Xenbase Image ID: 159184

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. Image published in: Zhu X et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue otx2.L otx-2, otx2-a, otx2-b, otxA, Xotx-2, Xotx2 X. laevis Sometime during NF stage 24 to NF stage 25 anterior hoxb9.L hox 2.5, xhoxb9, XlHbox-6, XlHbox6 X. laevis Sometime during NF stage 24 to NF stage 25 posterior posterior neural tube egr2.L EGR-2, Krox-20, krox20, XKr20, XKrox-20 X. laevis Sometime during NF stage 24 to NF stage 25 rhombomere R3 rhombomere R5 cranial neural crest Image source: Published Larger Image Printer Friendly View

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