Function of RNF12 in Zebrafish Embryos(A and B) Dose-dependent effects of overexpression of zRnf12. The phenotypes caused by zRnf12 overexpression included the following: type I, phenotype similar to wild-type embryo; type II, phenotype with a wider notochord and blood cell deposits; type III, phenotype with much more blood cells deposited. The red bracket represents the width of the notochord, and the white arrowhead indicates the deposited blood cells. The statistical values indicate the ratios of phenotypes in different groups of injected embryos. The underlined numbers indicate the total numbers of observed embryos. All embryos were observed at 25–26 hr postfertilization (hpf). (B) Dose-dependent induction of blood island caused by zRnf12 overexpression.(C) The expression patterns of no tail (ntl) and goosecoid (gsc) under conditions that caused zRnf12 knockdown (r12 MO) or/and squint (sqt) overexpression. All embryos are shown in the shield stage (middle gastrulation). All the embryo injections were carefully controlled with the same amount of control MO and control GFP mRNA.(D) (Da) The most common phenotype in zebrafish embryos injected with smad7 mRNA (200 pg); this phenotype was called “Sphere.” (Db) The phenotype caused by injecting zRnf12 mRNA. (Dc) Major phenotype in zebrafish embryos coinjected with smad7 and zRnf12 mRNAs; this phenotype was called "Little Tail." (Dd) The statistical ratios of phenotypes in embryos injected with different mRNAs and their combinations. All embryos were observed at 31 hpf with the head oriented to the left. The number of embryos showing the phenotype was indicated.(E) The shh expression pattern of the same types of embryos shown in (Da)–(Dc); (upper panel) the lateral view with the head oriented to the left; (lower panel) the dorsal view with the head oriented to the left. All embryos were fixed at 24 hpf. The number of embryos showing the phenotype is indicated.(F) Relative shh expression levels of the same types of embryos shown in (Da)–(Dc) as detected by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments.(G) Relative ntl expression levels of the same types of embryos shown in (Da)–(Dc) as detected by qRT-PCR. Values and error bars represent the mean ±SD of triplicates and are representative of at least two independent experiments.(H and I) Western blotting for Smad7-GFP fusion protein levels affected by zRnf12 MO or ectopic expression of zrnf12.(J) Western blotting for endogenous Smad1/5/8 and Smad2 C-terminal phosphorylation levels affected by zrnf12 MO or zRnf12mRNA.(K) The knockdown of zebrafish smad7 can rescue rnf12 morphants phenotype. The statistical bars are listed on the right. All phenotypes were monitored at 22–24 hpf. Arrow indicated bigger head and anterior phenotype caused by rnf12 MO; Fused-So indicated fused-somite defect caused by smad7 MO. The injection dose for every embryo was 4 ng of the MO mixture (2 ng of MO1 and 2 ng MO2 pooled).

Zebrafish Rnf12 expression pattern and controls labeled with morpholino-modified antisense oligonucleotides (MO)
(A) Expression profile of zRnf12. The embryonic stages were as follows: 8 cells, 30%
epiboly, 70% epiboly and 24 hours postfertilization (hpf). (B) zRnf12 expression in different embryonic stages was assayed by semi-quantitative PCR. Actin was included as a loading control. Different amount of zRnf12 plasmid template was set as positive controls. Template-free DEPC (diethylpyrocarbonate) water was set as a negative control; Bud-2S, Bud stage to 2 somite stage. Expression analysis of actin was included for loading control.
(C) Rescue by zrnf12 mRNA of rnf12 MO-induced effects on zebrafish embryo morphology and sonic hedgehog (shh) expression. The in vivo phenotype and shh marker indicated that zRnf12 mRNA injections could rescue embryos that were silenced for zRnf12 (zRnf12 morphants). The injection dose for every embryo was 4 ng of the MO mixture (2ng of MO1 and 2 ng MO2 that were pooled) and 300 pg of zRnf12 mRNA. hpf, hours postfertilization. The number of embryos showing the phenotype are indicated.
(D) Analysis of the rnf12 MO-induced downregulation of RNF12 expression. Endogenous zebrafish rnf12 was detected by Western Blot analysis. The last lane represents the positive control embryos that were injected with zRnf12 mRNA at the single-cell stage. Actin was included as a loading control. The injection dose is indicated.
(E) RNF12/Smad7 interaction is evolutionally conserved. HEK293T cells were transfected with Myc-tagged human RNF12 (h) or zebrafish RNF12 (zf) and Flag-tagged Smad7. Cells were treated with MG132 for 4 h before harvesting.
(F) Zebrafish smad7 MO induced a downregulation of Smad7-RFP fusion protein expression. 0.5 ng smad7 MO or/and 100 pg of RFP-Smad7 DNA were co-injected. RFP signal was detected by immufluorescence.
(G) Injection of zebrafish smad7 MO in zebrafish embryos upregulated the expression of phospho-Smad2 and phospho-Smad1/5/8 expression. No effects of zrnf12 MO on total Smad1/5/8 and Smad2 levels were observed. All of embryos were digested at 9 hpf and incubated with phspho-antibodies overnigh. Before digestion, all of yolks were removed.
(H) Analysis of specificity of zebrafish smad7 MO by injecting embryos with smad7 mRNA, or smad7 MO or both and scoring morphology as normal or defective. 0.5 ng smad7 MO or/and 150 pg smad7 mRNA were injected. 16
(I) Flag-Smad6 interacts with Myc-RNF12 and mutant derivatives. HEK293T cells transfected with the indicated plasmids were harvested for immunoprecipitation and Western blot analysis. Cells were treated with MG132 for 5 h before harvest.