Decision letter: Revised roles of ISL1 in a hES cell-based model of human heart chamber specification

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell state but rather serves to accelerate cardiomyocyte differentiation. Conversely, ISL1 depletion delays cardiac differentiation and respecifies nascent cardiomyocytes from a ventricular to an atrial identity. Mechanistic analyses integrate this unrecognized anti-atrial function of ISL1 with known and newly identified atrial inducers. In this revised view, ISL1 is antagonized by retinoic acid signaling via a novel player, MEIS2. Conversely, ISL1 competes with the retinoic acid pathway for prospective cardiomyocyte fate, which converges on the atrial specifier NR2F1. This study reveals a core regulatory network putatively controlling human heart chamber formation and also bears implications for the subtype-specific production of human cardiomyocytes with enhanced functional properties. https://doi.org/10.7554/eLife.31706.001 Introduction The four chambers of the mammalian heart are specified from the first and second heart fields (FHF/SHF) encompassing distinct precursor cell populations that give rise, respectively, to the left ventricle (FHF) and the mostly SHF-derived right ventricle, left and right atria, and outflow tract (Buckingham et al., 2005). The LIM domain transcription factor ISL1 (Islet-1) is a prime player in and marker of the SHF from which, accordingly, both ventricular and atrial cells originate (Cai et al., 2003). The view that the SHF not only gives rise to the right ventricle and outflow tract, but also to most cells of the atria, is a result of revised lineage-tracing experiments in the mouse embryo using improved Isl1-Cre driver lines (in Yang et al., 2006). Conversely, mouse embryos deficient for Isl1 show severe cardiac defects highlighting the functional importance of ISL1 in this context (Cai et al., 2003). Proliferating ISL1+ cells not only form cardiomyocytes (CMs) but they also bear multipotent differentiation potential for generating the smooth muscle and endothelial cell lineages, as shown in vitro and in vivo (Laugwitz et al., 2008; Moretti et al., 2010, 2006). Interestingly, human embryonic stem cells (hESCs) as well as induced pluripotent stem cells may give rise to ISL1+ cells with multipotent properties (Bu et al., 2009; Moretti et al., 2010). Subsequently, these findings, as well as the fact that ISL1+ cells undergo significant expansion in vivo (Cai et al., 2003), have stimulated efforts to stably propagate them in vitro and potentially pave the way for regenerative medical approaches. ISL1 has thus been used as a main self-renewal marker in these studies (Cao et al., 2013; Cohen et al., 2007; Qyang et al., 2007; Zhang et al., 2016). A prime morphogen playing into SHF development is retinoic acid (RA). It is synthesized by the somites of the mouse embryo, to then signal to the posterior part of the SHF (Duester, 2008). At around E7.5, RA restricts the cardiac progenitor pool marked by ISL1, to subsequently promote atrial specification of the posterior SHF. Conversely, in embryos deficient in synthesizing RA, the ISL1-expressing domain of the late SHF, the anterior SHF, is expanded and atrial induction compromised (Ryckebusch et al., 2008; Sirbu et al., 2008; Zaffran et al., 2014). Interestingly, human pluripotent stem cells undergoing cardiac induction are responsive to RA - much like in the in vivo situation. Hence, activation of RA signaling promotes atrial specification at the expense of a default ventricular cell fate (Ma et al., 2011). Importantly, Devalla and colleagues have recently shown that NR2F1 (also known as COUP-TFI) is a pivotal RA-induced transcription factor. It activates at least part of an atrial-specific gene expression program including, for instance, the potassium ion channel-encoding KCNA5 gene mediating atrial-specific action potential properties (Devalla et al., 2015; Marczenke et al., 2017b). Moreover, in a recently established differentiation protocol, hESCs homogeneously pass through a transient ISL1 stage before acquiring a terminally differentiated cardiomyocyte (CM) state (Rao et al., 2016; Zhang et al., 2015). This fact prompted us to functionally investigate the role of ISL1 in hESCs undergoing cardiac differentiation. By combining directed cardiac differentiation of hESCs, targeted genetic manipulation, and functional genomics analysis, we show that ISL1 does not sustain self-renewal of cardiac precursor cells. Rather, it acts as an accelerator of cardiomyocyte differentiation and concurrently takes on a central position in the cardiac subtype specification network. Hence, we find that ISL1 is negatively linked to known and previously unrecognized drivers of atrial induction, NR2F1 and MEIS2, and that it, thereby, acts as a functional opponent of retinoic acid signaling in competing for ventricular versus atrial specification. Results ISL1 accelerates pan-cardiac gene induction in hESCs without affecting proliferation Given its key role in vertebrate cardiogenesis and its implication in the cardiac precursor cell state, we sought to investigate the function of ISL1 upon cardiac induction of human ES cells. To this end, a functional knockout-causing deletion was induced in HuES6 cells using CRISPR/Cas9n (Figure 1A). Clonal ISL1 knockout (KO) hESCs were then differentiated using a high-efficiency monolayer protocol (Figure 1B; Zhang et al., 2015). At day 5, the approximate peak expression time point of ISL1 in this protocol (Rao et al., 2016), ISL1 was undetectable in KO cells, as expected (Figure 1C). Using time course gene expression analysis of various cardiac markers, we noticed with interest that ISL1 KO cells were not entirely deficient in undergoing differentiation into cardiomyocytes. Rather, they displayed a delayed induction of structural as well as regulatory cardiac genes but eventually, they also started to contract spontaneously (Figure 1D and Figure 1—figure supplement 1A, Videos 1 and 2). These results were confirmed at the protein level where wild-type (WT) controls showed robust abundance of cardiac markers by day 6, the usual time point of spontaneous beating initiation in the protocol, whereas ISL1 KO showed a still incomplete pattern by day 8 (Figure 1E). Hence, ISL1 is not absolutely required for CM differentiation in hESCs but its depletion slows down the process. Figure 1 with 1 supplement see all Download asset Open asset ISL1 accelerates cardiac differentiation of hESCs without affecting cell proliferation. (A) CRISPR-mediated knockout of ISL1 in hESCs. Left: Strategy to delete the intron 1/exon 2 splice junction using two pairs of CRISPR/Cas9 nickase vectors. Right: Validation of induced genomic deletion by genomic PCR in a positive cell line. (B) Schematic of differentiation protocol. The indicated signaling factor treatments underly all cardiac induction experiments throughout this study. (C) Immunoblot validating the absence of ISL1 protein in KO cells at day 5 following the protocol of panel B. (D) Time course gene expression analysis of cardiac genes by RT-qPCR (n = 2–7). (E) Immunoblot confirming the differentiation delay of ISL1 KO cells at the protein level. (F) Schematic of inducible expression vector (top) and doxycycline treatment protocol used in most experiments to mimic the temporal ISL1 expression pattern of WT cells during directed cardiac induction (bottom). (G) Immunoblot confirming ISL1 inducibility in clonal ISL1KO/I.TET-ON cells during directed cardiac differentiation. (H) Time course gene expression analysis of cardiac genes in ISL1KO/I.TET-ON cells (RT-qPCR, n = 2–6). (I) Western blot showing restored kinetics of cardiac differentiation following pulsed ISL1 induction in ISL1KO/I.TET-ON cells. (J) Immunostaining of cardiac troponin T at day 6 of directed differentiation. (K) Cell proliferation measured by cell counting (left, n = 4) and cell cycle analysis (right) of WT and ISL KO hESCs undergoing directed cardiac induction. https://doi.org/10.7554/eLife.31706.002 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Spontaneously beating WT hESC-CMs. Stereo microscopic view of WT cells at day 8 of cardiac differentiation (24-well format). https://doi.org/10.7554/eLife.31706.004 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Spontaneously beating ISL1 KO hESC-CMs. Stereo microscopic view of ISL1 KO cells at day 8 of cardiac differentiation. https://doi.org/10.7554/eLife.31706.005 Interestingly, different cardiac genes were affected to different degrees in ISL1 KO cells regarding their induction kinetics. To understand whether this may conversely imply an active role of ISL1 in driving the differentiation process, we generated a stable cell line on ISL1 KO background in which an ISL1 transgene could be selectively induced by doxycycline addition. This strategy would allow to investigate the function of ISL1 in a stage-specific manner, by mimicking the transient induction pattern of endogenous ISL1, for instance (Figure 1F; compare to ISL1 in Figure 1D). The resulting cell line, termed ISL1KO/I.TET-ON, showed robust ISL1 expression following a 1-day exposure to DOX (Figure 1G). Transient ISL1 induction in these cells during cardiac differentiation fully restored WT-like gene expression kinetics based on a panel of markers, which also confirmed the specificity of the underlying genetic manipulations (Figure 1H and Figure 1—figure supplement 1C). Importantly, some of these genes, notably the key cardiac regulators MEF2C and NKX2.5, showed immediate-early responses to the reintroduction of ISL1 as they became significantly upregulated after only 1 day of DOX addition (Figure 1H and Figure 1—figure supplement 1B–D). This acceleration effect regarding the upregulation of key cardiac genes also became apparent at the protein level as well as by visual inspection (Figure 1I and Figure 1—figure supplement 1J, Video 3). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Spontaneously beating ISL1 KO hESC-CMs rescued by transgenic ISL1 induction at days 3 and 4. Stereo microscopic view of ISL1KO/I.TET-ON cells treated with DOXd3,4 (day 8 of cardiac differentiation). https://doi.org/10.7554/eLife.31706.006 To obtain a more global insight, we recorded genome-wide expression time-series using WT and ± DOXd3,4-treated ISL1KO/I.TET-ON cells (Supplementary file 1). Stringent filtering of these data yielded a set of approximately 70 known cardiac and uncharacterized genes, all showing accelerated induction kinetics as driven by ISL1 (Figure 1—figure supplement 1E–G). Moreover, cell number as well as cell cycle stage quantification in differentiating WT and ISL1 KO cells revealed only marginal differences between these, which thus could not explain the pronounced differences in cardiac gene induction seen at days 5–6 (Figure 1K). These analyses therefore suggest that ISL1 accelerates CM formation in differentiating hESCs by activating downstream pro-cardiac genes, rather than through promoting proliferation or stability of the cardiac precursor cell state, for instance. ISL1 does not stabilize the cardiovascular progenitor cell state To further investigate this latter possibility and to illuminate the putative multipotency of ISL1+ cells, we screened a panel of candidate signaling factors for their ability to either stabilize the precursor state and prevent further differentiation, or to induce different differentiation outcomes, notably cardiomyocytes, smooth muscle, or endothelial cells (Figure 2A). Several molecules promoted the specific induction of one or two or these differentiation fates. For example, FGF2 and VEGF clearly stimulated endothelial differentiation from the ISL1+ intermediate stage (Figure 2—figure supplement 1A–C). Somewhat surprisingly, however, none of the molecules, including previously reported factors and cocktails, could significantly promote ISL1 expression which eventually declined under all conditions tested (Figure 2B and C). Figure 2 with 1 supplement see all Download asset Open asset ISL1 does not stabilize the cardiovascular progenitor cell state. (A) Assay design interrogating self-renewal and multipotent properties of ISL1-expressing cells which emerge by day 4/5 of directed cardiac differentiation. (B) RT-qPCR results from screening the indicated signaling molecules for sustaining cardiac progenitor-specific gene expression within the first passage using WT cells (n = 3–4 per sample type). (C) ISL1 immunostaining of samples treated in the indicated ways. (D) Schematic of inducible expression vector used to generate a WTI.TET-ON hESC line (top) and doxycycline treatment protocol used here to induce a continuous overexpression of ISL1 on ISL1WT background (bottom). (E) RT-qPCR analysis of ISL1 (left) and a set of 5 pan-cardiac genes (right) at day 8 of directed cardiac differentiation without or with continuous DOX treatment as shown in panel D (n = 2). (F) Immunostaining of ISL1 and early cardiomyocyte markers at day 8 of directed cardiac differentiation. Note that endogenous ISL1 has already declined in untreated cells, whereas transgenic ISL1 does not interfere with CM formation. (G) Schematic of DOX treatment protocol used here to induce ISL1 overexpression in maturating CMs. (H) RT-qPCR analysis in 3-week-old CMs following late DOX treatment of the indicated samples (n = 3). (I) Immunoblot confirming RT-qPCR analysis (H) at the protein level. https://doi.org/10.7554/eLife.31706.007 Next, we challenged the system by applying continuous ISL1 overexpression in a newly generated WTI.TET-ON line (Figure 2D). Sustaining robust ISL1 levels by continuous DOX administration from day 3 of directed cardiac induction onwards did not interfere with CM formation and did not further accelerate the differentiation process (Figure 2D and E). Somewhat surprisingly, these data imply that ISL1 plays no role in sustaining the cardiac precursor cell state. As a confirmation, ISL1 activation in ISL1KO/I.TET-ON cells did not affect the expression of independent precursor genes like PDGFRA (Figure 2—figure supplement 1D). We did, however, notice that a later induction of ISL1 in already-formed CMs specifically antagonized the expression of ventricular-specific genes, notably that of MLC2v, without compromising pan-cardiac CTNT expression (Figure 2G–I). Similar observations were made upon enforced long-term (3 wk) overexpression of ISL1 (Figure 2—figure supplement 1E). Collectively, these data reveal distinct stage-dependent effects of ISL1 overexpression, in agreement with the mouse system (Dorn et al., 2015). Significantly, however, they establish that at early differentiation stages, ISL1 acts as a pro-differentiation factor. ISL1 KO phenocopies an atrial wild-type CM phenotype induced by retinoic acid As already mentioned, ISL1 KO cells displayed a delay in beating initiation and also, at a later stage, appeared to have shorter contraction durations as compared to wild-type cells. These observations reminded us of the behavior of wild-type CMs differentiated in the presence of retinoic acid, which we coincidently studied in parallel efforts. RA treatment during CM induction is to apply a physiological signaling cue for promoting CM subtype specification into atrial cells, as opposed to ventricular CMs usually obtained by default (Devalla et al., 2015; Ma et al., 2011). Interestingly, a thorough optimization of atrial fate induction by RA in WT cells revealed that the optimal time window of exposure overlapped with that of endogenous ISL1 induction in our protocol. Hence, a 2-day treatment with RA (0.5 µM) on days 3 and 4 was sufficient to induce robust atrial specification while suppressing a ventricular fate (Figure 3—figure supplement 1A–D). Concomitantly, however, RA administration caused a differentiation delay similar to ISL1 KO cells differentiated with the standard protocol (Figure 3A and B). We therefore hypothesized that the ISL1 KO - instead of compromising CM formation in general - may cause a switch in CM subtype identity, from ventricular to atrial-like, similar to the effects of RA stimulation. Figure 3 with 1 supplement see all Download asset Open asset ISL1 KO phenocopies an atrial wild-type CM phenotype induced by retinoic acid. (A) Upon directed cardiac induction, ISL1 KO as well as RA-treated wild-type hESCs display delayed terminal CM differentiation reflected by a later initiation of spontaneous beating (semiquantitiative analysis, n = 3–14 per sample). ‘ISL1’ and ‘no RA’ denote different batches of WT HuES6 cells. (B) Immunofluorescence analysis of the early CM marker myosin heavy chain 6 upon directed cardiac differentiation of the indicated samples/cell lines. ‘ISL1’ cells are d 3/4 transgene-induced ISL1 KO hESCs. (C) Expression pattern of atrial and ventricular-enriched genes in the indicated in vivo and hESC-derived samples. Primary human heart samples served as specificity controls (microarray data). (D) Confirmation of ventricular and atrial-specific gene expression profiles by RT-qPCR in independent sets of experiments (n = 4–7 per sample type). (E) Immunoblots 3 wk after the initiation of cardiac differentiation for ventricular-specific myosin light chain and atrial natriuretic peptide. (F) Confirmation of cardiac subtype-specific phenotypes by immunostaining (~3 wk time point). (G) Model summarizing the opposing roles of ISL1 and RA signaling in cardiac subtype specification. (H) Enhanced atrial and further reduced ventricular gene expression in RA-treated ISL1 KO CMs as compared to RA-treated WT and untreated ISL1 KO cells (RT-qPCR data at ~2.5 wk, n = 3). (I) Spontaneous beating analysis of the indicated hESC-CM samples on multielectrode arrays. Left: Representative traces. Right: Beating rate quantification (ntech. = 3). Results were reproducible in independent experiments. (J) Representative action potential traces from patch clamp analyses of the indicated types of hESC-CMs. Note the additional action potential shortening upon combining ISL1 depletion with RA treatment. See Supplementary file 2 for averaged data from independent samples. In case of using ISL1KO/I.TET-ON cells, all ISL1+ data in this figure are based on a day 3–4 treatment with DOX. https://doi.org/10.7554/eLife.31706.009 A microarray analysis of later-stage ± DOXd3,4-treated ISL1KO/I.TET-ON cardiomyocytes, ± RAd3,4-treated CMs, as well as human ventricular and atrial heart tissue served to address this idea. A panel of accepted and newly identified markers such as atrial ANP, DHRS9, KCNA5, SLN (encoding sarcolipin) and the ventricular-specific MLC2v (Josowitz et al., 2014; Piccini et al., 2015) could well discriminate between the distinct human heart samples. Strikingly, these were also differentially expressed between DOX-treated ISL1KO/I.TET-ON and WT CMs on the one hand, and ISL1-deficient and RA-treated WT CMs on the other (Figure 3C). These data were confirmed in independent experiments, at RNA and protein levels: RA-treated WT CMs consistently showed a pronounced expression of atrial-specific markers and a strong decline in ventricular-specific gene expression - and this was phenocopied by the ISL1 knockout, with only some variation in the degree of the phenotypes from experiment to experiment (Figure 3D–F). These data establish a novel and crucial role of ISL1 at the cardiac precursor state, namely, to promote ventricular CM subtype specification and counteract an atrial fate, thereby competing with RA signaling for prospective CM identity (Figure 3G). Intriguingly, ISL1 depletion alone was apparently sufficient to promote an atrial-like expression pattern - without need for exogenous RA stimulation during cardiac induction. We next sought to investigate whether this change in gene expression would also translate into distinct functional properties. In addition, we wondered if RA stimulation and ISL1 depletion would synergize to yield an enhanced atrial phenotype. To this end, we compared untreated WT CMs, ISL1 KO CMs, RAd3,4-treated WT CMs, and RAd3,4-treated ISL1 KO CMs to one another. The latter type indeed showed enhanced expression levels of the atrial regulator NR2F1 and of KCNA5 which encodes a key atrial-specific potassium ion channel (Figure 3—figure supplement 1E and Figure 3H, respectively). KCNA5 accounts for the shortening of atrial CM-specific action potentials as compared to ventricular ones, which also translates into faster spontaneous beating of atrial-like hiPSC-CMs (Marczenke et al., 2017b). Likewise, ISL1 KO and RAd3,4-treated hESC-CMs showed increased beating frequencies as compared to ventricular-like controls. Interestingly, RAd3,4-treated ISL1 KO CMs contracted at even faster rates (Figure 3I). Furthermore, using single-cell patch clamp analysis, ISL1 KO CMs displayed action similar to In line with the increased KCNA5 expression however, action potential durations in RAd3,4-treated ISL1 KO CMs were even further reduced (Figure and Supplementary file 2A). to reveal a functional we KCNA5 using a (Devalla et al., 2015). As this treatment no effect on ventricular-like WT hESC-CMs but it significantly action potentials in the other in RAd3,4-treated ISL1 KO cells (Figure 3—figure supplement and Supplementary file These results show that ISL1 KO CMs functionally atrial cardiomyocytes and that this phenotype may be further enhanced by transient RA in line with the of Figure acid signaling ISL1 by we sought to the gene regulatory of this between RA signaling and RA signaling is known to the ISL1-expressing domain at the SHF stage in vivo (Ryckebusch et al., 2008; Sirbu et al., 2008). To investigate a potentially similar effect in the hESC we recorded gene expression time of differentiating RAd3,4-treated and untreated WT hESCs (Supplementary file 1). ISL1 indeed became by RA but it did with a delay of 1 an (Figure filtering of the data set as well as independent RA stimulation experiments revealed two genes that could potentially the the transcription and became upregulated by RA and became (Figure These kinetics two in the right part of Figure of their specific in cardiogenesis reported to we to both these using newly generated expression cell lines (Figure and Figure supplement 1A). induction during cardiac differentiation did not to an upregulation of ISL1, which us to the first (Figure supplement 1A). Figure 4 with 1 supplement see all Download asset Open asset acid signaling ISL1 by MEIS2. (A) time course gene expression analysis of untreated and RA-treated (0.5 on d WT hESCs to cardiac induction conditions Supplementary file 1). RT-qPCR analysis on day 3 of cardiac induction (n = expression at day 5 between ± RA and confirmed effect within 4 of RA treatment. Right: on of ISL1 by (B) Schematic of vector for expression Immunoblot on day 5 of cardiac differentiation confirming induction in clonal hESCs following DOX addition Immunostaining confirming (C) ± RA and ± DOX differentiation time using hESCs. Left: RT-qPCR analysis (n = Right: Western blot (D) analysis of HuES6 WT and KO hESC differentiation without or with RA addition Left: RT-qPCR analysis (n = 3 Right: By as to the optimal RA treatment window at days 3 and 4 of directed cardiac differentiation, significantly - and - ISL1, similar to RA treatment (Figure The downstream effects on the human ISL1 gene MEF2C were even more Importantly, induction also caused an upregulation of NR2F1 which is a key driver of atrial fate induction, with some delay compared to RA stimulation (Figure Devalla et al., 2015). Moreover, all these effects were RA signaling and gene (Figure supplement Hence, to be an player in atrial CM downstream of RA signaling to ISL1 and promote the induction of the atrial specifier gene NR2F1. To further this we next sought to the after with that for the of its (Figure supplement 1C). Clonal hESCs were to directed cardiac induction in to WT cells. differentiated cells to show spontaneous they positive for markers (Figure supplement 1D). We that in light of the of this the would still allow for the early regulatory of interest upon precursor cell formation. In WT RA administration at days 3 and 4 ISL1 and promoted NR2F1 induction, as seen before (Figure In cells to ISL1 as well as its gene MEF2C in the presence of RA, and the induction of NR2F1 was compromised (Figure These data establish as a of ISL1 by RA thereby atrial specification. ISL1 the early atrial specifier NR2F1 Conversely, we ISL1 may atrial induction promoted by RA In addition to the as well has been shown to be an atrial driver downstream of RA (Devalla et al., 2015). We time course expression analyses ISL1+ and ISL1-deficient hESCs undergoing cardiac induction, while to these two early or later atrial and ventricular markers did not consistently show an response to ISL1 activation by DOX in differentiating ISL1KO/I.TET-ON cells (Figure and Figure supplement 1A). This was also for which showed a significant but delayed response to ISL1 (Figure By NR2F1 became induced in ISL1 KO cells, in WT cells, and was antagonized by ISL1 induction as early as at day 4 (Figure pulsed ISL1 induction by DOX addition on days 3 and 4 of differentiation levels of NR2F1 protein in early CMs (Figure These data show that ISL1 serves to the key atrial regulator gene NR2F1. Figure 5 with 1 supplement see all Download asset Open asset ISL1 the early atrial specifier NR2F1. (A) Time course gene expression analysis of early atrial and ventricular genes by RT-qPCR (n = per data point). between WT and ISL1 KO cells and ISL1 KO versus pulsed ISL1 with ISL1KO/I.TET-ON Note the immediate-early effect on which is not seen in case of or (B) Immunoblot for NR2F1 at an early CM (C) RA does not the phenotype of ISL1 KO cells, as shown by RT-qPCR n = 4) and in the indicated we sought to whether endogenous RA signaling is in promoting NR2F1 induction in an ISL1-deficient We a