c-kit is expressed in the developing and adult mouse heart

We first generated a knock-in mouse model, c-kitH2B-tdTomato/+, by gene targeting (Fig. 1a and Supplementary Fig. 1). In this animal, the H2B-tdTomato cassette was inserted into the c-kit start codon without deleting any genomic sequences, thereby expressing tdTomato under the control of the full complement of endogenous c-kit regulatory elements. Since tdTomato is fused to histone H2B gene24, its expression is localized to the nucleus.

(a) Diagram of the c-kitH2B-tdTomato/+knock-in allele. (bi) Sections of c-kitH2B-tdTomato/+hearts at embryonic days (E) 8.5, 9.5, 12.5 and 14.5 (be) and at postnatal (P) days 1, 30, 60 and 120 (fi). c2, e2, g2 and i2 are high-magnification images (without DAPI) of the areas outlined in c1, e1, g1 and i1, respectively. c-kitH2B-tdTomato cells are denoted by arrows. LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle; VS, ventricular septum. n=3 for each stage. Scale bar, 100m.

To confirm the fidelity of the c-kitH2B-tdTomato signal to the endogenous c-kit expression pattern, we performed whole-mount RNA in situ hybridization on the wild-type mice from embryonic day (E) 9.5 to E13.5. By comparing c-kitH2B-tdTomato signals to c-kit mRNA expression, we found that the signals overlapped in all known regions of c-kit expression25, 26, such as the pharyngeal arches, liver, umbilical cord and melanocytes (Supplementary Fig. 2ac). Furthermore, H2B-tdTomato expression was detected in other organs, including the lung, stomach, intestine and spleen (Supplementary Fig. 2e), as well as the neural tube and yolk sac during embryogenesis. This finding is consistent with previous reports of c-kit expression in these organs25, 26. Immunostaining of sectioned c-kitH2B-tdTomato/+ mouse tissues revealed that the c-kitH2B-tdTomato-positive cells co-localized with c-kit antibody in the liver, lung and melanocytes (Supplementary Fig. 3). Further support for the sensitivity and fidelity of this reporter is the observation that cells with low c-kit expression detected by antibody exhibited bright H2B-tdTomato fluorescence (Supplementary Fig. 3b,c).

Next, we examined the location of c-kit+ cells in the hearts of c-kitH2B-tdTomato/+mice (Fig. 1). Endocardial cells with nuclear tdTomato expression were observed as early as E8.5 and 9.5 (Fig. 1b,c). Starting from E12.5, cells with strong c-kitH2B-tdTomato expression were dispersed throughout the heart, with the highest density in the inner layers of the atrial and ventricular chambers at all embryonic stages tested (Fig. 1d,e). At postnatal day (P) 1, P30, P60 and P120, c-kitH2B-tdTomatoexpressing cells were consistently detected in all chambers of the heart (Fig. 1fi). The broad distribution of c-kitH2B-tdTomato-positive cells in the heart from embryonic stages to adulthood is inconsistent with previous studies reporting that c-kit+ cells represent a small population of CSCs in the mammalian heart7, 12, 13, 14, 15, 27.

In the initial characterization of cardiac resident c-kit+ cells in the adult rat, c-kit+ cells were shown to contain a mixed population of cells exhibiting early stages of myogenic differentiation as demonstrated by the active expression of the early cardiac transcription factors Nkx2.5, Gata4 and Mef2c in the nucleus and of sarcomeric proteins in the cytoplasm of these cells7, 15. To determine whether c-kitH2B-tdTomato-positive cells express the cardiac progenitor marker Nkx2.5, we crossed Nkx2.5H2B-GFP/+knock-in mice28 with c-kitH2B-tdTomato/+mice to obtain compound heterozygotes (c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+). H2BGFP expression in Nkx2.5H2B-GFP/+mice faithfully recapitulates the endogenous Nkx2.5 pattern28. We examined cardiac tissues throughout the embryonic (E9.518.5) and postnatal (P1120) stages (Supplementary Fig. 4). All histological sections from E9.5 to 13.5 hearts and more than 30 sections from E14.5 to P120 hearts were inspected (n=3 for each stage). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found (Supplementary Fig. 4b,dg), except at E12.5, wherein only 11 double-positive cells were detected in the ventricular septum (Supplementary Fig. 4c, ~0.007% of total Nkx2.5H2B-GFP-positive cells).

To determine whether any c-kit+ cells produce sarcomeric or myocardial proteins7, 15, we applied a cTnTH2B-GFP/+ knock-in mouse model with insertion of an H2BGFP cassette into the start codon of cTnT (Tnnt2; Supplementary Fig. 5a). On examining heart sections from c-kitH2B-tdTomato/+;cTnTH2B-GFP/+ compound heterozygous animals at embryonic and postnatal stages (E8.5P120), we did not detect any cells in which both markers were co-localized (Supplementary Fig. 5), with the exception of E13.5, where an average of 15 double-positive cells were found within the ventricular septum (Supplementary Fig. 5d, ~0.009% of total cTnTH2B-GFP-positive cells). These observations reveal that c-kit+ cells in c-kitH2B-tdTomato/+mice very rarely co-express either Nkx2.5 or cTnT in the embryonic heart and do not co-express these markers in foetal or adult hearts.

To further determine the identity of c-kit+ cells, we performed immunostaining with antibodies against the endothelial marker PECAM (CD31) and the smooth muscle marker, -SMA. Surprisingly, at all the stages examined (E8.5P120), c-kitH2B-tdTomato-positive cells were PECAM+(Fig. 2a-f) but -SMA (Fig. 2g,h). This finding suggests that cardiac c-kitH2B-tdTomato-positive cells are endothelial cells. Quantitative flow cytometric analysis of 4-month-old hearts demonstrated that ~43% PECAM+ cells in the ventricles were also c-kit+ (Supplementary Fig. 6). Thus, our results indicate that c-kitH2B-tdTomato-positive cells represent a subset of cardiac endothelial cells.

(a,b) At E8.5 and E9.5, c-kitH2B-tdTomato cells are endocardial (PECAM+). (cf) c-kitH2B-tdTomato cells express PECAM at E16.5 (c) and at P1120 (df). Arrows indicate PECAM+ and tdTomato+ double-positive cells. Arrowheads indicate PECAM+ and tdTomato cells. (g,h) Cardiac smooth muscle cells (-SMA+) are tdTomato at P120 (arrowheads). a2h2 are high-magnification images of the areas outlined in a1h1 (without DAPI), respectively. n=3 for each stage. Scale bar, 100m.

tdTomato is a bright fluorescent protein29, 30. We were concerned that the long stability of tdTomato could complicate the detection of transient c-kit expression. To confirm the identity of c-kit+ cells identified by c-kitH2B-tdTomato/+, we generated another reporter line, c-kitnlacZ-H2B-GFP/+, by inserting a LoxP-nlacZ-4XPolyA-LoxP-H2BGFP cassette into the c-kit start codon (Fig. 3a and Supplementary Fig. 7). H2BGFP is not detected in this line unless the nlacZ-4XPolyA stop cassette is removed by Cre-mediated recombination. We performed whole-mount X-gal staining on c-kitnlacZ-H2B-GFP/+ embryos and found that the c-kitnlacZ signal was not only reliably recapitulated by c-kit mRNA expression, but also consistent with the H2BtdTomato expression patterns in c-kitH2B-tdTomato/+mice (Supplementary Fig. 2). Furthermore, X-gal staining of whole-mount and sectioned hearts at E15.5P90 readily detected a broad distribution of c-kitnlacZ-positive cells throughout the heart (Fig. 3b,d,f,h, and j), including the endocardium (Fig. 3b,h), similar to the pattern observed in c-kitH2B-tdTomato/+mice. X-gal staining of compound heterozygous littermate hearts bearing an endothelial-specific Tie2-Cre allele (c-kitnlacZ-H2B-GFP/+;Tie2Cre) could not detect c-kitnlacZ-positive cells (Fig. 3c,e,g,i and k; less than 10 randomly distributed c-kitnlacZ-positive cells were found in the adult heart, representing ~0.0002% of total c-kit+ cells). Consistent with the endothelial nature of c-kit+ cells in the heart, c-kitH2B-GFP-positive cells generated by Tie2Cre excision were all co-stained with anti-PECAM antibody (Supplementary Fig. 8). Thus, the c-kitnlacZ-H2B-GFP/+ reporter line confirms the endothelial identity of cardiac c-kit+ cells.

(a) Diagram of the c-kitnlacZ-H2B-GFP/+reporter allele (a1). The c-kitH2B-GFP/+ allele is generated when the nlacZ cassette is removed by Cre excision (a2). (bk) X-gal staining of c-kitnlacZ-H2B-GFP/+ and c-kitnlacZ-H2B-GFP/+;Tie2Cre littermate hearts at E15.5 (b,c, sections) and at P190 (dk). Arrows indicate comparable regions to X-gal+ or X-gal staining. Arrowheads indicate rare X-gal+ cells on c-kitnlacZ-H2B-GFP/+;Tie2Cre hearts, suggesting that most c-kit+ cells lose the nlacZ gene because they are in the Tie2Cre lineage. f2k2 are high-magnification images of the areas outlined in f1k1, respectively. n=35 for each stage. Scale bar, 400m (black) and 200m (white).

To further address the issue of stability of both H2BtdTomato and nlacZ proteins, we analysed cardiac c-kit cells with the third reporter allele c-kitMerCreMer/+, in which an inducible MerCreMer cassette was inserted into the c-kit start codon (Fig. 4a and Supplementary Fig. 9). c-kitMerCreMer/+;ROSA26RtdTomato/+mice were subsequently generated by crossing with ROSA26RtdTomato/+ mice. In the absence of tamoxifen treatment, no tdTomato-expressing cells were detected in the adult hearts. To confirm whether c-kit is actively expressed in the postnatal heart, we injected tamoxifen at P30, P60 or P90 for 3 consecutive days (days 1, 2 and 3), and immediately collected cardiac tissues for analysis at day 4 (P3034, P6064) or 14 (P90104). This treatment consistently resulted in tdTomato labelling of a large number of cells in the heart (Fig. 4b,d,e) that also expressed PECAM (Fig. 4c). This result further confirms that cardiac c-kit+ cells are endothelial (Figs 2 and 3), and supports the previous observation that cardiac c-kit+ cell progeny are endothelial19.

(a) Diagram of the c-kitMerCreMer/+ allele. c-kitMerCreMer/+ animals were crossed to the ROSA26RtdTomato reporter line to obtain c-kitMerCreMer/+;ROSA26RtdTomato/+. (be) Cre activity was transiently induced in c-kitMerCreMer/+;ROSA26RtdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 13. Hearts were harvested on days 4 and 14. Many tdTomato+ cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato+ cells were PECAM+ (c2, arrows, P3034). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. (f) Diagram of the cTnTnlacZ-H2B-GFP/+allele and lineage tracing using c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). (g) cTnTH2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100m (white).

c-kitH2B-tdTomato/+, c-kitnlacZ-H2B-GFP/+ and c-kitMerCreMer/+ animals are heterozygous null for c-kit (c-kit+/). Haploinsufficiency of c-kit could affect c-kit regulation in vivo20, 31, 32, 33, possibly leading to ectopic cardiac expression. To determine whether ectopic c-kit expression occurs in the reporter mouse hearts, we performed immunostaining at embryonic (E11.515.5) and postnatal stages (P160) using c-kit antibody on mice of four different genotypes: wild type, c-kitH2B-tdTomato/+ (c-kit+/), c-kitH2B-tdTomato/MerCreMer(c-kit/) and c-kitMerCreMer/MerCreMer(c-kit/). Using c-kit antibody, we frequently detected cells in wild-type hearts that were dually labelled with c-kit and PECAM (Supplementary Fig. 10a4,d4,g2 and Supplementary Fig. 11a,f,h,i). In c-kitH2B-tdTomato/+ animals, c-kit antibody immunoreactivity co-localized with c-kitH2B-tdTomato (Supplementary Fig. 10b2, e2,h2 and Supplementary Fig. 11b,c), although the immunofluorescence was decreased compared with that in wild-type animals. Reduced c-kit immunoreactivity in c-kitH2B-tdTomato/+ tissues is consistent with the c-kit+/ genetic background (theoretically 50% c-kit protein reduction in c-kit+/). Importantly, c-kit antibody staining was completely undetectable in c-kit/mutant hearts or lungs, even with Tyramide Signal Amplification (TSA) amplification (Supplementary Figs 10c,f and 11d,e), demonstrating the specificity of the antibody staining. Therefore, immunostaining with c-kit antibody also reveals that cardiac c-kit+ cells are endothelial and indicates that no ectopic cardiac c-kit expression occurs in the new knock-in mouse models employed.

To further determine the myogenic potential of c-kit+ cells during heart formation, we applied cTnTnlacZ-H2B-GFP/+ cardiomyocyte-specific reporter mice with the LoxP-nlacZ-4XPolyA-LoxP-H2B-GFP cassette targeted into cTnT start codon. cTnTH2B-GFP expression is detected in cardiomyocytes when Cre is expressed in the myocardium or myogenic precursor cells (Fig. 4f). We crossed c-kitMerCreMer/+ mice with cTnTnlacZ-H2B-GFP/+mice and injected tamoxifen in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animals. After two doses of tamoxifen administration (days 1 and 2) to pregnant mice (E11.5 embryos) or four doses (days 1, 2, 3 and 5) to P30, P60 and P90 mice, we collected hearts for analysis at E13.5 or at P37, P67 and P97, respectively. All cardiac sections were assessed for cTnTH2B-GFP-positive cells. On average, approximately 50, 324, 156 and 66 cells were found in hearts (n=3 for each group) at E13.5, P37, P67 and P97, respectively (Fig. 4g), representing <0.04% of cardiomyocytes at corresponding stages (<0.007% after P90). This finding demonstrates that the myogenic potential of c-kit+ cells, if any, is extremely low in both embryonic and postnatal hearts.

Previous studies have reported that within 4 weeks of myocardial infarction in adult mouse hearts, the number of c-kit/Nkx2.5 double-positive myogenic precursors significantly increased in the injured region, and some of these myogenic precursors transformed into proliferative cardiomyocytes7, 15. To directly investigate the differentiation potential of cardiac c-kit+ cells post myocardial infarction, we ligated the left anterior descending (LAD) coronary artery of c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+ mice (25 months old, n=12, Fig. 5a,b). Examination of cardiac sections at 1, 3, 7, 21, 30 and 60 days post-surgery (dps) revealed many c-kitH2B-tdTomato-positive cells in the infarcted region (Fig. 5cf). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found in the injured area at any stage tested (Fig. 5c1f1). To further determine the cell identity of these c-kit+ cells, we performed LAD ligation on Tie2Cre;c-kitnlacZ-H2B-GFP/+ mice (24 months old, n=3). c-kitH2B-GFP-positive cells were readily detected in the infarcted region, demonstrating that they retained their endothelial nature after injury (Fig. 6a).

(a) Diagram of LAD ligation. (b) Masson trichrome staining shows the infarcted region of a c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+heart at 60 days post-surgery (dps). b1 and b2 are high-magnification images of the numbered outlined areas in b. (cf) No c-kitH2B-tdTomato/Nkx2.5H2B-GFP double-positive cells were found in the infarcted regions at 3 (c), 21 (d), 30 (e) and 60dps (f). c1/c2, d1/d2, e1/e2, and f1/f2 are high-magnification images of the numbered outlined areas in c, d, e and f, respectively. Scale bar, 500m (black) and 50m (white).

(a) c-kitH2B-GFP-positive cells were present in the infarcted region of Tie2Cre;c-kitnlacZ-H2B-GFP/+ hearts at 30dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. (b) Masson trichrome staining of cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ hearts at 60dps shows the infarcted region. (c) Adjacent section of b. ROSA26RtdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kitH2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. (d) Masson trichrome staining of c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts at 60dps. (e) Adjacent section of d shows a few cTnTH2B-GFP cells (<20) that were found in the infarcted zone (e1, arrowhead). cTnTH2B-GFP cells were also present in a remote, uninjured region (e2, arrowhead). Scale bar, 100m.

A recent study reported that a subpopulation of endothelial cells yields progeny with CSC characteristics in the adult mouse heart34. This subpopulation purportedly arises from endothelialmesenchymal transition and gives rise to cardiomyocytes that contribute to heart renewal34. To determine whether c-kit+ endothelial cells produce CSCs that further differentiate into cardiomyocytes following cardiac injury, we performed LAD ligation on cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ mice (24 months old, n=4, Fig. 6b). cTnTMerCreMer/+ mediates specific and effective myocardial recombination after tamoxifen induction35. If c-kitnlacZ-H2B-GFP/+ cells become cardiomyocytes and if c-kit expression is maintained in these cells, then c-kitH2B-GFP-positive cells would be detected. However, after tamoxifen was injected at 37dps and 3135dps (three tamoxifen treatments for each period), we detected no c-kitH2B-GFP-positive cells in the infarcted region (Fig. 6c), although myocardial recombination was widely detected in and adjacent to the infarcted region (as revealed by ROSA26RtdTomato staining, Fig. 6c). Furthermore, examination of adult c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ mice after LAD ligation (35 months old, n=3, Fig. 6d) revealed <20 cTnTH2B-GFP-positive cells per heart (~0.002% of total myocardial cells) throughout the injured region (Fig. 6e). cTnTH2B-GFP-positive cells could also be detected in remote uninjured regions (~30 cells, ~0.003% of total myocardial cells, Fig. 6e), suggesting that the cTnTH2B-GFP-positive cells found in the injured region are likely not a response to cardiac injury. These cardiac injury mouse models revealed that the myocardial potential of c-kit+ endothelial cells, if any, is extremely low. However, these data do not preclude the possibility that c-kit cardiac endothelial cells may have the potential for endothelialmesenchymal transition and myocardial differentiation.

In the lineage tracing experiments used to determine the myocardial potential of c-kit+ cells during development and after cardiac injury in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animal models, very small number of cTnTH2B-GFP-positive cells was detected (Fig. 4g, ~66156 cells; and Fig. 6e, ~20 cells). In all cases, the number was extremely low when compared with the total number of c-kitH2B-tdTomato-positive cells (<0.005%) or myocardial cells (<0.015%) in whole hearts. The origin of these rare cells is unknown. These cells may be derived from uncommitted cells originally expressing c-kit, or they could be cardiomyocytes that express both c-kit and cTnT due to a rare stochastic event. To explore these possibilities, we examined cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ adult mouse hearts (24 months old, uninjured) after tamoxifen injection for 2 consecutive days (days 1 and 2). At days 3, 7 and 30, we detected ~2030 c-kitH2B-GFP-positive cells per heart after examining all the heart sections (n=3, Supplementary Fig. 12). This result suggests that a very small number of resident c-kit cells are cardiomyocytes (~0.005% of total c-kit+ cells and ~0.002% of total myocardial cells in the heart). Notably, the number of c-kitH2B-GFP-positive cells detected in cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ hearts (~2030, Supplementary Fig. 12) is less than the number of cTnTH2B-GFP-positive cells in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts (~66156, Fig. 4g3). This is probably due to much higher levels of cTnT expression than c-kit expression and/or to differential sensitivity of the reporters to Cre-mediated recombination.

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Resident c-kit+ cells in the heart are not cardiac stem ...

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