Quantitative proteomics identify DAB2 as a cardiac developmental regulator that inhibits WNT/β-catenin signaling Peter Hofsteena,b,c,1, Aaron M. Robitaillec,d,1, Daniel Patrick Chapmana,b, Randall T. Moonc,d,e,2, and Charles E. Murrya,b,c,f,g,2 aDepartment of Pathology, University of Washington, Seattle, WA 98109; bCenter for Cardiovascular Biology, University of Washington, Seattle, WA 98109; cInstitute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109; dDepartment of Pharmacology, University of Washington, Seattle, WA 98109; eHoward Hughes Medical Institute, Chevy Chase, MD 20815; fDepartment of Bioengineering, University of Washington, Seattle, WA 98109; and gDivision of Cardiology, Department of Medicine, University of Washington, Seattle, WA 98109 Contributed by Randall T. Moon, December 11, 2015 (sent for review September 19, 2015; reviewed by Loren J. Field and Sean P. Palecek) To reveal the molecular mechanisms involved in cardiac lineage endocardial-like endothelial cells to differentiate toward the cardiac determination and differentiation, we quantified the proteome of lineage (3). Thus, understanding which proteins are involved in pro- human embryonic stem cells (hESCs), cardiac progenitor cells (CPCs), moting or repressing the WNT/β-catenin signaling pathway is crucial and cardiomyocytes during a time course of directed differentiation for resolving the ambiguities associated with cardiac development. by label-free quantitative proteomics. This approach correctly iden- Here, using label-free quantitation (LFQ) proteomics, we tified known stage-specific markers of cardiomyocyte differentiation, measured protein expression patterns during a time course of including SRY-box2 (SOX2), GATA binding protein 4 (GATA4), and hESC-derived cardiomyocyte differentiation. LFQ proteomics is myosin heavy chain 6 (MYH6). This led us to determine whether a robust technology for quantifying differences in protein ex- our proteomic screen could reveal previously unidentified mediators pression, which have been shown to correlate with differentiated of heart development. We identified Disabled 2 (DAB2) as one of the cell types (9, 10). Furthermore, quantifying unmodified protein most dynamically expressed proteins in hESCs, CPCs, and cardiomyo- expression has the advantage of eliminating the unknown effects cytes. We used clustered regularly interspaced short palindromic of posttranscriptional regulation. Using this approach, we identified repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis in regulators of cardiac development, including Disabled 2 (DAB2). zebrafishtoassesswhetherDAB2plays a functional role during car- β diomyocyte differentiation. We found that deletion of Dab2 in zebra- We found that DAB2 negatively regulates WNT/ -catenin sig- fish embryos led to a significant reduction in cardiomyocyte number naling and promotes cardiomyocyte differentiation from mesoderm- and increased endogenous WNT/β-catenin signaling. Furthermore, derived progenitors. the Dab2-deficient defects in cardiomyocyte number could be sup- Results pressed by overexpression of dickkopf 1 (DKK1), an inhibitor of WNT/β-catenin signaling. Thus, inhibition of WNT/β-catenin sig- Quantification of the Proteome During hESC-Directed Differentiation naling by DAB2 is essential for establishing the correct number of Toward Cardiomyocytes. To reveal molecular mediators and markers cardiomyocytes in the developing heart. Our work demonstrates of cardiac development, we quantified the proteome of hESCs as that quantifying the proteome of human stem cells can identify previously unknown developmental regulators. Significance quantitative proteomics | cardiomyocyte | zebrafish | embryonic stem cell | The directed differentiation of human embryonic stem cells into WNT/β-catenin cardiomyocytes provides a tool for understanding human heart development and disease. During the process of cardiomyocyte irected differentiation of human embryonic stem cells (hESCs) differentiation, tight regulation of the WNT/β-catenin signaling Dtoward definitive cardiomyocytes provides a platform for un- pathway is required. Thus, understanding which proteins are derstanding human heart development and disease (1). To this end, involved in promoting or repressing the WNT/β-catenin signal- genome-wide screens focusing on transcriptional regulation and ing pathway is crucial for identifying positive and negative RNA expression during time course staging of cardiac develop- regulators of cardiac development. Here we measured protein ment have identified regulators of cardiac development (2, 3). expression during a time course of cardiomyocyte differentia- Although these studies have increased our understanding of the tion. We identified a regulator of cardiac development, Disabled transcriptional mechanisms active during heart development, 2, and found that in zebrafish embryos, it negatively regulates there remains little information regarding regulation of the pro- WNT/β-catenin signaling to promote cardiomyocyte differentia- teome in this same context. This led us to quantify the proteome of tion. Thus, our work reveals a highly conserved, previously un- hESCs during a stage-specific differentiation to definitive car- identified process relevant for human heart development. diomyocytes, a primary functional cell of the heart muscle. It is well known that tight regulation of the WNT/β-catenin Author contributions: P.H., A.M.R., D.P.C., R.T.M., and C.E.M. designed research; P.H., A.M.R., and D.P.C. performed research; P.H., A.M.R., and D.P.C. analyzed data; and P.H., A.M.R., D.P.C., signaling pathway during the process of cardiomyocyte differen- R.T.M., and C.E.M. wrote the paper. tiation is imperative (4, 5). Temporal overactivation or inhibition Reviewers: L.J.F., The Riley Heart Research Center; and S.P.P., University of Wisconsin– of the WNT/β-catenin signaling pathway has been shown to result Madison. in cardiac null phenotypes in vivo (4, 6, 7), because WNT/β-catenin The authors declare no conflict of interest. is required to form mesoderm, and it subsequently must be re- Data deposition: Proteins identified by mass spectrometry in the datasets have been de- pressed to form cardiomyocytes. Furthermore, overactivation of posited in the UniProt database (www.uniprot.org). WNT/β-catenin signaling during hESC-derived cardiomyocyte dif- 1P.H. and A.M.R. contributed equally to this work. ferentiation results in a shift in mesoderm patterning to specify 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. endothelium and early blood cells, but not cardiomyocytes (8). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Conversely, inhibition of WNT/β-catenin has been shown to direct 1073/pnas.1523930113/-/DCSupplemental. 1002–1007 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1523930113 Downloaded by guest on September 30, 2021 they differentiated toward cardiac progenitor cells (CPCs) and efficiency, and verified that >80% of the cardiomyocyte population definitive beating cardiomyocytes. hESCs were induced to differ- was cTnT-positive (Fig. 1B). entiate with activin A and bone morphogenetic protein 4 (BMP4), As a readout of normal cardiac differentiation, we assessed in combination with sequential small molecule activation and in- markers of pluripotency, [SRY-box2 (SOX2)], mesoderm formation hibition of WNT/β-catenin signaling. Cells were then harvested as [Brachyury T (BryT)], CPCs (GATA4), and cardiomyocytes (MYH6) by quantitative reverse-transcriptase PCR (qRT-PCR) pluripotent hESCs (day 0), CPCs (day 5), or cardiomyocytes (day C A over the time course of differentiation (Fig. 1 ). SOX2 is highly 14)(3,5,8)(Fig.1 ). We have previously shown that as cells expressed in hESCs, whereas expression decreases to baseline differentiate toward CPCs, they express cardiac transcription fac- levels as cells differentiate toward precardiac mesoderm. As cells tors, such as GATA binding protein 4 (GATA4), T-box 5 (TBX5), exit pluripotency, the pan-mesodermal marker BryT is expressed and NK2 homeobox 5 (NKX2.5) (3, 8) (Fig. 1A). CPCs then in on day 2, whereas during specification of CPCs, BryT expression turn differentiate predominately toward cardiomyocytes and ex- returns to baseline levels. The increased expression of the car- press the cardiac structural genes [cardiac troponin T (cTnT)] and diac transcription factor GATA4 is observed at day 5, followed [myosin heavy chain 6 (MYH6)], with a minority of cells showing by cardiac structural proteins MHY6 and cTnT at day 14 (Fig. B characteristics of the fibroblast/smooth muscle (2, 3). Thus, we 1 ). These data indicate successful differentiation of hESCs assessed our cultures by fluorescence-activated cell sorting (FACS) toward cardiomyocytes with normal transitioning among hESCs, mesoderm, CPCs, and definitive cardiomyocytes. analysis to determine cardiomyocyte purity and differentiation Using LFQ proteomics, we measured changes in protein ex- pression during a time course of hESC cardiomyocyte differen- tiation by quantifying protein extracted from hESCs (day 0), D ABCPCs (day 5), and cardiomyocytes (day 14) (Fig. 1 ). Normalized CCM Isotype control quantities of protein were tryptically digested and fractionated RPMI + B27 minus insulin cTnT-PE using reverse-phase chromatography before measurement by mass RPMI + B27 plus insulin spectrometry. Protein quantification was reproducible, with an >80% cTnT+