Smurf ubiquitin ligases regulate development processes in embryonic stem cells

Authors

• JH Shin
• X Xu
• T Chen
• Y Tang
• Y Du
• C Liu
• Y Zhang

Abstract

Smurfs are HECT domain containing E3 ubiquitin ligases that have Smurf1 and 2 in mammals. They are originally found to be negative regulators of TGFb/BMP signaling, and which have been shown to play key roles in many biological processes.
Mouse lacking Smurf1 and Smurf2 are embryonic lethal, and they displayed gastrulation defects. To understand the role of Smurfs in early embryonic development, we established ESC lines using E3.5 blastocytes for WT, SF1KO, SF2KO, and SF1/2KO (DKO) from mouse. We found that Smurf-deletion had no effect on ESC morphology, alkaline phosphatase staining, or pluripotent marker expression as assessed by RT-qPCR or immunofluorescence. Moreover, WT and Smurf-deficient ESCs supported teratoma formation to a similar degree in vivo, confirming that Smurf1 and Smurf2 are not required for maintaining pluripotent potential of ESCs.
However, when these ESCs formed embryoid body (EB), we observed that the size of DKO EBs were considerably smaller than that of WT, SF1KO or SF2KO EBs. H&E staining revealed that the DKO EBs were less differentiated than that of WT EBs. Further RNAseq and RT-qPCR assays revealed that depletion of both Smurf1 and Smurf2 causes the developmental delay and arrests cells at the gastrulation stage. These observations were also confirmed using monolayer neuronal differentiation assay. Besides higher level of Smad-dependent TGFb signaling in DKO EBs or differentiated cells, several other developmental signaling pathways were also affected by Smurf DKO. These results indicating that Smurfs regulate developmental process in embryonic stem cells by affecting multiple signaling pathways, including TGFb signaling.

Scientific Focus Area: Stem Cell Biology

This page was last updated on Monday, September 25, 2023