Abstract
How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2), a homeobox transcription factor of msh family, as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs, while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs, and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore, MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription, while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly, SOX2 can promote the degradation of MSX2 protein, suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together, our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs.
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Acknowledgements
This work was supported by the National Basic Research Program of China (2012CB966403 and 2015CB964902 to JZ), the National Natural Science Foundation of China (31171431 to JZ, 31301206 to YB and 81370600 to DL) and Tianjin Natural Science Foundation (11JCYBJC27300 to JZ). We thank Drs. Tao Cheng, Dangsheng Li and Lihong Shi for their suggestions during manuscript preparation. We thank members of the Zhou lab for their insightful discussion during the course of this work. We thank Drs Xiaoqing Zhang and Baoyang Hu for providing plasmids for this study.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
MSX2 suffices to induce hESC mesendoderm differentiation. (PDF 394 kb)
Supplementary information, Figure S2
hESCs monolayer mesendoderm differentiation model was established successfully. (PDF 281 kb)
Supplementary information, Figure S3
MSX2 is necessary for hESC mesendoderm specification. (PDF 345 kb)
Supplementary information, Figure S4
MSX2 is knockout in hESCs successfully and MSX2 suppresses SOX2 expression by direct binding to its promoter. (PDF 279 kb)
Supplementary information, Figure S5
MSX2 suppression of SOX2 is linked to its mesendoderm-inducing function. (PDF 417 kb)
Supplementary information, Figure S6
SOX2 rescues MSX2 mesendoderm induction by promoting its degradation. (PDF 430 kb)
Supplementary information, Figure S7
Nodal signaling is an essential downstream effector of MSX2. (PDF 320 kb)
Supplementary information, Figure S8
MSX2 induces NODAL expression by directly binding to and activating the NODAL promoter. (PDF 134 kb)
Supplementary information, Data S1
Material & Methods (PDF 203 kb)
Supplementary information, Table S1
The soluble factors used in this paper (PDF 58 kb)
Supplementary information, Table S2
The primers used for amplifying genes (PDF 100 kb)
Supplementary information, Table S3
The primers used for CRISPR sgRNA guide sequences and the genotyping (PDF 53 kb)
Supplementary information, Table S4
Sources and dilutions of the antibodies (PDF 53 kb)
Supplementary information, Table S5
The primers used for real-time PCR (PDF 103 kb)
Supplementary information, Table S6
The primers used for amplifying gene fragment and luciferase reporter plasmids construct (PDF 56 kb)
Supplementary information, Table S7
The primers used for PCR after ChIP (PDF 57 kb)
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Wu, Q., Zhang, L., Su, P. et al. MSX2 mediates entry of human pluripotent stem cells into mesendoderm by simultaneously suppressing SOX2 and activating NODAL signaling. Cell Res 25, 1314–1332 (2015). https://doi.org/10.1038/cr.2015.118
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DOI: https://doi.org/10.1038/cr.2015.118
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