Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion. DOI: http://dx.doi.org/10.7554/eLife.02164.001


Introduction
Embryonic cardiac progenitor cells (CPCs), identified from early embryos or differentiating pluripotent stem cells, hold tremendous regenerative potential with their unique capability to expand and differentiate into nearly all cell types of the heart (Parmacek and Epstein, 2005;Kattman et al., 2006;Moretti et al., 2006;Kwon et al., 2007). Over the past decade, significant progress in developmental cardiology led to the identification of CPC markers and lineages (Cai et al., 2003;Kattman et al., 2006;Moretti et al., 2006;Kwon et al., 2009). However, CPCs are highly heterogeneous and it is unknown if they can undergo self-renewal without differentiation. Consequently, understanding the precise mechanisms of CPC self-renewal and maintenance remains a fundamental challenge.
Numb and Numblike (Numbl)-mammalian Numb homologs sharing collinear topology and extensive sequence identity with functional redundancy-are evolutionarily conserved proteins that are required for the self-renewal of neural progenitors and mediate asymmetric cell divisions in various contexts of cell fate decisions (Zhong et al., 1997;Petersen et al., 2002Petersen et al., , 2004Roegiers and Jan, 2004), but their role in CPC development has not been explored. In the current study, we sought to identify and investigate CPCs affected by Numb and Numbl. By taking combinatorial approaches, we demonstrate that Mesp1 + progenitor-derived Isl1 + Nkx2.5 − cells renew and expand without cardiac differentiation in the second pharyngeal arch (PA2) and that PA2 serves as their microenvironment during mammalian heart development.

Results
Numb and Numbl are required for heart development Numb is expressed ubiquitously in developing mouse embryos (Zhong et al., 1997;Jory et al., 2009; Figure 1-figure supplement 1). To quantitatively examine the expression of Numb and Numbl in developing CPCs, we used the embryonic stem (ES) cell differentiation system that recapitulates early cardiogenesis (Kattman et al., 2011;Van Vliet et al., 2012;Figure 1A). Numb levels were relatively low at day 4, when Mesp1 was induced, but upregulated at day 6, when Isl1 appeared ( Figure 1B). Numbl levels were also increased at day 6 ( Figure 1B), implying that Numb and Numbl may have a role in CPC development after Mesp1 induction. To test this possibility, we deleted Numb in Mesp1 + progenitors, the earliest mesodermal progenitors giving rise to the entire heart and vasculature eLife digest Human embryos contain cells called 'cardiac progenitor cells' that serve as the building blocks to make the heart. Cardiac progenitor cells, or CPCs for short, initially move into areas of the embryo called the first and second heart fields, and then undergo a change to become specific types of heart cells: such as cardiac muscle cells. However, it is not known if CPCs are maintained during the development of the heart. Now, Shenje, Andersen et al. have shown that Numb and Numblike-two proteins that are needed for the development of nerve cells-are also involved in the development of the heart. Mouse embryos without the genes for Numb and Numblike failed to develop hearts normally; and these mutants also had fewer CPCs in the 'second pharyngeal arch': a part of the embryo that becomes the sides and front of the neck. Experiments on wild-type mice showed that the CPCs multiplied within this arch, and then changed into specific heart cells as they left this structure. Furthermore, mixing CPCs in a petri dish with cells taken from this arch encouraged the CPCs to multiply without changing into specific cell types.
To investigate the importance of these two proteins further, Shenje, Andersen et al. engineered 'chimeric' mice in which some CPCs contained the Numb and Numblike genes and other CPCs did not. In most of these chimeric mice, the hearts developed normally, but the CPCs without the Numb or Numblike genes failed to multiply in the second pharyngeal arch. This shows that these genes must be present within an individual CPC to regulate the multiplication of that cell within this arch.
By uncovering how problems with the maintenance of CPCs can lead to heart defects-a very common birth defect in humans-this work may lead to new ways to prevent or treat congenital heart disease. Furthermore, identifying the other factors or mechanisms that can allow the longterm maintenance of CPCs in the laboratory will be crucial for research into heart regeneration, and for CPC-based treatments to repair the heart. DOI: 10.7554/eLife.02164.002 (Saga et al., 2000), by crossing Mesp1 Cre mice with Numb flox mice (Zhong et al., 2000). The deletion did not affect LV formation, but resulted in a hypoplastic OT/RV and PA2 ( Figure 1C, C′,D,D′). While the phenotype appeared to be confined to the OT, RV, and PA2, the penetrance was variable. Since Numbl-null mice are viable and fertile, but Numbl can compensate for loss of Numb function (Petersen et al., 2002), we extinguished Numb and Numbl expression in Mesp1 + progenitors by deleting Numb in the Numbl-null background (Petersen et al., 2002). The resulting Numb and Numbl double knockout (Numb/Numbl DKO) embryos showed a hypoplastic OT/RV and PA2 with complete penetrance (n = 12 embryos, Figure 1E,E′) and uniformly lethal by embryonic day (E) 10.0. These data suggest that Numb and Numbl are required for the formation of the PA2 and OT/RV.

Deletion of Numb and Numbl depletes Mesp1 + cell progeny in PA2
Based on the Numb/Numbl DKO phenotype, we focused on investigating Mesp1 progeny contributing to the PA2 and OT/RV. To visualize Mesp1 progeny affected by Numb and Numbl, we introduced a Cre reporter (Ai9) into the DKO mouse embryos, in which red fluorescent protein (RFP) permanently  marks Mesp1 + progenitors, and traced the RFP + cells (Figure 2A). The resulting RFP + cells showed a near complete deletion of Numb, confirmed by fluorescence-activated cell sorting (FACS) and quantitative PCR (qPCR) ( Figure 2B). Interestingly, control and DKO littermates showed no noticeable morphological defects at E8.5 (8-12 somite stage) and were histologically indistinguishable (Figure 2-figure supplement 1). RFP + cells were normally expressed the transient CPC marker Isl1 (Cai et al., 2003) from the primordial PA2 to the bulbus cordis (BC), and there was no significant difference in the number of RFP + Isl1 + cells or the percentage of RFP + Isl1 + cells positive for the mitosis marker phospho-histone H3 (PH3) (Figure 2-figure supplement 1B,B′,G,G′,K, Figure 2M). The RFP + Isl1 + cells in PA2 primordia were continuous with the endo-, myo-and peri-cardial cell layers of the heart tube, and expressed the cardiac transcription factors Nkx2.5 and Mef2c from the distal BC and the sarcomeric protein α-Actinin from the proximal BC in both control and mutant embryos (Figure 2-figure supplement 1B-E′,G-J′). Thus, Numb/Numbl DKO does not appear to affect CPC and heart development at E8.5.
Mesp1 progeny expand in PA2 and migrate out to become heart cells While PAs contain SHF progenitors (Kelly et al., 2001;Xu et al., 2004), it is unknown if Mesp1 + cellderived Isl1 + Nkx2.5 − cells in the PA2 give rise to cardiac cells. Based on the Numb/Numbl DKO phenotype, cardiac gene expression patterns, and ES cell data, we hypothesized that the Isl1 + Nkx2.5 − cells expand without differentiation in the PA2 and migrate out of the arch to differentiate into cardiac cells. To test this, we examined proliferating cells in cardiac regions of E9.0 embryos by performing wholemount staining with 5-ethynyl-2′-deoxyuridine (EdU), a nucleoside analog of thymidine. Remarkably, EdU + cells were concentrated in the PA2 and rarely detected in other cardiac regions including the growing heart ( Figure 3A,B). Consistently, about four percent of Mesp1 + cell-derived (RFP + ) Isl1 + Nkx2.5 − cells were PH3 + in the PA2, whereas PH3 + cells were nearly absent in RFP + Isl1 +/− Nkx2.5 + OT and α-Actinin + RV cells ( Figure 3C). Since DNA analogues are commonly used to show actively dividing stem cells and their lineages (Eisenhoffer et al., 2008;Snippert and Clevers, 2011), we labeled the proliferating cells by administering a single pulse of EdU, and monitored EdU + cells in RFP + cells ( Figure 3D). EdU + cells were first identified in the outer layer of the RFP + cell cluster in PA2 after 2 hr, but not in the OT/RV ( Figure 3D). By 4-8 hr, the RFP + EdU + cells were abundant in the PA2 and OT, and eventually found in the RV after 24-48 hr of chase ( Figure 3D). RFP + cells in the PA2 remained EdU + ( Figure 3D,E), implying their renewal in the PA2. The purdurance of the EdU pulse, determined by a sequential injection of EdU and 5-bromo-2′-deoxyuridine (BrdU, another thymidine analog), was shorter than 4 hr (Figure 3-figure supplement 1). These data suggest that Mesp1 + cell-derived Isl1 + Nkx2.5 − cells in the PA2 migrate out of the arch and differentiate into cardiac cells.
To directly test the potential of RFP + cells in the PA2, we labeled proliferating cells with EdU for 2 hr at E9.0, dissected out PA2s from embryos (asterisks, Figure 3D), and cultured the arches in 3D Matrigel culture. RFP + cells were monitored in real-time with fluorescent time-lapse movies. After 2 days, the cluster of RFP + cells expanded and exhibited multidirectional migration   . Although the range of their migration was somewhat limited, likely due to the extracellular environment of Matrigel, RFP + cells continued to expand in the PA2 and differentiated into cardiomyocytes. When cultured in an uncoated dish, RFP + cells expanded in the PA2 and progressively migrated out of the arch in a unidirectional fashion soon after being attached to the surface. However, no beating activity was observed on day 2 ( Figure 3F). They migrated out of the arch progressively soon after being attached to the surface ( Figure 3F). The migrated RFP + cells started to spontaneously contract by day 4 ( Figure 3F´). The expansion and migration of RFP + cells appear to continue over 2 weeks and the beating area was correspondingly expanded ( Figure 3F″; Video 3). The beating cells were positive for EdU, Nkx2.5, and cTnT/α-Actinin and exhibited a periodic Ca 2+ oscillation pattern similar to that of adult cardiomyocytes ( Figure 3G-I). To ascertain that the resulting cardiac cells were not derived from contamination from the adjacent OT, we isolated PAs from Nkx2.5 GFP embryos (Biben et al., 2000) that express GFP in cardiac cells including the OT, but not in the PAs. Freshly dissected PA2s did not contain GFP + cells, but cells expressed GFP 2-3 days after migration from the arch (Figure 3-figure supplement 3). We concluded that RFP + Isl1 + cells expand in the PA2 and differentiate into cardiac cells as they migrate out of the arch. It is worth noting that PA1 cells also migrated out from the arch, but they remained GFPand eventually formed myotube-like structures (Figure 3figure supplement 3, Figure 3J). This is consistent with the previous finding that pharyngeal mesoderm is also the source of head skeletal muscles (Nathan et al., 2008).

PA2 cells promote the renewal and expansion of CPCs
To determine if the PA2 affects CPC expansion, we isolated Isl1 + Nkx2.5 − CPCs from ES cells and cultured them with PA2 cells or heart cells derived from E9.0-9.5 embryos or without feeders. The CPCs were obtained by differentiating Isl1 Cre ; Ai9 ES cells  into Mesp1 + precardiac mesoderm (Kattman et al., 2011) and purifying RFP + Isl1 + Nkx2.5 − cells by FACS at day 5 ( Figure 4A). The RFP + cells spontaneously differentiate and form a sheet of beating cardiomyocytes . Strikingly, the co-cultured RFP + cells formed distinct colonies ( Figure 4A), which were never observed in control culture conditions, and their number was greatly increased over time ( Figure 4B). The increase was inversely correlated with the appearance of Nkx2.5 + /cTnT + cells ( Figure 4C, Figure 4figure supplement 1), indicating PA2 cells promote expansion of Isl1 + Nkx2.5 − CPCs and suppress their cardiac differentiation. This is unlikely due to the altered 3D environment as the effect was mimicked by PA2 cell-conditioned medium, but not by embryonic heart cells ( Figure 4A,B, Figure 4-figure supplement 2).
To test if the colonies maintain differentiation potential, we established an Isl1 Cre ; Ai9; Myh6-GFP ES cell line, in which green fluorescent protein (GFP) is expressed when cells differentiate into cardiomyocytes (Ieda et al., 2010). RFP + Isl1 + Nkx2.5 − CPCs were FACS-purified from the line at day 5 of differentiation and co-cultured with PA2 cells to form colonies. In the presence of PA2 cells, the colonies continued to grow without GFP expression. However, when PA2 cells were removed after a week of co-culture, they began to express GFP from day 3 and differentiated into beating cardiomyocytes ( Figure 4D). These data suggest that PA2 cells provide a cellular environment for the renewal and expansion of Isl1 + Nkx2.5 − CPCs and suppress their cardiac differentiation.

Generation of Mesp1 + cell lineagespecific mosaicism
Although the Cre-mediated conditional deletion revealed a crucial role of Numb and Numbl for the development of Mesp1 progeny in the PA2, it was unclear if the Numb/Numbl DKO phenotype reflected the intrinsic role of Numb and Numbl. To address this question, we generated mosaic animals lacking Numb and Numbl in Mesp1 lineages ( Figure 5A). We first established an Numb/Numbl DKO ES cell line, in which conditional deletion of Numb/Numbl occurs in Mesp1 + cells with resultant expression of RFP, by directly deriving ES cells from the DKO embryos. The ES cells were injected into host blastocysts of UBI-GFP/BL6 mice (Schaefer et al., 2001), which ubiquitously express GFP, to generate chimeras. In this system, Numb and Numbl deletion occurs in donor-derived Mesp1 + cells, and the DKO cells are traced by RFP expression. Donor-derived RFP + cells were formed exclusively within Mesp1 lineages and distinguished from GFP + host cells and RFP − GFP − donor progeny ( Figure 5B-D). No cells were found double positive for RFP and GFP ( Figure 5C,D), excluding the possibility of cell fusion, which potentially could rescue the phenotype of DKO cells. Therefore, we used wildtype blastocysts as hosts for further chimera generation. Consistent with the earlier mRNA analysis, Numb was absent in donor-derived RFP + cells ( Figure 5E).
The phenotype of DKO chimeras depended on the contribution of the donor RFP + cells. Major donor contribution caused a phenotype similar to DKO embryos (12.5% 2/16, Figure 5F,G). In most cases (87.5% 14/16), the chimeras were indistinguishable from wild-type embryos ( Figure 5H,I). Donor RFP + cells were normally populated in the PA2 and contributed to the OT, cardiomyocytes and endocardial cells ( Figure 5J-M, Figure 5-figure supplement 1), indicating deletion of Numb and Numbl did not affect the migration or cardiac differentiation of CPCs.

NumbNumbl DKO CPCs fail to expand in PA2
In control embryos, the Isl1 + Nkx2.5cells expanded dramatically after E8.5 in the PA2 ( Figure 2C′,D,M). Proliferating cells, marked by PH3, were mostly found within the border of the cluster in control embryos, but were depleted in Numb/Numbl DKO embryos ( Figure 6A-C‴). Likewise, the chimeras formed the Isl1 + cell cluster with PH3 + cells in the PA2 ( Figure 6E). However, none of the RFP + Isl1 + Nkx2.5 − donor cells was found positive for PH3 in the PA2s of chimeric embryos analyzed at E9.0-9.5 (n = 10, Figure 6E-I), implying that Isl1 + Nkx2.5 − cells in the PA2 lose their expansion potential in the absence of Numb and Numbl. There was no evidence of apoptosis in Numb/Numbl DKO cells in the PA2 (not shown). Consistent with the in vivo data, knockdown of Numb and Numbl in ES cell-derived Isl1 + Nkx2.5 − CPCs, but not in Mesp1 + or Nkx2.5 + CPCs, resulted in a significant reduction of cell proliferation ( Figure 6J, Figure 6-figure supplement 1). Conversely, increased levels of Numb in the Isl1 + Nkx2.5 − CPCs promoted their proliferation ( Figure 6K). These suggest that Numb and Numbl are cell-autonomous factors regulating the expansion of Isl1 + Nkx2.5 − cells in the PA2.

Discussion
Through the use of mouse genetics, lineage-specific mosaicism, embryonic stem cell systems, and ex-vivo organ culture, we have shown the existence of an undifferentiated and expansive population of CPCs and their microenvironment during development (Figure 7), which may provide a stem cellniche paradigm in cardiovascular biology. Given that the heart grows rapidly in size and cell number at E8.5-10.5, the CPCs may serve as a renewable source to supply the number of cardiac cells needed to sustain the ensuing heart growth.
Although highly heterogeneous, CPCs are considered as multipotent cells that are destined to become heart cells. However, it remains unclear if they are capable to self-renew without differentiation. In mice, their precursors are identified as early as E5.75 by expression of the T-box transcription factor Eomesodermin in the epiblast (Russ et al., 2000) and specified to Mesp1 + cells at the onset of gastrulation at E6.5 (Costello et al., 2011). Mesp1 + cells are further specified to CPCs, vascular progenitors and some of the head mesenchyme that contribute to the entire heart, vasculature and subsets of head muscle cells, respectively (Saga et al., 2000). CPCs giving rise to the RV migrate through the OT from the SHF and express the transcription factors Isl1, Mef2c or Nkx2.5. These factors, however, are also expressed in neighboring cells including pharyngeal ectoderm and endoderm, foregut endoderm, neural progenitors and neural crest cells that are not originated from mesoderm (Lints et al., 1993;Edmondson et al., 1994;Cai et al., 2003), making it difficult to precisely discern CPCs. Moreover, it is unclear when and where CPCs are specified from Mesp1 + cells or their progeny. Thus, we traced Mesp1 lineages and examined the expression of CPC markers in Mesp1 progeny. Unexpectedly, heart cells rarely proliferated during early cardiogenesis, implying the existence of an alternate cell source for the ongoing growth of the heart. Indeed, we found a proliferative cluster of Mesp1 + cell-derived Isl1 + Nkx2.5 − cells in the PA2-directly linked to the Isl1 + Nkx2.5 + OT of the growing heart-that migrated to the heart and became heart cells. The Isl1 + Nkx2.5 − CPCs continued to expand in vivo, ex vivo, and in vitro without cardiac differentiation in the PA2, suggesting that the CPCs may serve as a renewable cell source for the developing heart. This cell renewal system may provide a parsimonious and efficient way to quickly generate a large number of cells used to build the heart during embryogenesis, which can be advantageous over local proliferation of differentiated cardiac cells. Further characterization of the CPCs will be necessary to provide quantitative information on their cellular contribution to the developing heart. Recent studies suggested that subsets of  head and cardiac muscles share their progenitors (Lescroart et al., 2010). While the progenitor has not been identified, it will be interesting to investigate if this progenitor is derived from Isl1 + Nkx2.5 − cells.
PAs are transient, segmented bulges that appear on the craniolateral side of developing embryos (Grevellec and Tucker, 2010). Each PA is composed of a mesodermal core and neural crest cellderived mesenchymal cells that are surrounded by ectoderm outside and endoderm inside. At E8.5, the PA1-positioned most cranially among PAs-is structurally distinct, while the PA2 becomes noticeable after E9.0. Mesp1-derived Isl1 + Nkx2.5 − cells proliferate in PA2s and initiate expression of Nkx2.5 + soon after exiting the arch. This suggests that PA2s function as a microenvironment to maintain the CPCs in an undifferentiated and expanding state and likely contain cells secreting paracrine factors that control the CPC number and fate. In fact, numerous signaling molecules are secreted from PA endoderm, ectoderm, and mesenchyme including Wnts, bone morphogenetic proteins, sonic hedgehog, and fibroblast growth factors (Rochais et al., 2009), and dysregulation of these signals is often associated with OT/RV defects (Frank et al., 2002;Stottmann et al., 2004;Washington Smoak  (H and I) Percentage of donor-derived Isl1 + Nkx2.5 − cells in PA2 is shown in (H) and number of PH3 + RFP + Isl1 + cells per 12-micron PA2 section is shown (I). Data are mean ± SD; n = 10; *p<0.05. (J and K) Relative percentage of EdU + cells in ES cell-derived Mesp1 + progenitor, Isl1 + Nkx2.5 − CPCs or Nkx2.5 + CPCs transfected with control or Numb/Numbl DKO siRNA (J) or Isl1 + Nkx2.5 − CPCs transfected with control (CTV) or Numb overexpression construct (CTV-Numb) (K). Data are mean ± SD; n = 3; *p<0.05. The Mesp1 + , Isl1 + Nkx2.5 − , or Nkx2.5 + cells were FACS-purified from day 4 Mesp1 Cre ; Ai9, day 5 Isl1 Cre ; Ai9, or day 6 Nkx2.5 GFP ES cells, respectively. Dapi (blue) was used to counterstain the nuclei. p values were determined using the paired Student t test. Scale bars, 10 μm (B, C, F, G). 50 μm (A, D, E). fe, foregut endoderm; pa, pharyngeal arch ec, endocardial layer; mc, myocardial layer; pc, pericardial layer. DOI: 10.7554/eLife.02164.021 The following figure supplements are available for figure 6:  Kwon et al., 2007). Thus, it will be important to identify the extrinsic factors and cell types that provide key signals for the CPC maintenance and PA2 development.
Although the heart phenotype (hypoplastic OT/RV) caused by Numb/Numbl DKO may not result entirely from CPC depletion in the PA2, our findings together with published literatures suggest that the CPCs in the PA2 might be a major source of cells contributing to the OT/RV. For instance, the SHF-giving rise to the entire OT/RV-is located in PAs (Kelly et al., 2001;Rochais et al., 2009) and we found that proliferating cells are present predominantly in the PA1 and PA2 and rarely detected in the rest of the PAs. Our ex-vivo culture further showed that PA2 cells robustly differentiate into cardiac cells, whereas PA1 cells appear to give rise to myotubes without cells expressing Nkx2.5. It is also worth noting that nearly all, if not all, embryos showing severely hypoplastic OT/RV exhibit hypoplastic PA2s (Srivastava et al., 1997;Gottlieb et al., 2002;Cai et al., 2003;Kwon et al., 2007Kwon et al., , 2009.
Numb and Numbl are highly conserved proteins that participate in cell fate determination by mediating asymmetric division, endocytosis and recycling of specific proteins, ubiquitination and cell migration (Santolini et al., 2000;Cayouette and Raff, 2002). Classic studies of Drosophila demonstrated Numb's spatio-temporal segregation to one pole of the mitotic cell as the primary mechanism by which cell fate is determined in single organ precursors (Uemura et al., 1989). In mammals, Numb and Numbl are required for the self-renewal of neural progenitors to maintain their number during development; while in the other settings they promote a neuronal fate by neural progenitor specification (Petersen et al., 2002(Petersen et al., , 2004. Similarly, Numb and Numbl were required for the renewal of CPCs during cardiogenesis, suggesting a conserved role in progenitor maintenance. It is unlikely that disruption of the yolk sac or angiogenesis contributed to the restriction of cardiac growth because there was no discernable difference in the phenotype of embryos or yolk sacs at E8.5. Furthermore, the results from somatic mosaicism demonstrated that Numb/Numbl DKO CPCs were unable to proliferate in normal PA2 environment, suggesting a cell-autonomous role of Numb and Numbl. Curiously, the deletion of Numb and Numbl in CPCs appears to affect the growth of neighboring PA2 cells as well. This suggests that Numb and Numbl may also influence CPC renewal and expansion by regulating PA2 development in a non-cell autonomous manner. Numb and Numbl may affect CPC renewal and expansion via Notch, a conserved transmembrane receptor, given that Numb and Notch are mutually antagonistic (Schweisguth, 2004). In fact, Notch1 deficiency causes CPC expansion in the OT (Kwon et al., 2009). The expansion of CPCs is at least in part mediated by accumulation of the Wnt signaling effector β-catenin that are negatively regulated by membrane Notch (Kwon et al., 2011). Membrane Notch appears to require Numb and Numbl for the negative regulation of β-catenin (Kwon et al., 2011;Andersen et al., 2012), suggesting Numb and Numbl may be essential regulators of Notch and Wnt signaling during CPC development. With our current study, it will be necessary to re-examine the roles of Notch and Wnt signals at the level of renewing CPCs in the PA2.

Ex-vivo culture and PA2 co-culture
For explant culture, PA1s or PA2s were carefully dissected from embryos at E9.0-9.5. Absence of contamination from the adjacent outflow tract was confirmed by absence of Nkx2.5 + cells. The explants were cultured in standard serum free medium supplemented with ascorbic acid at 37°C in 5% CO 2 . For 3D cultures, the PA explants were cultured in Matrigel. For PA2 cell co-culture, PA2s were isolated from E9.0-9.5 Isl1 Cre ; Ai9 embryos and cultured in gelatin-coated plate with PA media (DMEM: F-12, 7.5% FBS, 1X penicillin-streptomycin, 1X Glutamax). RFP − cells from PA's with significant outgrowth were isolated and further passaged. The PA2-derived or control (embryonic heart) cells were plated at a density of 10,000-50,000 cells/cm 2 in multi-well plates and ES cell-derived CPCs were plated on top of the PA2 cells at a density of ∼10,000 cells/cm 2 in SFD medium.

Flow cytometry and time-lapse imaging
For flow cytometry, cells were dissociated and analyzed with Accuri C6 Flowcytometer (BD Biosciences, San Jose, CA) and FlowJo software (TreeStar, Ashland, OR). For intracellular-flow cytometry, cells were stained with indicated antibodies after dissociation as previously described (Uosaki et al., 2011). For FACS, dissociated cells were resuspended in PBS containing 0.1% FBS, 20 mM Hepes and 1 mM EDTA and sorted with FACSAria II (BD Biosciences) and SH800 sorter (Sony Biotechnology, Japan). Timelapse imaging was done with a BZ9000 All-in-One Fluorescence microscope (Keyence, Japan).

Ca2 + transient measurement and analysis
PA2 explants were incubated with 3 µM Fura-2 AM (Invitrogen, Molecular Probes, Carlsbad CA) for 20 min at 37°C. After washing and de-esterification for 20 min the explants were placed in an imaging chamber and electrical field stimulated at 1 Hz, 37°C, pH 7.4 with Ca2 + . The change in intracellular Ca2 + was measured with an inverted fluorescence microscope (TE2000, Nikon, Japan) and Myocam (IonOptix, Milton, MA) by Fura-2 AM fluorescence intensity ratio at 340 nm and 380 nm.

Statistical analyses
Differences between groups were examined for statistical significance using the paired Student's t test. A p-value <0.05 was regarded as significant. Error bars indicate standard error of the mean.
was conducted under the animal protocol (MO10M444) approved by the Animal Care and Use Committee at Johns Hopkins University.