Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm formation via suppressing TGF-β signaling

Eaf1 and Eaf2 mediate zebrafish dorsal-ventral axis patterning via suppressing Wnt/β-Catenin activity

“…CRISPR/Cas9 genome editing system is reported as an effective tool for gene editing in organisms 26 , 27 . Cas9 system was used to constructed F0 fish for editing eaf1 or eaf2 , and the sequences of gRNA for eaf1 or eaf2 were reported in our current published paper 24 , but only eaf1 mutants were constructed successfully 24 . Expression of ved , vent , or vox was tested in embryos from in-crossed eaf1 F1 heterozygous mutants, and the represented embryos with increased expression or normal expression were genotyped by primers in Table S2 .…”

“…The primers used for amplifying zebrafish ved , vox , vent , bmp2b , wnt8a , and 18s (used as an internal control) were listed in Supplemental Table 2 . Semi-quantitative RT-PCR (qRT-PCR) was performed as described previously 5 , 24 , 25 . Each sample was run in triplicate and repeated at least three times.…”

“…Each sample was run in triplicate and repeated at least three times. Differences were calculated by the △△Ct comparative quantization method using 18s as an internal control, and the data were analyzed with one-way analysis of variance (ANOVA) and post hoc Tukey's test (*** represents P < 0.001; ** represents P < 0.01; * represents P < 0.05) 24 , 28 , 29 .…”

“…For detecting the interaction abilities between Tcf and β-catenin1/2, cells were transfected with the indicated combination of plasmids and then incubated with conditional media. Western blot analysis was performed as described previously 5 , 24 using the following antibodies: anti-Myc antibody (Santa Cruz) with a 1:2000 dilution, anti-HA antibody (Covance) with a 1:5000 dilution, anti-Flag antibody (Santa Cruz) with a 1:2000 dilution, anti-β-actin antibody (Abcam) with a 1:2000 dilution. The blots were photographed using a FujiFilm LAS4000 mini luminescent image analyzer.…”

“…Eaf1 and Eaf2 are originally identified as partners of ELL (Eleven-nineteen lysine-rich leukemia), a fusion protein frequently associated with myeloid leukemia, and the two genes are found to suppress prostate cancer and leukemia 19 - 21 . In our previous studies, during zebrafish embryogenesis, both eaf1 and eaf2 have been revealed to mediate effective convergence and extension movements 22 , form a negative feedback loop with wnt4 23 , and function as novel factors antagonizing canonical Wnt/β-catenin signaling in forebrain induction 5 and as novel factors suppressing TGF-β signaling in germ layers specification 24 . We have also found dorsal-ventral axis patterning defects in eaf morphants 5 , but how maternal and zygote eaf1/2 act in patterning ventral cells is still unclear.…”