note that Wnt11b mRNA was present in the superficial mesoderm (sm; white arrowheads) on the dorsal side, as shown in bisected embryos in (A′) and (B′), while expression on the ventral side extended into deeper tissue (B′). Note also expression in the circumblastular collar (cbc) of involuting cells, as demonstrated in a histological section (D′). (E, F) Altered Pitx2c expression in Wnt11b-manipulated embryos. (E) Representative specimens. (F) Quantification of results. Green arrowheads, wild-type expression; red arrowheads, ectopic or absent expression. Dashed lines in (A, B, D′) indicate planes of section. *** Very highly significant (p<0.001). a = anterior, an = animal, d = dorsal, l = left, n = number, p = posterior, r = right, uninj. = uninjected, v = ventral, veg = vegetal. doi:10.1371/journal.pone.0073646.g001
	Fig. S1. Foxj1 expression requires Wnt signaling through Fz8, but is largely independent of Wnt11b. (A, A′) Foxj1 expression in the superficial mesoderm at stage (st.) 10.5. in whole mount (A) and bisected specimens (A′). (B, C) Marginal effects on Foxj1 mRNA expression levels and localization in Wnt11b morphants (quantification in C). (D, E) Wildtype expression of Xnr3 (D) and Not (E) in Wnt11b morphant embryos. (F, G) Foxj1 expression requires Fz8. (F) Summary of results. (G) Altered Foxj1 expression in Fz8 morphants is partially rescued by co-injection of β-catenin (β-cat). Green arrowhead, wild-type expression; red arrowhead, reduced expression; gray arrowhead, absent expression. Dashed line in (A) indicates plane of bisection. Highly significant (p<0.01), * Very highly significant (p<0.001). a = anterior, an = animal, d = dorsal, l = left, n = number, p = posterior, r = right, v = ventral, veg = vegetal.
	Figure 3. Wnt11b is required for cilia polarization and GRP morphogenesis.Embryos were injected at the 4-cell stage into the prospective dorsal marginal zone and dorsal explants were prepared at stage 17. Specimens were processed for immunohistochemistry (IHC) to assess cilia polarization, ciliation rate and cell surface area. (A–D) Presence and polarization of cilia, as shown by acetylated tubulin IHC to stain cilia (red) and phalloidin to stain actin (green) in order to outline cell boundaries. (A) Control uninjected (uninj.) specimen. (B) Wnt11b morphant. (C) Specimen injected with dominant-negative Wnt11b DNA (dnWnt11b). (D) Specimen injected with wild-type Wnt11b DNA. (A′–D′) Evaluation of ciliation and polarization. Green = posterior localization of cilia, yellow = other localization, red = cells without cilia. (E, F) Evaluation of results. (E) Cilia polarization. (F) Apical cell surface area. ***Very highly significant (p<0.001). a = anterior, l = left, p = posterior, r = right.
	Figure 4. Altered Xnr1 and Coco expression in Wnt11b manipulated embryos.Embryos were injected at the 4-cell stage into the DMZ and analyzed for Xnr1 (A–E) or Coco expression (F–J) by whole mount in situ hybridization. Wildtype expression patterns (A, F) were reduced (B, G) or absent/strongly reduced (C, H) in Wnt11b morphants and in embryos injected with a dnWnt11b DNA construct, while signal intensities were unaltered upon ectopic wildtype Wnt11b expression from a DNA construct (D, I). (E, J) Quantification of results. Note that co-injection of wild-type Wnt11b DNA was sufficient to partially rescue Xnr1 expression at the GRP of Wnt11b morphants. ***Very highly significant (p<0.001). a = anterior, l = left, p = posterior, r = right.
	Figure 2. Wnt11b is required for leftward flow at the GRP.Flow analysis in wildtype and manipulated specimens. (A–C) Frequency distribution of trajectory angles in representative explants of uninjected control embryos (uninj., A), Wnt11bMO (B) and Wnt11b DNA (C) injected specimens. Dashed circles indicate maximum frequency (in %), n represents the number of tracked particles above threshold. a = anterior, l = left, p = posterior, r = right, v = average velocity of particles, ρ = quality of flow. (D, E) Compiled results of all embryos analyzed for flow directionality (D) and velocity of fluorescent beads added to GRP explants at stage 17 (E). Note that both parameters were significantly reduced in Wnt11b morphants or Wnt11b DNA injected embryos. In (D, E), n represents number of analyzed explants. * Significant (p<0.05), ***Very highly significant (p<0.001), n = number of analyzed explants.
	wnt11b (wingless-type MMTV integration site family, member 11B) gene expression in Xenopus laevis embryo NF stage 13, in (D) vegetal/blastoporal view (D') dorsal view and (D") sectioned through blastoporel, assayed via in situ hybridization, showing expression in mesoderm of the circumblastoporal collar (cbc). KEY: GRP,gastrocoel roof plate.
	Still from Movie S1 Flow defects in GRP explants from Wnt11b manipulated embryos. Movie shows time-lapse sequences of dorsal explants to which fluorescent beads were added (cf. Figure 2A-C). Specimens were mounted dorsal side down and viewed from the ventral side, anterior to the top. Movie represents a total length of 500 frames taken at a rate of 2 frames/sec and runs at 40×real time. Opening frame displays bright field images and indicates orientation of GRP (dashed lines). Videos were processed to yield gradient time trails (GTTs), i.e. color-coded tracks of beads which revealed direction of transport and velocity of particles (from green to red; 25 s). Note that robust leftward flow (uninjected controls) was impaired in Wnt11b morphant and upon injection of wild-type Wnt11b DNA.

References [+] :

Adams, Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. 2006, Pubmed, Xenbase