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β-Arrestins are multifaceted proteins that play critical roles in termination of G protein-coupled receptor (GPCR) signaling by inducing its desensitization and internalization as well as in facilitation of many intracellular signaling pathways. Here, we examine using Xenopus embryos whether β-arrestin 1 might act as a mediator of β-catenin-independent Wnt (non-canonical) signaling. Xenopus β-arrestin 1 (xβarr1) is expressed in the tissues undergoing extensive cell rearrangements in early development. Gain- and loss-of-function analyses of xβarr1 revealed that it regulates convergent extension (CE) movements of mesodermal tissue with no effect on cell fate specification. In addition, rescue experiments showed that xβarr1 controls CE movements downstream of Wnt11/Fz7 signal and via activation of RhoA and JNK. In line with this, xβarr1 associated with key Wnt components including Ryk, Fz, and Dishevelled. Furthermore, we found that xβarr1 could recover CE movements inhibited by xβarr2 knockdown or its endocytosis defective mutant. Overall, these results suggest that β-arrestin 1 and 2 share interchangeable endocytic activity to regulate CE movements downstream of the non-canonical Wnt pathway.
Fig. 1. Expression pattern of xbarr1 in Xenopus early development. (A) Amino acid sequence comparison of Xenopus b-arrestin 1 and its homologues identified in human and
mouse. The most conserved residues are indicated by the shaded background. (B) RT-PCR analysis showing the temporal expression pattern of xbarr1. ODC serves as a loading
control. Stages (St.) are indicated above the lane. (C–L) Whole-mount in situ hybridization assays. (C and D) Lateral view with animal to the top. (E) Dorso-vegetal view with
animal to the top. (F) Anterior view with dorsal to the top. (G and H) Lateral view with anterior to the left. Inset in (J) shows the dorsal view of head region. A dotted circle in
(L) denotes heart region.
Fig. 2. xbarr1 regulates CE movements of dorsal tissue with no effect on cell fates. (A) Gain- and loss-of-function phenotypes of xbarr1. (B) DMZ elongation assays. (C)
Quantitation of convergent extension of DMZ explants shown in (B). (D and E) Whole-mount in situ hybridization analysis. (A–E) Two blastomeres of four-cell stage embryos
were injected in the dorsal equatorial region as indicated with xbarr1 RNA (1 ng), xbarr1 MO (10 ng) and Co MO (10 ng) and then cultured to stage 26 (A), 10.25 for in vitro
elongation assays (B) or 10.5–14 for in situ hybridization (D and E).
Fig. 3. xbarr1 acts downstream of the Wnt/PCP pathway to control CE. (A–K) DMZ elongation assays. (F) and (K) show the quantitation of elongation of DMZ explants in (A–E)
and (G–J), respectively. (L) Immunoblotting (IB) analysis of the effect of xbarr1 on JNK activation. Total JNK serves as a loading control. (A–L) Two blastomeres of four-cell
stage embryos were injected in the dorsal marginal zone with the indicated combination of DN XWnt11 (2 ng), DN XFz7 (2 ng), xbarr1 (1 ng), XJNK (200 pg), XRhoA (200 pg),
xbarr1 MO (10 ng) and Co MO (10 ng) and then DMZ explants were excised at stage 10.25 and cultured to stage 18 for observation of CE (A–K) or stage 12 for western blotting
(L). CTL, uninjected control DMZ explants. (M) HEK 293T cells were transfected with DNA constructs, either alone or in combination as indicated. Cell lysates were
immunoprecipitated with anti-GFP antibody and the immunocomplexes were blotted with anti-myc or anti-GFP antibodies. Arrows denote interacting proteins.
Fig. 4. xbarr1 substitutes for xbarr2 in regulation of CE. (A–E) In vitro elongation assays. (F) Quantitation of convergent extension of DMZ explants shown in (A–E). Four-cell
stage embryos were injected in the dorsal marginal region of two blastomeres as indicated with xbarr2 MO (10 ng), xbarr2 AAEA (1 ng) and xbarr1 (1 ng) and DMZ explants
were dissected at stage 10.25 and cultured to stage 18.
arrb1 (arrestin, beta 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up.
arrb1 (arrestin, beta 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, horizontal view, animal up.
arrb1 (arrestin, beta 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
arrb1 (arrestin, beta 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
arrb1 (arrestin, beta 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 37, lateral view, anteriorleft, dorsal up.
