During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.https://doi.org/10.7554/eLife.30668.001
We all start life as a single cell, which – over the course of nine months – multiplies to generate the billions of cells that can be found in a newborn. As an embryo develops, the cells need to achieve two major tasks: they need to diversify into different types of cells, such as blood cells or muscle cells, and they need to organize themselves in space to form tissues and organs.
The heart of an embryo, for example, first forms a simple structure called the heart tube that can pump blood and later develops into the four chambers that we see in adults. The tube is made up of cells from two different origins, known as the first and second heart fields. Unlike other organs, the heart has to start beating while it is still developing, and until now, it was unclear how the heart manages this difficult task.
Here, Ivanovich et al. studied mouse embryos grown outside the womb by using a combination of advanced microscopy and genetic labeling to track how single cells turn into beating cells and move while the heart forms. The results showed that specializing into beating cells and forming the heart tube shape happened during alternating phases. The first heart-field cells turned into beating cells and began to contract at an early stage before the heart tube was formed. Next, the cells of the second heart field did not instantly develop into beating cells, but instead, helped the first heart-field cells to acquire the shape of a heart tube. Once this was completed, the second heart-field cells started to specialize into beating cells and created the additional parts of the more complex adult heart.
This research shows that the second heart field plays an active role in helping the heart tube form. The alternating phases of cell specialization and tissue formation allow the heart to become active whilst it is still developing. A better insight into how the heart forms may help us to create new treatments for various genetic heart conditions. The methods used here could also help to study how cells build other organs.https://doi.org/10.7554/eLife.30668.002
The heart is the first organ to form and function during embryonic development. At embryonic day (E) 7.5, cardiac precursors in the splanchnic mesoderm (mesoderm apposed to the endoderm) differentiate into cardiomyocytes by assembling the contractile sarcomere machinery (Tyser et al., 2016) and form a bilateral structure known as the cardiac crescent (cc) in the mouse. Concomitant with foregut invagination, the cc swings inwards to become placed underneath the developing head folds. By a complex morphogenetic process, the cc subsequently transforms into an early heart tube (HT) initially opened dorsally, which by E8.25 has transformed into a closed and beating linear HT, also known as the primitive HT (Evans et al., 2010; Kelly et al., 2014).
The cc and early HT mainly give rise to the left ventricle (Zaffran et al., 2004). The right ventricle (RV), the outflow tract and most of the atria derive instead from cardiac progenitors located dorso-medially to the cc in the splanchnic mesoderm, that are progressively recruited at the poles of the HT at subsequent developmental stages (Cai et al., 2003; Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001; Galli et al., 2008). These findings led to the concept that cardiac mesodermal progenitors contain two populations of cells: the first heart field (FHF) precursors, recruited early in development to form the initial HT and mostly containing the LV primordium, and the second heart field (SHF), recruited later and elongating the HT (Buckingham et al., 2005).
Clonal analysis (Devine et al., 2014; Lescroart et al., 2014; Meilhac et al., 2004a) supports the idea that FHF and SHF precursors are two independent developmental fields with dedicated molecular pathways. Clonal analysis also showed that the SHF shares a common origin with the skeletal muscles of the head and neck within the pharyngeal mesoderm (Lescroart et al., 2010; Lescroart et al., 2015), further supporting differences between FHF and SHF populations. However, the existence of a common precursor between FHF and SHF was also reported in the early mouse embryo (Meilhac et al., 2004a) and other views suggest that the heart would form by a continuous differentiation process from a single population of cardiac precursors and only timing of recruitment would distinguish cells of the FHF and SHF (Abu-Issa et al., 2004; Moorman et al., 2007). In support of the latter, the typical marker of the SHF, Islet1 (Cai et al., 2003), is also transiently expressed in FHF precursors and must therefore be considered as widespread cardiac progenitor marker instead (Cai et al., 2003; Prall et al., 2007; Yuan and Schoenwolf, 2000). Whether the recruitment of cardiomyocytes from progenitors is a continuous process and how this coordinates with morphogenesis, however, has not been directly studied. This is partly because the spatial arrangement of progenitors and differentiated cardiomyocytes has so far been analyzed on fixed embryos (Cai et al., 2003; Später et al., 2013) and the expression dynamics of genes reporting differentiation together with cell movements during HT morphogenesis have not been captured so far (Abu-Issa, 2014).
Here, we report the live-imaging and 3D+t cell tracking of HT formation in whole mouse embryos. Using this method, in conjunction with an Nkx2.5eGFP reporter line, which provides high level of GFP in differentiated cardiomyocytes, we studied the dynamics of cardiac field differentiation. During an initial phase, FHF cardiac precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. During a second phase, no differentiation events are detected and extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. Finally, using an Isl1-Cre lineage-tracing assay combined with live-imaging, we show that during a third phase, cardiac precursor differentiation resumes and contributes not only to the known SHF-derived regions but also to the dorsal aspect of the HT. These results show essential properties of FHF and SHF contribution to heart development and reveal tissue-level coordination between alternating phases of differentiation and morphogenesis during HT formation.
To assess how the initial cardiogenic region transforms into a HT and differentiates, we first analyzed Nkx2.5cre/+; Rosa26Rtdtomato+/-embryos, in which both cardiac precursors and cardiomyocytes are labeled (Stanley et al., 2002). Before cc differentiation, at early head fold stage (EHF, ~E7.5), the cardiogenic region is visualized as a flat horse shoe-shaped tdtomato+ mesodermal layer at the rostral border of the embryo (Figure 1A,A’, and Video 1). Figure 1—figure supplement 1 shows the criteria for embryo staging (Downs and Davies, 1993; Lawson and Wilson, 2016). In the Nkx2.5cre/+; Rosa26Rtdtomato+/- embryos, tdtomato labeling is also observed in the endocardium and endothelial cells (Stanley et al., 2002) but not in the endoderm (Figure 1—figure supplement 2A,A’). We next studied the distribution of Cardiac troponin T (cTnnT), one of the first evident sarcomeric proteins to appear in the cardiac crescent (Tyser et al., 2016). At EHF stage (Figure 1B), while most embryos are negative for cTnnT expression, some embryos show weak cTnnT localization in subsets of cells (Figure 1—figure supplement 3A,A’). At a subsequent embryonic stage (~E7.7), cTnnT signal reveals the cc, which is folding inwards. During folding, the cTnnT signal increases. cTnnT+ cells are initially columnar epithelial cells and show apical localization of the tight junction component zona-occluden-1 (ZO-1) (Figure 1—figure supplement 3B,B’). During differentiation, cardiac precursors switch to a rounded shape (Linask et al., 1997) (Figure 1C,D) and separate from the endoderm, while maintaining a basal lamina at the endocardial side (inset in Figure 1D and Figure 2D). Morphogenetic changes starting at ~E8 subsequently lead to the formation of a hemi-tube whose major axis is transversal to the embryo A-P axis. We will refer to this stage as transversal HT (Figure 1E). Later, the tube adopts a more spherical shape, very similar to the linear HT but still open dorsally. We will refer to this stage as open HT (Figure 1F). The HT eventually closes dorsally (Figure 1G, red arrows in Figure 1G’’) and a prominent arterial pole (prospective RV) (Zaffran et al., 2004) becomes visible, completing linear HT formation by ~E8.25 (yellow arrows in Figure 1G’’, Figure 1H, see also Video 2).
To assess the overall growth of the forming HT, we measured cTnnT+ tissue volume in segmented z-stacks, at the stages described above (Figure 1I and Figure 1—source data 1). During the first phase of cardiomyocyte differentiation, the cTnnT signal expands resulting in a cardiac crescent rapidly doubling in volume (Figure 1C’, D’, I). During the subsequent phase of morphogenesis, from cc to open HT stage, growth is less pronounced despite extensive morphological changes (Figure 1E’, F’, I). The volume of the HT appears to increase again upon addition of the RV primordium to the arterial pole and dorsal HT closure (Figure 1G’, I). HT growth likely reflects an increase in cell number occurring during the formation of the heart tube. Cardiomyocytes are proliferative at this stage (de Boer et al., 2012), and we can indeed observe mitotic figures in the HT (Figure 1J). From this analysis, however, it is unclear how much of the growth observed is due to cardiomyocyte proliferation versus addition of new cardiomyocytes from cardiac progenitor cells located in the splanchnic mesoderm.
To visualize the boundary where cardiac progenitors abut differentiating cardiomyocytes during HT morphogenesis, we immunostained Nkx2.5cre/+Rosa26Rtdtomato+/- embryos at the transversal-HT stage with the differentiation marker cTnnT and acquired whole-mount images (Figure 2A). Cre activation of tdtomato was detected in both the FHF and SHF, as well as in the endoderm and endocardium (see optical sections in Figure 2A’, A’’, F,F’ and Figure 2—figure supplement 1A) (Stanley et al., 2002). Cardiomyocytes are separated from the endoderm by the endocardium. In contrast, undifferentiated cardiac precursors lie medio-dorsally in direct contact with the endoderm in areas where endocardial cells are not detected (Figure 2A’, A’’). This is confirmed by the absence of the endothelial marker CD31 (Figure 2—figure supplement 2). We then rendered in 3D the Nkx2.5cre-labeled lineages, including both FHF and SHF, and the cTnnT +tissues, including only the FHF/cc, which allowed visualizing the boundary between SHF and cc at the tissue level (Figure 2A,B,B’, Figure 2—figure supplement 1, Video 3 and see Materials and methods – non-cardiac tdtomato signal was removed manually).
To identify the changes associated to cardiomyocyte differentiation at the cellular level, we labeled single cells with membrane-GFP (see Materials and methods). cTnnT- progenitors have an epithelial-like columnar cell shape, while the differentiated cTnnT+ cardiomyocytes are rounder and have lost the columnar epithelial organization (Figure 2C–E and Figure 2—source data 1). This is reminiscent of the cell shape transition observed in the distal outflow tract (OFT) at later stages of heart development, when SHF progenitor-to-cardiomyocyte differentiation takes place (Francou et al., 2014; Ramsbottom et al., 2014; Sinha et al., 2012). Interestingly, some cells at the boundary zone exhibit weak cTnnT localization and yet show columnar shapes typical of mesodermal cardiac precursors (Figure 2F–F’’’’ and red arrows in Figure 2F’’’, F’’’’). Unlike differentiated cardiomyocytes, these cells do not show rounded shapes, and therefore they may represent a transient state between progenitors and differentiated cardiomyocytes; however, the nature of such state cannot be addressed by static analysis. Differentiation of cardiac progenitors is thus accompanied by changes in cell shape and detachment from the endoderm.
We next assessed the expression pattern of the Nkx2.5eGFP enhancer reporter line, in which GFP expression is restricted to cardiomyocytes (Lien et al., 1999; Prall et al., 2007; Wu et al., 2006). To characterize this reporter line in detail, we immunostained Nkx2.5eGFP embryos against cTnnT, ( )(Figure 3A,B and Figure 3—figure supplement 1A,B) and compared the relative intensities of cTnnT and GFP in manually segmented single cells (Figure 3C,D Figure 3—figure supplement 2A and Figure 3—source data 1). We found that the GFP level varied linearly with cTnnT level (Figure 3D), although considerable variability of GPF levels was observed within each cTnnT+ and cTnnT- cell populations. Scoring of a large number of cells allowed to reproducibly identify the top 50% GFP-expressing cells as positive for cTnnT+ (Figure 3—source data 1). Genetic tracing experiments using the Nkx2.5cre/+;Rosa26Rtdtomato+/- line, instead show strong tdtomato level in both the FHF and SHF (Figure 3—figure supplement 3A).
We next characterized the boundary between cardiomyocytes and cardiac precursors in transversal HT stage embryos of the Nkx2.5eGFP reporter line. We measured mean fluorescent intensity in manually segmented cells at the boundary zone and found that GFP level and cTnnT signals correlate at the individual-cell level (Figure 3B’, E, Figure 3—figure supplement 2B,C and Figure 3—source data 1). Altogether, these results indicate that the Nkx2.5-eGFP reporter is suitable for tracking cardiomyocytes in live-imaging and reliably identifies the top 50% GFP-expressing cells as cTnnT-positive.
We next established a live-imaging method to dynamically characterize the formation of the HT in the mouse embryo (Figure 4A) (Chen et al., 2014). We adapted a previously reported culture system (Nonaka, 2009; Nonaka et al., 2002) in which the whole mouse embryo is immobilized by inserting the extraembryonic region in a holder (Figure 4C). After culture, embryos showed normal morphology, their hearts were beating and circulation was initiated (Figure 4D,E and Video 4). This culture system in combination with two-photon microscopy enabled the generation of high-resolution 3D+t videos (Figure 4B and see Materials and methods). Maximum culture time and imaging achieved was 24 hr (Figure 4—figure supplement 1A, n = 3). However, typical acquisition times varied in most cases between 3 and 13 hr. The rate of acquisition varied in most cases between 4 and 9 min, with some exceptions depending on the specific aim of the recording (see video legends).
Imaging Mesp1cre/+; Rosa26Rtdtomato+/- embryos allowed tracking the anterior mesoderm (Saga et al., 1996; Saga et al., 1999), including cardiac lineages, from cc stage up to HT stage (Figure 4F,F’ and Videos 5–6). The time-lapse analysis provided insight on the formation of the endocardial lumen (Figure 4F’, F’’). The endocardium is initially observed as a bilayer of cells and eventually splits into dorsal and ventral layers, which move apart from each other allowing the lumen to become visible between the layers (see Videos 6 and 7 for confocal views and Video 8 for a bright field view). Thin cytoplasmic bridges between the endocardial layers persist and extend as the endocardial layers separate from each other (see white arrows in Figure 4F’ and Video 6). Heartbeat becomes detectable around this stage and circulation in the embryo is then initiated.
Imaging Nkx2.5cre/+; Rosa26RmT/mG+/- embryos, in which Cre-recombined cells activate membrane-bound GFP, we could track cells during differentiation, as they transit from a columnar to a round shape and start contracting (Figure 4G–G’’, Video 7 and bright-field Video 8). We found that the endocardial lumen started to appear while cc cells still remained columnar (compare time points 2 hr 13 m and 3 hr 48 m in Figure 4G’). Cell rounding is therefore unlikely to initiate the formation of the cardiovascular lumen.
Interestingly, this analysis also showed that the transformation of the cc into the HT involves the antero-medial displacement of the splanchnic mesoderm (white arrows in Figure 4F,F’). This displacement promotes the folding of the cardiac crescent into a hemi-tube by bringing closer the future borders between the HT and the mesocardium (see yellow lines in Figure 4F’ indicating the distance between the borders at different times). These borders coincide with the frontiers between cardiomyocytes and undifferentiated splanchnic mesoderm (see details in Videos 7, 17 and Figure 7A’, A’’). Interestingly, the movement of the splanchnic mesoderm is not coherent with the endoderm but a relative displacement is detected between the two layers (Video 9). Estimation of splanchnic mesoderm displacement speed toward the midline from time-lapse analyses (Figure 4F’, and Video 6) indicates a range of average speeds from 12 to 20 µm/hr (15.8 ± 2.4 µm/hr, mean ±SD, n = 3, Figure 4—figure supplement 2 and Figure 4—source data 1). These measurements estimate that midline convergence of the splanchnic mesoderm takes approximately 5–7 hr from the late cc stage until the open HT stage (Figure 4—figure supplement 2).
Our results show the feasibility of live time-lapse analysis of mouse HT formation and reveal that splanchnic mesoderm displacement, at least in part by sliding over the endoderm, is an essential aspect of HT morphogenesis.
Next, to specifically track cardiac differentiation, we used the Nkx2.5eGFP live reporter. We first studied the general activation pattern of this reporter in live analysis. At E7/bud stage, a faint and scattered GFP signal is detected in proximity to the yolk sack, at the anterior border of the embryo (Figure 5—figure supplement 1A). At neural plate stage, just prior to the ventral folding of the embryo, the GFP signal remains weak but spreads to delineate a crescent in the anterior region of the embryo (Figure 5—figure supplement 1A and Video 10). During about 5–6 hr starting at the EHF stage, the GFP signal increases in intensity, which correlates with the previous observations on cTnnT activation (Figure 4H, Figure 1C and Video 11). From transversal to open HT (5–7 hr) and from the open HT to HT (2–3 hr) the GFP signal remains stable (Figure 5—figure supplement 1B,B’, C,C’ and Video 12). We conclude that an increase in GFP level in Nkx2.5eGFP embryos reports cardiomyocyte differentiation. In addition, these results reveal the timing of the main phases of linear HT development; cc differentiation, formation of the open HT and dorsal closure (Figure 4I).
We next sought to track the trajectories and differentiation of individual cardiac precursors within the entire cardiogenic region by 3D+t live imaging. To this end, we used the Polr2aCreERT2 (RERT) allele (Guerra et al., 2003), which provides ubiquitous tamoxifen-inducible Cre activity in combination with a Rosa26Rtdtomato reporter. We then titrated the tamoxifen dose for a labeling density that would allow single cell tracking during prolonged time-lapse analysis and combined this with the Nkx2.5eGFP reporter (see materials and methods). Typically, for each video, we acquired z-slices every 3–5 μm achieving a total z-depth of 200 μm – with some variations depending on the stage considered- and manually tracked in 3D+t for several hours an initial population of ~50 to ~100 cells per video, which represents around 5–10% of the total number of cells present in the cc (de Boer et al., 2012).
We first tracked cells of the cardiac forming region starting at EHF stage – when cardiac precursors are undifferentiated – up to stages in which cardiomyocytes have differentiated in the cc but the transversal HT stage has not been reached yet (Figure 5A and Figure 5—source data 1). At the onset of cardiac differentiation, the cardiac crescent swings ventrally concomitant with foregut pocket formation. During this movement, we found that the relative positions of the cardiac progenitors are maintained from the initial stage through the differentiated cc (Video 13). Relative cell positions therefore remain mostly coherent as the embryonic tissues undergo this initial global movement. Differentiation events are detected in some of the tracked cells by cell shape change from columnar to rounded and by the increase in GFP signal (see example in Figure 5B,D and Figure 5—source data 1). In contrast, other tracked cells located in the splanchnic mesoderm remain in contact with the endoderm, retain a columnar shape and show low GFP level throughout the videos (Figure 5C). These cells are likely to be SHF cardiac progenitors and not endocardial cells, which are not present in this area (Figure 2—figure supplement 2A,B,B’). Endocardial cells are instead present in the cardiac crescent and have typical elongated spindle-like shapes (Figure 4F and Figure 5—figure supplement 2A).
Next, in order to establish a fate map of the cardiac forming region at the EHF stage, we tracked back in time the population of cells that showed high GFP intensity (top 50%) at the end of the video (Figure 5F,H and Figure 5—source data 1). According to our previous analysis (Figure 3D), these cells can reliably be assigned to cardiomyocytes. The initial location of this cell population fated to become cc cardiomyocytes delineates a crescent-shaped domain at EHF stage (Figure 5H). Cells retaining lower GFP intensity level throughout the video initially localize posteriorly and medially to this crescent. Most of the cells that have high GFP levels at the final time point show low GFP levels at the initial time point and increase their GFP level over time (Figure 5E–G and Figure 5—source data 1). These results suggest that cardiomyocytes of the cc differentiate during 5–6 hr starting at the EHF stage, which is consistent with the onset of detectable cTnnT at that stage (Figure 1B and Figure 1—figure supplement 3).
Cells in the cardiac mesoderm do divide during the observation time, so we identified cell division events and tracked the descendant cells. 43% of the tracked cells underwent one division during the 4–5 hr videos. To determine whether cell fate (differentiation versus progenitor) is allocated in the cardiogenic mesoderm at the EHF stage, we tracked GFP levels in dividing cells and their descendants. We found that most sister cell pairs show matched high or low GFP intensity levels at the end of the observation period (38 out of 39, Figure 5I, Figure 5—figure supplement 3A–D and Figure 5—source data 1). This observation suggests that commitment of cardiac precursors to differentiation is already established by the EHF stage and largely transmitted by lineage.
We next studied cardiac differentiation dynamics during subsequent stages when the cc transforms into the HT by extensive morphogenesis. To do so, we tracked cells located in the splanchnic mesoderm and cc in Nkx2.5-eGFP embryos at successive periods of around 3 hr covering the 5–7 hr during which the transversal HT transforms into the open HT (Figure 6A,B,E, Video 14 and Figure 4—source data 1). This analysis revealed that the trajectories of cc cells move apart from each other over time as the tissue expands during the transition from the transverse HT to the more spherical open HT. The HT starts to beat during the observation period, especially at the later stages, and therefore, in some cases, cardiomyocyte cell shape appears distorted in single optical sections (Figure 6F); however, the GFP level could be determined. As mentioned above, antero-medial movement of the splanchnic mesoderm can be observed concomitant with the transformation of the transversal HT into the open HT (visible also in Videos 5 and 12). We found that cells with high GFP level at the initial time points –differentiated cardiomyocytes – retain rather stable GFP levels (green tracks in Figure 6C,D,G and Figure 6—source data 1). In addition, all cells that initially showed low GFP levels did not increase GFP intensity during time-lapse; thus new events of cardiac differentiation were not detectable by this approach (red tracks in Figure 6C,D). Tracking cells for longer periods of time, covering from cc differentiation continuously up to the open HT stage, confirmed that cc differentiation is followed by a period of time in which no differentiation events occur (Figure 6—figure supplement 1, n = 19 cells tracked in one embryo).
To confirm the absence of detectable cardiac differentiation events during this period, we next focused on the live analysis of cells located at the boundary between cardiomyocytes and undifferentiated splanchnic mesoderm. As expected, we observed GFP-low cells located adjacent to GFP-high cells in the boundary zone (Figure 6H and Video 14). Those cells retain stable GFP levels throughout the tracking time and did not increase their GFP level (Figure 6I, boundary imaged 20 times in different locations and in six independent embryos). Importantly, they retain a columnar shape typical of weak cTnnT+ and cTnnT- cells located at the boundary zone (see Figure 2F’’ and Figure 3B’). Although these cells migrate antero-medially relative to the underlying more static endodermal cells (see endodermal cell highlighted by the red arrow from t = 69 m in Video 9), they do not contribute to the HT during the observation period. We confirmed this observation in longer time-lapse videos spanning 7 hr that covered the whole transition from transversal to open HT stage. Again, progenitors strictly respected the boundary with the HT throughout the entire time-lapse video (Figure 7A–A’’’ and Video 15, boundary imaged five times in different locations and in two independent embryos). All together, these data suggest that during the transformation of the cc into the dorsally open HT no cardiomyocytes are added to the HT from the SHF. These observations suggest two distinct phases of early HT formation: a first phase of differentiation of the FHF into the cc, lasting around 5 hr, and a second phase of HT morphogenesis in which the SHF progenitors remain undifferentiated, lasting around 5–7 hr. During this second phase, extensive remodeling of the cardiac crescent is concomitant with the antero-medial splanchnic mesoderm displacement.
The LIM domain transcription factor Islet1 (Isl1) is a cardiac progenitor marker. Its expression is transient in the precursors of the cc, while it remains expressed in SHF progenitors for an extended period (Brade et al., 2007; Prall et al., 2007; Yuan and Schoenwolf, 2000). Cells of the Isl1-expressing lineage detected with Cre reporters therefore contribute only scarcely to the cc, while extensively to the SHF and its derivatives (Cai et al., 2003; Ma et al., 2008). To test these observations in live imaging, we combined Nkx2.5eGFP with tracing of the Isl1 cell lineage using the Isl1cre driver and the Rosa26Rtdtomato reporter. We found that tdtomato labeling is first detectable in scarce isolated cells of the GFP+ cc during the period when the cc swings ventrally and differentiates (from t = 2 hr 36 m to t = 4 hr in Figure 8A,B). In contrast, a dense tdtomato labeling appears in the GFP-low cells of the splanchnic mesoderm as the cardiac crescent fully differentiates (from t = 2h24 m to t = 4 hr in Figure 8A and C and Video 16). tdtomato is as well detected in the endoderm and endocardium (not shown). Consistently with previous reports (Cai et al., 2003), Isl1cre-induced recombination detected in live analysis is thus low in the cc and complete in the SHF.
Once the cc is formed, if cells of the SHF would continuously differentiate, then regions of the forming heart tube contributed by the SHF precursors should appear densely co-labeled with both GFP and tdtomato. Live imaging shows instead that tdtomato+ cells establish a boundary with GFP+ cells, confirming no signs of differentiation of SHF precursors during the observation period (Figure 8A,C and Video 17). The live imaging, however, did not allow to unambiguously identify all cells located deep inside the live tissue at the final stages recorded. The arterial pole in particular is located deep in the embryo. It is therefore challenging to accurately track cardiac differentiation by the increase of GFP levels there (Figure 8—figure supplement 1A–A’’, from around 200 μm depth, see next section). To overcome these limitations, we fixed and immunostained embryos against cTnnT after completion of the live-imaging experiments, and imaged them by 3D confocal microscopy. No solid domains containing double-labeled cells were detected, indicating that progenitors located in the SHF did not undergo differentiation in the boundary zone from cc to open HT stage (Figure 8D). These results are consistent with the single-cell tracking analysis and confirm that the SHF does not differentiate during linear HT morphogenesis.
We next wanted to determine when cardiac progenitors located in the SHF start to differentiate. In order to address the timing of SHF contribution to the arterial pole, we next fixed and optically cleared Nkx2.5eGFP; Ils1cre/+; Rosa26Rtdtomato+/- embryos at different stages from cc up to heart looping (n = 10) and assessed the appearance of GFP and tdtomato double-positive domains in the HT. In agreement with our previous observations, we found that SHF cells do not differentiate up to the open HT stage, when the dorsal seam of the heart is still open. In contrast, massive appearance of solid domains of double positive cells is observed subsequently in the fully closed HT, reinforcing our previous interpretation (Figure 9A,B and Videos 18,19). At this stage, the primordium of the RV has been added at the arterial pole (Zaffran et al., 2004; Laugwitz et al., 2005; Moretti et al., 2006) and is fully composed of double-positive cells. The dorsal seam of the HT is also densely populated by double-positive cells, indicating a contribution of precursors from the splanchnic mesoderm to the cardiomyocyte population that finalizes the dorsal closure of the linear HT.
In order to capture the initiation of SHF contribution to the HT by live imaging, we first focused on the venous pole, as this region is more directly exposed than the rest of the HT and therefore more suitable for live-imaging. In videos that captured the transition from the open HT to the closed linear HT, some cells at the border between the FHF and SHF maintain low GFP levels during the first part of the recording and upregulate GFP to the level of the cardiomyocyte population as the HT forms (Figure 8—figure supplement 2). We next aimed to live-track the activation of SHF differentiation at the arterial pole. Because of the imaging limitations in this area, quantitative analysis of the GFP signal was not possible and we instead used the qualitative detection of SHF cells addition to the HT. To achieve this, we imaged an Nkx2.5eGFP; Ils1cre/+; Rosa26Rtdtomato+/- embryo at successive time points throughout the transition from open to linear HT and tracked tdtomato+ cells incorporation to the HT at the arterial pole. This study further confirmed that SHF differentiation at the arterial pole is initiated when the HT is about to close dorsally but not before (Figure 9C). Thus, SHF cells do not differentiate during the 5–7 hr period when morphogenesis of the open HT takes place, but coordinately start differentiation at different regions of the HT during dorsal closure. Interestingly, these regions do not only include arterial and venous poles but also the dorsal seam of the HT.
Here, we established a whole-embryo live-imaging method based on two-photon microscopy that allows whole-tissue tracking at cellular resolution. By combining various genetic tracing tools, we labeled progenitor and differentiated cardiomyocytes and performed 3D cell tracking over time combined with 3D reconstruction of the HT at multiple stages. We report three distinct temporal phases of HT formation (Figure 10). During the first phase, the cc differentiates rapidly and morphogenesis, in terms of changes in the relative position of cells, is minimal. During the second phase, differentiation is not detected and morphogenetic remodeling gives rise to a dorsally open HT. During the third phase, cardiac precursor recruitment and differentiation resumes, contributing to the formation of the RV and the dorsal closure of the HT. Our results support the early establishment of distinct FHF and SHF cell populations and show that the morphogenetic changes that transform the cc into a HT largely take place in the absence of cardiac precursor differentiation. These observations indicate tissue-level coordination of differentiation and morphogenesis during early cardiogenesis in the mouse.
The series of 3D reconstructions from fixed embryos was important to establish a reference staging of HT formation. This allowed us to accurately stage embryos in live experiments based on morphology and it will also be useful in the future for gene expression mapping and accurate phenotypic analysis of mutant embryos. The tissue growth pattern observed in static 3D reconstructions was insightful to suggest variability in growth rates during different phases of HT formation. Growth of the differentiated cardiac tissue is relatively paused when the cc undergoes morphogenesis to form the open HT during the second phase. This is consistent with previous studies in mouse, chick and human models showing that proliferation drops in the differentiated myocardium of the forming HT, while proliferation remains high in the splanchnic mesoderm (van den Berg et al., 2009; de Boer et al., 2012; Sizarov et al., 2011). Our live analysis further showed that SHF cells do not contribute to the forming HT during the differentiation pause, which correlates with the growth rate reduction during this phase. This period coincides as well with the onset of cardiac contractility in the embryo (Tyser et al., 2016).
Following the phase of differentiation pause, growth of the HT is reinitiated by incorporation of new cells as the HT closes dorsally and the RV precursors are added at the arterial pole during the third phase. During this third phase, similarities were found in the differentiation dynamics of SHF precursors and splanchnic precursors contributing to the dorsal regions of the linear HT. The dorsal aspect of the linear HT gives rise to the inner curvature of the looped heart, which has an important contribution to non-chamber myocardium, including atrio-ventricular canal and parts of the conduction system (Christoffels et al., 2000). Our results suggest that the late recruitment of progenitors to the dorsal HT could contribute to differences between inner curvature cardiomyocytes and the rest of the heart tube.
While the live-imaging experiments were essential for the identification of the 5–7 hr hiatus between FHF and SHF differentiation, live imaging of the arterial pole during the SHF differentiation phase was challenging and was complemented by 3D reconstructions based on fixed and optically cleared embryos. These experiments confirmed the pause in differentiation during open HT formation and its reactivation during linear HT closure. In the future, it will be interesting to explore whether novel non-toxic index-matching media compatible with embryo viability (Boothe et al., 2017) may alleviate the limitations for deep cardiac imaging during late HT formation.
Regarding the specification of FHF and SHF populations, previous prospective clonal analyses showed that these lineages diverge around gastrulation (Devine et al., 2014; Lescroart et al., 2014). In agreement with this, our tracking of cell lineages in the cardiac forming region shows that sister cells share fates to either the cardiac crescent or the SHF. In addition, the fact that cells contributing to the SHF do not differentiate during the period when the cardiac crescent transforms into the primitive heart tube may contribute to the establishment of the sharp boundary observed between left and right ventricles later in development (Devine et al., 2014). Further studies will be required to assess how this temporal pause of cardiac differentiation is regulated. The molecular analyses of early FHF and SHF precursors suggest that intrinsic molecular differences between the two lineages appear around or shortly after gastrulation (Lescroart et al., 2014). These intrinsic differences may contribute to the regulation of the two distinct differentiation schedules described here. These studies and our observations, however, cannot discriminate whether this lineage allocation results from intrinsic differences between these lineages or it is due to their exposure to position-specific environments, especially as in our studies sister cells remain close neighbors. Environmental cues thus could also control the sequential differentiation of FHF and SHF precursors. The Wnt and BMP pathways are well known regulators of cardiac differentiation (Ai et al., 2007; Jain et al., 2015; Klaus et al., 2007; Kwon et al., 2007; Marvin et al., 2001; Qyang et al., 2007; Tirosh-Finkel et al., 2010; Ueno et al., 2007) and specific mechanisms affecting these pathways could be operating during the formation the HT, whereby the differentiation pathways could be temporally restrained. Finally, the endoderm may also play a key role in mediating FHF differentiation. Indeed cardiac mesoderm differentiation is affected in the absence of Sox17 -a transcription factor required for maintenance of the definitive endoderm- and this is accompanied by the formation of a morphologically abnormal HT (Pfister et al., 2011). It remains, however, unclear whether this effect is secondary to an initial defect in foregut development that is observed in this mutant.
A recent study reported spontaneous calcium transients propagating laterally thought the cardiac crescent (Tyser et al., 2016). Left/Right (L/R) asymmetry therefore exists within the cardiac crescent, prior to any detectable cardiac contraction, and BMP/SMAD1 signaling in the lateral plate mesoderm may be involved in this asymmetry (Furtado et al., 2008). In our studies, however, we did not detect any L/R difference in cTnnT expression by whole-mount immunodetection or Nkx2.5GFP activation by live analysis in the cc.
Regarding the possible conservation of the temporal differentiation sequence described here, elegant experiments in zebrafish using a cardiac myosin light chain reporter line and a Kaede photo-conversion assay, addressed the temporal order of cardiac differentiation in live embryos (de Pater et al., 2009; Liu and Stainier, 2012). Two distinct phases of cardiomyocyte differentiation were also observed. During a first phase, cardiomyocytes were recruited first into the ventricle and atria at the venous pole. During a second phase, cardiomyocyte differentiation was observed at the arterial pole of the HT. These pulse-chase experiments, however, do not address whether cardiac differentiation is continuous or includes a differentiation-paused phase, so further studies will be required to establish the conservation of the observations made here for the mouse embryo.
Finally, an important question to address is the functional relevance of the three distinct phases described here; more specifically, what is the role of the observed differentiation pause. An interesting possibility is that this pause would be functionally related to the extensive morphogenetic events that transform the cardiac crescent into the HT. Our study demonstrates coordinated splanchnic mesoderm movements during this phase. These movements involve a very active antero-medial displacement of the splanchnic mesoderm surrounding the cc -mostly fated to the SHF-. This behavior of the splanchnic mesoderm appears essential for transforming the cc into a dorsally closed HT. Importantly, this displacement involves the sliding of the splanchnic layer over the endoderm, suggesting an active role of SHF precursors in this morphogenetic movement. Such displacement of the splanchnic mesoderm over the endoderm during cardiogenesis had been suggested by classical time-lapse studies in the chick embryo (Dehaan, 1963), and it is tempting to speculate that a similar phenomenon contributes to the incorporation of SHF cells to both poles of the HT at later stages of HT development (van den Berg et al., 2009; Kelly et al., 2001; Zaffran et al., 2004). These observations suggest that SHF cells do not represent just a reservoir of cardiac precursors but play a morphogenetic role essential for heart tube formation. The main consequence of the displacement of the splanchnic mesoderm layer over the endoderm is the medial convergence of the left and right frontiers between the cc and SHF to form the dorsal mesocardium and close the HT. An important consequence of the differentiation pause described here is the stability of the cc/SHF frontiers during these morphogenetic movements. This stability prevents further spreading of the differentiation wave from the cc into the SHF/splanchnic mesoderm, which could interfere the ability of the latter to efficiently displace over the endoderm. We therefore hypothesize that the stability of the cc/SHF frontiers – and thus the differentiation pause – would be essential to allow the effective displacement of SHF/splanchnic mesoderm and elicit HT formation. The temporal allocation of the morphogenetic phase after cc differentiation allows the formation of the HT while simultaneously providing an incipient cardiac function essential for the organization of embryonic circulation. This hypothesis poses a functional basis for the alternation of differentiation and morphogenesis phases during HT formation.
Our study applies whole-embryo live analysis of cardiac development at tissue level and with cellular resolution. We expect that extending this experimental approach will allow to further uncover unexpected and novel mechanisms of organogenesis. While limited attention had been paid so far to the temporal dynamics of differentiation during embryonic development, this is an essential aspect of organogenesis (Gogendeau et al., 2015; Parchem et al., 2015; Yang et al., 2015). Here, we show the relevance of differentiation timing regulation during heart tube formation and its coordination with morphogenesis at the tissue level. Further understanding of the molecular and cellular mechanisms underlying these phenomena will help us expanding pools of cardiac progenitors in vitro or directing them towards differentiation.
Mouse alleles used in the manuscript are listed including bibliographic references and allele identities at the ‘Mouse Genome Informatics’ data base (MGI, http://www.informatics.jax.org/). Mesp1cre (Saga et al., 1999, MGI:2176467), Isl1cre (Cai et al., 2003, MGI:3623159), Nkx2.5cre (Stanley et al., 2002, MGI:2448972), Rosa26Rtdtomato (Madisen et al., 2010, MGI:3809524), Rosa26RmTmG (Muzumdar et al., 2007, MGI:3716464), Nkx2.5eGFP (Wu et al., 2006, MGI:5788423), Polr2aCreERT2 (RERT) (Guerra et al., 2003, MGI:3772332) and C57BL/6 (Charles River). Mice were genotyped as previously described. All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Ref. PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
Embryos dissected in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) were fixed overnight in 2% PFA at 4C, then permeabilized in PBST (PBS containing 0.1% Triton X-100) and blocked (5% goat serum). Embryos were incubated overnight at 4°C with antibodies diluted in PBST: mouse anti-cTnnT (1:250, MS-295 Thermo Scientific), rabbit anti-PH3 (1:250, 06–570 Millipore), CD31 (553370 BD Pharmingen clone MEC 13.3), SMA (C6198 Sigma) and rabbit anti-Laminin1 (1:500, Sigma, L9393). After washing in freshly prepared PBST at 4°C, embryos were incubated with secondary antibodies (Molecular Probes, A21121, A21141, A11035) coupled to 488, 549 or 649 fluorophores as required at 1:250 and DAPI at 1:500 (Molecular Probes, D3571) overnight at 4°C. Before imaging, embryos were washed in PBST at room temperature and cleared with focus clear (Cell Explorer, FC-101) to enhance the transparency of the embryo. Confocal images were obtained on a SP8 Leica confocal microscope with a 20X oil objective (0.7 NA) at a 1024 × 1024 pixels dimension with a z-step of 2–4 μm. Embryos were systematically imaged throughout the entire heart tube from top to bottom.
For 3D rendering, fluorescent signal in confocal z-stacks was first segmented by setting intensity thresholds using the trainable Weka segmentation tool plugin available in Fiji (Arganda-Carreras et al., 2017; Schindelin et al., 2012). The resulting z-stacks were then corrected manually on a slide-by-slide basis to eliminate segmentation mistakes. In case of the cTnnT immunofluorescence images (Figure 1), background signal from the yolk sack was manually masked. The volume of the cTnnT positive myocardium was then computed by multiplying the total segmented area by the z-stack interval using a custom Fiji macro. In the Nkx2.5cre/+; Rosa26tdtomato+/- and Nkx2.5eGFP embryos, fluorophore signal present in the endothelium, endocardium and endoderm cells was manually masked prior to segmentation (Figure 1A, Figure 1—figure supplement 1, Figure 2B, Figure 2—figure supplement 2A,A’ and, Figure 6A’’). For 3D visualization of the 3D segmented image stacks, Imaris software (Bitplane) was used.
Embryos were dissected at E7.5 in pre-equilibrated DMEM supplemented with 10% foetal bovine serum, 25 mM HEPES-NaOH (pH 7.2), penicillin (50μml21) and streptomycin (50mgml21). Embryos were staged on the basis of morphological criteria (supplementary Figure 1) (Downs and Davies, 1993; Lawson and Wilson, 2016), and those between the bud and early somitogenesis stages were used for culture and time-lapse imaging. To track the early phase of cardiac differentiation and subsequent phases of morphogenesis, we used embryos at EHF to transversal HT stage. Embryos were cultured in 50% fresh rat serum, 48% DMEM without phenol red, 1% N-2 neuronal growth supplement (100X, Invitrogen 17502–048) and 1% B-27 supplement (50X Thermo Fisher Scientist 17504044) filter sterilised through a 0.2 mm filter. To hold embryos in position during time-lapse acquisition, we made special plastic holders with holes of different diameters (0.5–3 mm) to ensure a good fit of embryos similarly to the traps developed by Nonaka, 2009, Nonaka et al. (2002). Embryos were mounted with their anterior side facing up. To avoid evaporation, the medium was covered with mineral oil (Sigma-Aldrich; M8410). Before starting the time-lapse acquisition, embryos were first pre-cultured for at least 2 hr in the microscopy culture set up. The morphology of the embryo was then carefully monitored and if the embryos appeared unhealthy or rotate and move, they were discarded, otherwise, time-lapse acquisition was performed. For the acquisition, we used the Zeiss LSM780 equipped with a 5% CO2 incubator and a heating chamber maintaining 37°C. The objective lens used was a 20X(NA = 1) dipping objective, which allowed a long working distance for imaging mouse embryos and tissues. A MaiTai laser line at 1000 nm was used for two-channel two-photon imaging. Acquisition was done using Zen software (Zeiss). Typical image settings were: output power: 250 mW, pixel dwell time: 7μs, line averaging: two and image dimension: 610 × 610 μm (1024 × 1024 pixels). To maximize the chance of covering the entire heart tube during long-term time lapse videos, we allowed 150–200 μm of free space between the objective and the embryo at the beginning of the recording.
For labeling single cells, tamoxifen was administered by oral gavage (2–4 mg/mL) in RERT;Rosa26R-tdtomato (cell tracking) or RERT;Rosa26RmTmG mice (cell shape study) at E7. Cell shape measurements were done on single cells, imaged in mosaic-labeled, fixed and immunostained embryos and analyzed with Fiji software (Figure 2D,E). Tracking of tdtomato-labeled cells was done on single cells located within the cardiogenic mesoderm -excluding endothelial, pericardial and endodermal cells- and their GFP intensity was measured over time. To track cells manually in 4D stacks, the MTrackJ Fiji plugin (Meijering et al., 2012) was used. A local square cursor (25 × 25 pixels) on the cell of interest snaps according to a bright centroid feature on a slice-by-slice basis. Only tracks lasting for the entire length of the video were kept. When an ambiguity arises in the tracking between consecutive time points, the track was discarded. Tracks split at cell divisions. A cell division event is normally clearly distinguishable over at least two time points. In case one of the two daughter cells is not tractable, the other daughter cell is still tracked. Each track is assigned an ID number and excel files with all the tracks coordinates in x, y, z and t was generated. Coordinates of each track were converted into 8-bit 4D images using a custom Fiji macro in which each cell was represented by a sphere of specific pixel intensity, from 1 to 255, while pixels corresponding to background were set to zero. The 4D images were then opened with Imaris to perform visualization of the 3D trajectories of each cell using the ‘spots’ tool, where each object were identified according to pixel intensity. GFP intensity measurement is performed by segmentation of cell shape. A Gaussian filter whose radius is adjusted to the typical size of a cell was first applied, followed by a Laplacian filter. The resulting 32 bits image was next converted to a mask by thresholding. When objects touched each other, a watershed on the binary mask and manual corrections was applied. Each segmented cell was checked and tracked manually for accuracy. In Figure 3B’ nuclei segmentation was performed manually. The mean GFP signal intensity of the segmented objects was then measured using the ‘analyze particle’ tool in Fiji. To quantify the GFP level of tracked cells through time, four to five successive time points were arbitrarily chosen in each video (Figure 5F, Figure 6C,D and Figure 5—figure supplement 1C) except in Figure 5D and Figure 6I, where GFP intensity level was measured in every time point. Background intensities were measured in neural tube cells, which are known to be negative for GFP and cTnnT. Tables containing ID number of tracked cells and GFP intensities were generated and plotted using Prism statistical software.
For comparisons of two groups, a Mann–Whitney U-test was used using Prism statistical software. To find a correlation between GFP and cTnnT levels of 0.8 with an alpha-level f 0.05 and a power of 0.2 at least 10 cells per embryo were required (Figure 3D and Figure 3—figure supplement 2C). Many more cells were computed for each experiment. The linear fit was done using ‘lm’ function from R statistical software (https://www.r-project.org/). To calculate the average speed of splanchnic mesoderm displacement, the shortest distance between the left and right splanchnic mesoderm was calculated in two z-level, using the measure tool in Fiji, and the variation in this distance by time unit was divided by 2, to determine the speed of movement of each sliding side of the splanchnic mesoderm.
Heart fields: spatial polarity and temporal dynamicsThe Anatomical Record 297:175–182.https://doi.org/10.1002/ar.22831
A tunable refractive index matching medium for live imaging cells, tissues and model organismseLife, 6, 10.7554/eLife.27240, 28708059.
Building the mammalian heart from two sources of myocardial cellsNature Reviews Genetics 6:826–837.https://doi.org/10.1038/nrg1710
Chamber formation and morphogenesis in the developing mammalian heartDevelopmental Biology 223:266–278.https://doi.org/10.1006/dbio.2000.9753
Migration patterns of the precardiac mesoderm in the early chick embrvoExperimental Cell Research 29:544–560.https://doi.org/10.1016/S0014-4827(63)80016-6
Kaufman's Atlas of Mouse Development Supplement3 – A revised staging of mouse development before organogenesis, Kaufman's Atlas of Mouse Development Supplement, Academic Press.
Zebrafish in the study of early cardiac developmentCirculation Research 110:870–874.https://doi.org/10.1161/CIRCRESAHA.111.246504
Inhibition of Wnt activity induces heart formation from posterior mesodermGenes & Development 15:316–327.https://doi.org/10.1101/gad.855501
Methods for cell and particle trackingMethods in Enzymology 504:183–200.https://doi.org/10.1016/B978-0-12-391857-4.00009-4
Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesisThe Journal of Cell Biology 164:97–109.https://doi.org/10.1083/jcb.200309160
The outflow tract of the heart is recruited from a novel heart-forming fieldDevelopmental Biology 238:97–109.https://doi.org/10.1006/dbio.2001.0409
Modification of mouse nodal flow by applying artificial flowMethods in Cell Biology 91:287–297.https://doi.org/10.1016/S0091-679X(08)91015-3
Sox17-dependent gene expression and early heart and gut development in Sox17-deficient mouse embryosThe International Journal of Developmental Biology 55:45–58.https://doi.org/10.1387/ijdb.103158sp
Fiji: an open-source platform for biological-image analysisNature Methods 9:676–682.https://doi.org/10.1038/nmeth.2019
Right ventricular myocardium derives from the anterior heart fieldCirculation Research 95:261–268.https://doi.org/10.1161/01.RES.0000136815.73623.BE
Richard P HarveyReviewing Editor; Victor Chang Cardiac Research Institute, Australia
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis" for consideration by eLife. Your article has been favorably evaluated by Marianne Bronner (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Isabelle Migeotte (Reviewer #2).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
This is an elegant paper that tracks in real time the movements and differentiation status of cells contributing to the heart, specifically discriminating between the behaviours of the first and second heart field. The study used fluorescent reporters and confocal microscopy to obtain high resolution 3 and 4D images of early mouse hearts, including highly informative time-lapse movies of heart tube formation in wildtype embryos. It brings together many disparate and mostly 2D findings into a more coherent concept and provides a way to visualise the formation of what is called the transverse heart tube. The authors careful observations make a number of new points, including defining distinct stages of heart tube morphogenesis based on detailed 3D reconstructions that will be extremely useful for staging embryos and defining gene expression and function. The most significant finding is the discovery of a differentiation hiatus during heart tube formation that corresponds to a phase of heart tube morphogenesis separating first and second heart progenitor cell differentiation. This pause also covers the onset of cardiac function. This is an important observation that further highlights the distinction between the early and late differentiating populations of cardiac progenitor cells. Although the authors did not investigate how this pause might be regulated, for example by studying heart tube formation in mutant embryos, their approach could be adapted for such analysis in the future, with the challenge of dealing with complex genetic crosses. The transition between progenitor and differentiation has been observed as an increase in the levels of Nkx2-5-GFP and cTnT. Importantly, this is not continuous and differentiation of FHF and SHF progenitors is separated by a time window of ~5-7 hours. This point has some considerable implications for conceptualising this transition in light of the various findings to date and the difficult issues surrounding the use of Cre reagents and Cre reporters of varying efficacy upon which the concept of FHF and SHF are in part built. How the situation in mouse relates to that in Zebrafish will remain unclear until more careful studies allow a more direct comparison between the two species. To our knowledge, live Imaging of mouse heart morphogenesis has never been performed for such long period with such high degree of cellular resolution and quantitative analysis of individual cells fate. The data nicely complement previous studies showing temporal waves of precursors at gastrulation corresponding to the FHF and SHF, and provides a credible explanation for the long period between the exit of those populations with different profiles from the streak, and their allocation to the proper anatomical position. The data is of high quality, and the health of the embryos is remarkable after quite a long imaging.
1) The data suggesting that FHF and SHF differentiation and morphogenetic behaviours are set within the cardiac crescent, does not formally address whether there are meaningful functional differences in lineage state between these populations. Geographical issues rather than lineage issues may determine the timing of differentiation and morphogenesis. This should be made very clear in the Discussion.
2) Subsection “Imaging the Isl1-expressing cell lineage confirms absence of cardiac progenitor differentiation during early heart tube morphogenesis”, first paragraph. With respect to the activation of the Isl1Cre x reporter, I think it is the efficacy of the Cre not the frequency of activation that is the issue. Please clarify.
3) The reviewers disagree with the statement in the second paragraph of the Discussion that the contribution of the Isl1+ cells/SHF cells to the dorsal part of the primary HT will define these as non-chamber myocardium. This is not shown and lineage tracing by Christoffels with Tbx-Cre reagents show that non-working myocardium of the AVC and contribute significantly to chamber myocardium. Furthermore, it follows that the mosaic contribution of the SHF to the inflow/atrial region would also have to remain non-chamber myocardium. This is not shown and previous lineage tracing with Isl1 Cre and other reagents do not show exclusion form the atrial appendages (working myocardium). Please qualify appropriately.
4) Technical information about the time period between frames would be useful.
5) The Cre activation of Tomato also labels endoderm as can be seen in Figure 2A, especially in the sections. This should be commented on early in the Results, as it is later in the Isl1 section. Are endodermal cells also seen in Figure 1A? Have they been removed in the reconstructions? Please comment on the basis for the differences between the different Nkx2-5 alleles (Cre versus eGFP).
6) The authors describe the major axis at the "transversal" hemitube stage as being transversal to the AP axis. The authors should clarify whether their data suggest that this axis is realigned or simply replaced by an AP axis during the transition to the linear HT.
7) The authors are encouraged to elaborate in the Results section on what has been learnt from the dynamic imaging shown in Videos 5-8. For example, in the second paragraph of the subsection “2-photon live-imaging of early cardiac development in the mouse embryo” they state that this has provided information concerning the formation of the endocardium and lumen formation, yet they do not say what this information is. Similarly, can they expand on any new findings concerning the anterior movement of splanchnic mesoderm? Given that even the striking observation of the differentiation pause is validated using confocal imaging of fixed embryos it is important to highlight exactly what can be gained by time-lapse imaging. This clearly goes beyond concluding that increased Nkx2.5 expression reports cardiomyocyte differentiation (as stated as the conclusion of the next paragraph).
8) The Discussion is short and could be extended, in part by elaborating on some of the points mentioned above. The observation that there is a growth pause coincident with the differentiation pause also merits discussion. The coincidence of this pause with the onset of cardiac function could also be further discussed. Also, in the Discussion, the authors state that addition of the SHF to the zebrafish heart is seen as a continuum. However this is unclear, and the original work of de Pater et al. suggest SHF addition demarcates a distinct late differentiation phase, not dissimilar to the authors' new observations in the mouse.
9) Panel 4A explaining the live imaging set-up is slightly confusing. Was imaging of the second phase carried out by continuing imaging of embryos from the first phase or in separate experiments? Given the embryos can be cultured for 24 hours, were embryos imaged continuously through these two phases and if so were the results then the same for the second phase? Please clarify. It would be helpful to add a little more technical information to the first Results paragraph dealing with live imaging (subsection “2-photon live-imaging of early cardiac development in the mouse embryo”), mentioning maximal culture and imaging times and frequency of time-lapse acquisitions.
10) Can the authors provide live imaging data of the third phase during which SHF cells initiate differentiation? This would be most interesting to document in time-lapse images and would significantly reinforce their hypothesis that this phase involves recovery from the differentiation hiatus.
11) Concerning Figure 5, please clarify the significance of cells that remain columnar with low levels of GFP expression. Can the authors conclude that these are SHF cells versus endocardial cells?
12) Making conclusions about lineage commitment by following the GFP status of sister cells seems a relatively weak argument. For example, do the authors envisage that cells would move from a GFP high to low state? It would potentially be more interesting to highlight that differentiation is not associated with a cell division event.
13) It is unclear how the cells in Figure 2D and E were labeled. Please explain this more fully in the Results section.https://doi.org/10.7554/eLife.30668.057
- Miguel Torres
- Miguel Torres
- Kenzo Ivanovitch
- Kenzo Ivanovitch
- Miguel Torres
- Miguel Torres
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank the CNIC Microscopy unit for help with the live confocal analysis, Fatima Sanchez Cabo for help with the statistical analyses and Florencia Cavodeassi, Miguel Manzanares, Briane Laruy and members of the Torres lab for helpful comments on the manuscript. This work was supported by grants BFU2015-71519-P, BFU2015-70193-REDT and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC). KI was supported by a Human Frontiers Science Program (LT000609/2015) and EMBO (ATL1275-2014) postdoctoral fellowships. The CNIC is supported by the Spanish MEIC and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015–0505). The authors declare no conflicts of interest.
Animal experimentation: All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Ref. PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.
- Richard P Harvey, Reviewing Editor, Victor Chang Cardiac Research Institute, Australia
© 2017, Ivanovitch et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.