Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation Peng Li1, Ying Chen2, Kinglun Kingston Mak3,4,5, Chun Kwok Wong1,4, Chi Chiu Wang2,3,4,6, Ping Yuan1,2,3,4* 1 Department of Chemical Pathology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China, 2 Li Ka Shing Institute of Health Sciences, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China, 3 School of Biomedical Sciences, the Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China, 4 CUHK Shenzhen Research Institute, the Chinese University of Hong Kong, Shenzhen, Guangdong, China, 5 Key Laboratories for Regenerative Medicine, Ministry of Education, the Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China, 6 Fetal Medicine Unit, Department of Obstetrics and Gynaecology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China Abstract The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation. Citation: Li P, Chen Y, Mak KK, Wong CK, Wang CC, et al. (2013) Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation. PLoS ONE 8(11): e79867. doi:10.1371/journal.pone.0079867 Editor: Robert Dettman, Northwestern University, United States of America Received May 23, 2013; Accepted September 26, 2013; Published November 5, 2013 Copyright: © 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction pathway promotes apoptosis and inhibits tumorigenesis in mammals [7-10]. The Hippo pathway was first discovered in Drosophila. Mst1 and Mst2 (Mammalian sterile 20-like kinases 1 and 2) Through genetic mosaic screens, core components of the are the core components of the Hippo pathway. They play Hippo pathway, such as Warts (Wts), Hippo (Hpo) and important roles in early embryonic development, cell Salvador (Sav) were identified as tumor-suppressor genes proliferation, apoptosis and organ size control. Mst1 null mice [1-4]. These components restrict cell proliferation and promote are viable and fertile but have a reduced number of mature apoptosis by repressing the downstream effector Yokie (Yki) in naive T cells, while Mst2 null mice are also fertile but exhibit no Drosophila. Depletion of core components of the Hippo developmental or immunological defects [11]. However, depletion of both Mst1 and Mst2 resulted in embryonic lethality pathway or overexpression of Yki results in enhanced cell at embryonic day 8.5, suggesting redundant roles of Mst1 and proliferation and reduced apoptosis respectively [5]. This Mst2 [12]. One functional copy of either Mst1 or Mst2 is pathway is highly conserved in mammals. Serine/threonine necessary and sufficient for early embryonic development kinases Mst1/Mst2 and Lats1/Lats2 in mammals are homologs [11,13]. of Hippo and Wts in Drosophila respectively. Together with an Like other components of the Hippo pathway that promote adaptor protein hMob1, they transmit signals to downstream apoptosis, Mst1/Mst2 are pro-apoptotic kinases [14,15]. Under effectors [6]. Through inhibiting the transcriptional co-activators oxidative stress, Mst1/Mst2 activate transcription factor Foxo and oncoproteins Yap (Yes kinase-associated protein) and Taz and promote neuronal cell death [16,17]. Heart specific (transcriptional coactivator with PDZ-binding motif), the Hippo expression of Mst1 leads to dilated cardiomyopathy with PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e79867 Differentiation Distortion of Mst-/- ES Cells reduction in cell density in heart [18]. Liver specific removal of further address this question, we generated Mst-/- ES cells Mst1/Mst2 in newborn mice results in liver enlargement and from Mst1 and Mst2 mutant mice [12]. We found that the formation of hepatocellular carcinoma and cholangiocarcinoma phosphorylation level of Yap was decreased in Mst-/- ES cells, [12,19-21]. Similarly, in mouse intestines and pancreas, whilst the pluripotency marker Nanog was increased inactivation of Mst1/Mst2 leads to intestinal stem cell significantly compared to wild type ES cells. Mst-/- ES cells overproliferation, colonic tumorigenesis and pancreas also showed differentiation resistance for a relatively longer overgrowth [22-24], suggesting important roles of Mst1/Mst2 in time compared to wild type ES cells under differentiation organ size control and tumorigenesis. conditions. Consistent with the developmental defects of Mst1/ Mst1/Mst2 activate Lats1 and Lats2 by phosphorylation, and Mst2 double knockout mice, Mst-/- ES cells showed lineage in turn phosphorylate Yap and inhibit it from translocating into development distortion during embryoid body (EB) formation the nucleus [25]. Unphosphorylated Yap can be translocated and obvious defects to differentiation to cardiac progenitor into the nucleus to activate TEA-domain (TEAD) family cells. Microarray analysis revealed that the non-canonical Wnt members. The Yap/Taz-Tead complex further activates pathway ligands Wnt2b and Wnt5a, which are critical for proliferation by a genome wide transcriptional program [26-28]. cardiac progenitor cell differentiation, were significantly Ectopic expression of Yap in mammalian cells leads to a downregulated in Mst-/- EBs. Unlike wild type ES cells, Mst-/- phenotype resembling that from ablation of core components of ES cells could not form teratoma after subcutaneously injected the Hippo pathway. Similar to the simultaneous removal of into nude mice. Taken together, our data suggest that Mst1/ Mst1/Mst2, overexpression of Yap in mice results in a dramatic Mst2 are required for teratoma formation. Their functions are increase of liver mass with subsequent tumor formation. In also critical for proper cardiac lineage cell formation. addition previous research reveals that Yap is an important pluripotent factor. Expression of Yap enhances reprogramming Results of differentiated cells to induced pluripotent stem (iPS) cells [26,29,30]. In adults Yap is enriched in organs such as the Derivation of mouse Mst-/- embryonic stem cells small intestine and the developing brain and its expression is Based on the schematics of null alleles of Mst1 and Mst2 highly restricted to the progenitor or stem compartments, generated in a previous study [12], we crossed the Mst1+/- whereas in other tissues, such as skin and skeletal muscle, the Mst2-/- male and female mice, harvested the E3.5 embryos and expression of Yap is gradually decreased with regard to derived ES cells on MEF feeder (Figure S1A and S1B). These differentiation status [10,26,31,32]. Yap is therefore, a cells were further adapted to form feeder-free ES cell lines stemness gene in mammalian cells, while key components of under the 2i+LIF condition for genotyping. Exon 4 and 5 which Hippo pathway such as Mst1/Mst2, function to constrain this encode kinase domain are deleted in Mst1 knockout, while stemness gene in restricted compartments. exon 5 and 6 which encode kinase domain are deleted in Mst2 Mst1/Mst2 double knockout mice die at E8.5 with knockout (Figure S1C). With primers targeted to the adjacent abnormalities in the placenta, vascular patterning and primitive sequence of the deleted regions of Mst1 (exon 4 and 5) and hematopoiesis, suggesting that Mst1/Mst2 are not required for Mst2 (exon 5 and 6), PCR confirmed that respective regions of pluripotent inner cell mass (ICM) formation but are required for Mst1 and Mst2 genomic DNA were deleted in Mst1/Mst2 subsequent organ and tissue development. As an in vitro double knockout (Mst-/-) ES cell lines respectively (Figure 1A). derivative of the pluripotent inner cell mass (ICM), ES cells Two Mst1/Mst2 double knockout lines, Mst-/-1 and Mst-/-2 ES retain the developmental characteristics of ICM and can self- cell lines were selected for further studies (Figure 1A). The renew and differentiate to all three