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The RING finger protein Rnf146 encodes an E3 ubiquitin ligase capable of targeting poly-ADP-ribosylated substrates for proteasomal degradation. Rnf146 has been identified as a critical regulator of Axin1 and thus of Wnt/β-catenin signaling. However its physiological significance in vertebrate embryonic development remains to be demonstrated. In this study, we take advantages of early Xenopus embryos to demonstrate that Rnf146 is essential for embryonic pattern formation. Depletion of zygotic Rnf146 using a translation blocking morpholino oligo (MO) results in anteriorized development and increased expression the anterior marker gene Otx2, consistent the notion that Rnf146 is a positive regulator of Wnt/β-catenin signaling through negatively regulating Axin1 expression. This notion is further supported by examination of the role of maternal Rnf146 in the context of Spemann organizer formation and dorsal axis development. Depletion of maternal Rnf146 using an antisense oligodeoxynucleic acid (ODN) leads to ventralized development and diminished expression of organizer genes. Together, we have provided evidence for the first time that Rnf146 is a critical regulator of embryonic pattern formation in vertebrates.
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28807725
???displayArticle.link???Mech Dev
Fig. 1.
Expression of Rnf146 during early Xenopus development. (A) Peptidyl sequence alignment of Rnf146 from humans and Xenopus. (B) A schematic comparison of Rnf146 from humans and Xenopus. (C) RT-PCR examination of Rnf146 expression in early Xenopus development. odc serves as loading controls. (D–G) Embryos at different stages in situ hybridized with rnf146. ani.: animal pole view. dor.: dorsal side view. lat.: lateral view. bis.: bisection view.
Fig. 2.
MO-mediated depletion of Rnf146 and its effects on Wnt signaling. (A) Western blotting assessment of the blocking effect of Rnf146 MO on translation of an rnf146-myc mRNA. β-Actin serves as a loading control. (B) Western blotting detection of Axin1 and β-catenin in cMO and/or Rnf146 MO injected embryos. 40–80 ng Rnf146 MO was injected into both cells at the 2-cell stage. 80 ng cMO was injected as a control for non-specific effects. Injected embryos were then collected at the stage 12 and subjected to western blotting analysis using antibodies against Axin1 or β-catenin. β-Actin serves as loading controls. (C) Results from TOPFlash dual-luciferase assay in cMO, Rnf146 MO-injected embryos and embryos injected with mRNA encoding Rnf146 (2 ng). cMO or Rnf146 MO in indicated doses together with 20 pg TOPFlash reporter DNA and 20 pg renilla luciferase DNA was injected into both cells at the 2-cell stage. Injected embryos were then harvested at stage 12 and subjected to Dual-Luciferase assay.
Fig. 3.
Anteriorized development from zygotic Rnf146 depletion. (A) Embryos at stage 24/25 showing that Rnf146 MO injection causes defects in axis formation and body length shortening. (B) Western blotting detection of myc-tagged Rnf146 translated from the Rnf146 MO-resistant mRNA in the presence of Rnf146 MO, indicating that MO-resistant rnf146-myc mRNA used in rescue experiment shown in (A) could partially restore the expression of Rnf146 protein in Rnf146 morphant embryos. (C) Measurement of the relative body length of cMO-, Rnf146 MO-, and Rnf146 MO + mRNA (‘rescue’) injected embryos at stage 24/25. Results were summed from three independent experiments with numbers of embryos from each group indicated on the top of each histogram. *P < 0.05 by Student t-test. (D) Embryos at stage 18 in situ hybridized with otx2 (anterior view). For experiments shown in (A, C, and D): 60 ng cMO or Rnf146 MO was injected into both cells at the 2-cell stage. To rescue Rnf146 depletion, Rnf146 MO-resistant mRNA (200 pg), Rnf146-δRing mRNA (200 pg), or β-catenin mRNA (200 pg) was subsequently injected into both cells at the 2-cell stage after MO-injection. (E) Embryos in situ hybridized with egr2/hoxb9 (dorsal view). 60 ng cMO or Rnf146 MO was injected into both cells at the 2-cell stage; embryos were processed for WISH at stage 24. (F) Embryos in situ hybridized with egr2/hoxb9 (dorsal view). 30 ng Rnf146 MO together with the RLDx tracer was injected into one cell at the 2-cell stage and then fixed at the early tailbud stage for WISH. Red fluorescent photographs shown as insets indicate the side received Rnf146 MO injection. Black arrows indicate the anterior limit of hoxb9 expression domains in the control side; red arrows the anterior limit of hoxb9 expression domains in the MO-injected side. Note that embryos represented in (F) were cultured with vitelline membrane removed at the late gastrula stage (st13), resulting in these embryos a more extended appearance along AP axis than those embryos shown in (E), even though embryos were fixed at stage 24/25 in both occasions.
Fig. 4.
Ventralized development from depletion of maternal Rnf146. (A) RT-qPCR results showing that a specific antisense ODN (AS3m) depleted the maternal store of rnf146 mRNA in oocytes in a dose dependent manner, but had limited effects on the expression of rnf146 mRNA in early gastrula stage (stage 10) embryos. (B) Representative embryos at stage 22/23 from a typical host experiment where 6–10 ng Rnf146 AS3m were injected. DAI: dorsal anterior index. Normal embryos are scored as DAI 5; the fully ventralized embryos were scored as DAI 0; partially ventralized embryos were scored DAI ranging from > 0 lesser than 5. (B′) Histographic presentation of DAI scores from maternal Rnf146 depletion experiments representatively shown in (B). ***P < 0.001 by Student t-test. Embryos from three repeated experiments were pooled together for DAI scoring per the scales depicted in (B). Numbers of embryos for each treatment group are shown on the top of each histogram. (C) RT-qPCR examination of organizer gene expression in control and 6–10 ng AS3m injected embryos at stage 10. Data shown as mean + SD. Error bars were obtained through the statistical treatment of technical replicates. (D, D′) Assessment of specificity of maternal Rnf146 depletion using the antisense ODN AS3m. (D) Representative embryos at the early tadpole stage with indicated treatments. 200 pg mRNA encoding Rnf146 was injected into AS3m (8 ng)-injected oocytes before progesterone treatment. 200 pg mRNA b-catenin was injected into subequatorial zone of maternal Rnf146-depleted embryos at the 2-cell stage. (D′) Histographic presentation of DAI scores from maternal Rnf146 depletion experiments representatively shown in (D). ***P < 0.001 by Student t-test.
Fig. 5.
Functional interaction between maternal Rnf146 and Axin1. (A) Western blot detection of Axin1 and β-catenin. Embryos fertilized from oocytes injected with 6–10 ng AS3m were collected at the mid-blastula stage (st8) and subjected to western blotting analysis. (B) qPCR analysis of expression of siamois and nodal3.1 in early gastrula stage embryos depleted of maternal Rnf146, Axin1 or both Rnf146 and Axin1. (C) Schematic summary of the function of Rnf146 in regulating Wnt signaling via controlling Axin levels. Panel a, the expression level of cytosolic β-catenin is balanced by normal expression levels of Axin, which is regulated by Rnf146 and the TNKS poly-ADP-ribosylase. Panel b, depletion of Rnf146 results in more Axin to be stabilized, leading to reduced expression of β-catenin.
rnf146 (ring finger protein 146) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4, animal view.
rnf146 (ring finger protein 146) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
