Custos controls β-catenin to regulate head development during vertebrate embryogenesis

Abstract

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of β-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited β-catenin/TCF-dependent transcription. Custos binds to β-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of β-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-β-catenin signal transduction cascade.

Fig. 1.

Custos is required for anterior development. (A) Schematic representation of Custos and its domains. Arrowheads indicate the two clusters of dual CKI phosphorylation sites. (B) Custos-MO blocks translation of tagged-Custos protein and depletes endogenous Xenopus Custos protein levels. Myc-tagged Custos RNA containing the MO binding site was coinjected with control MO or Custos-MO, and tagged-protein was visualized by SDS/PAGE and Western blotting (Left). Endogenous Custos protein was assessed using anti-Custos antibody (Right). (C) Depletion and overexpression of Custos induce anterior head defects. The indicated RNA or MO was injected into the two dorsal blastomeres of 4-cell embryos. (D) Suppression of head development was scored by the dorso-anterior index (DAI). The numbers above each bar indicate the number of injected embryos.

Fig. 2.

Dysregulation of Custos inhibits the canonical Wnt pathway. (A and B) Overexpression or depletion of Custos reduced organizer gene expression (A) and anterior markers (B). Custos RNA (1.5 ng) or Custos-MO (50 ng) was injected with LacZ (250 pg) RNA into one dorsal cell of four-cell embryos. At stage 10.5, the expression level of organizer genes Xnr3 and Goosecoid (Gsc), or at stage 26, the expression of anterior markers Rx1, N-CAM and Wnt1, was examined by in situ hybridization. Red staining indicates the injected side. The number of embryos scored is shown on each panel. (C) Custos regulates nuclear accumulation of β-catenin at stage 10.5. Custos-MO (50 ng) or myc-Custos RNA (1 ng) was injected into one dorsal blastomere of four-cell embryos. mCherry RNA (250pg) was coinjected and red fluorescence was used as tracer for the injected side. Embryos were fixed at stage 10.5, and immunohistochemistry was performed with anti–β-catenin and anti-myc antibodies. Arrowheads indicate cells with nuclear accumulation of β-catenin.

Fig. 3.

Custos inhibits Wnt signal transduction. (A) Topflash reporters were cotransfected into HEK-293T cells with expression plasmids for Wnt1, Dvl or β-catenin and Custos, as indicated. (B) Topflash reporter was coinjected with Wnt8, Dsh, or β-catenin and Custos RNAs into all cells of two-cell embryos. Cell or embryo lysates (stage 10.5) were subjected to luciferase assay and normalized to reporter-only samples.

Fig. 4.

Custos binds to β-catenin and regulates its nuclear translocation. (A) HA-Custos was transfected into HEK-293T cells, the cells were treated with 250 ng/mL recombinant mouse Wnt3a, and lysed at different time points. HA-Custos was immunoprecipitated from cell lysates, and coimmunoprecipitated β-catenin was detected by SDS/PAGE and Western Blotting. The α-ABC antibody detects the active form of β-catenin. (B) Overexpression of Custos and truncated forms of Custos inhibits nuclear accumulation of β-catenin in HeLa cells. Myc-Custos transfected cells were treated with 250 ng/mL recombinant mouse Wnt3a for 3 h and then subjected to immunocytochemistry with the indicated antibodies. DAPI staining was used to show the cell’s nucleus. (C) Quantification of the studies in B; nuclei positive for β-catenin staining (as in the first panel of B) were counted. (D) Colocalization of myc-Custos with laminA/C. HeLa cells were transfected with myc-Custos and then subjected to immunocytochemistry with the indicated antibodies.

Fig. 5.

Custos is required for anterior development in zebrafish. (A and B) 25–100 pg of Myc-tagged Custos RNA was injected into the one-cell embryo and scored at 28 h postfertilization (hpf) for suppression of head development by the V1–V4 index. The numbers above each bar indicate the number of injected embryos. (C and E) Injection of Custos MO1 (C) or Custos MO2 (E) suppressed head and eye development which was rescued by coinjection of Custos Rescue RNA (2–4 pg). MOs (5–15 ng) were injected at the one-cell stage and embryos were scored at 24 hpf. (D and F) Quantitation of the results in C and E, respectively. The numbers above each bar indicate the number of injected embryos.