Fig. 2

Fig. 2

Overexpression of phosphorylation-deficient mutant UHRF1 (UHRF1^S661A) phenocopies UHRF1 overexpressing embryos eliciting dramatic gastrulation defects. (A) Progression of embryonic development in uninjected and embryos injected with 100 pg of UHRF1^S661A mRNA. At 50% epiboly (5.25 hpf), arrows indicate asymmetric epiboly. At Shield stage (6.0 hpf) arrows point to the missing shield and cells that did not undergo proper epiboly cell movements. At bud stage (10.0 hpf) arrows indicate failure to complete epiboly and premature development of tail bud. At 7-8-Somite stage (~12.0 hpf) arrows indicate disrupted anterior-posterior axis, somite formation, and irregular tail formation. At Prim-5 stage (24.0 hpf) arrows indicate defects in head and tail. Scale bar=500 µm. (B) Curve displays the percent of embryos injected with 100 pg of WT-UHRF1 or UHRF1^S661A or uninjected controls that were scored as normal; the stages set by the progression of uninjected control embryos (top) and hpf (bottom). Significance was determined by Log-rank Test (p<0.0001). (C) Distribution of phenotypes of embryos injected with 25, 50 and 100 pg of WT-UHRF1 and UHRF1^S661A mRNA. Penetrance of the asymmetric epiboly phenotype is statistically significant compared to uninjected controls at each indicated concentration by Fisher′s exact test. Asterisks (*) indicate p<0.001. n.s.=not significant. Number of biological replicates (clutches), number of embryos per treatment, and percent of embryos displaying the asymmetric epiboly phenotype are indicated. (D) Western blot using the Myc tag to detect UHRF1 in WT-UHRF1 and UHRF1^S661A overexpressing embryos. Mean levels of Myc-UHRF1 protein per treatment are quantified relative to tubulin and were determined to be equivalent by Student’s t-test (p=0.36). n.s.=not significant.