Blue dotted line represents the median intensity value a. Note that when cells divide, only one of the two daughter cells was represented for simplicity. E Distribution of the tracked cells from the time-lapse video shown in A at the initial and final times of the tracking period. Data collected from tracked cells from four independent videos of 2h52m to 3h12m duration. Average increase: 1. Cells show stable GFP levels and do not differentiate.
Interval between frames: 7 m 40 s. Duration of the video: 3 hr 12 m. Related to Figure 6A. 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. 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. We confirmed this observation in longer time-lapse videos spanning 7 hr that covered the whole transition from transversal to open HT stage.
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. Lower doses of tamoxifen were injected in order to label only a very small proportion of cells in red. Interval between frames: 19 m 19 s. Duration of the video: 7 hr 24 m 35 s.
Related to Figure 7A. Its expression is transient in the precursors of the cc, while it remains expressed in SHF progenitors for an extended period Brade et al. 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. To test these observations in live imaging, we combined Nkx2. Consistently with previous reports Cai et al. B Inset from A yellow frame. C Inset from A Red frame ; increase of the tdtomato intensity in the splanchnic mesoderm over time.
Note that tdtomato signal is also detectable in the endoderm. D Same embryo as in A post-fixed and immunostained against cTnnT after live-imaging, showing that the red cells located in the splanchnic mesoderm are undifferentiated. Interval between frames: 6 m. Duration of the video: 7 hr 18 m. Related to Figure 8A. 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.
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.
Drosophila tissue and organ development: Heart
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. 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 , At this stage, the primordium of the RV has been added at the arterial pole Zaffran et al.
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. The presence of double-positive cells in these areas reveals the differentiation of Isl1 lineage cells into cardiomyocytes A, B. Images are 74 and optical sections acquired every 2.
Cardiac patterning and morphogenesis in zebrafish
White arrows show the dorsal mesocardial regions of the linear HT. The embryo was imaged continuously every 13 m for the first 6 hr of culture and then imaged once again at time point 9h30m. Note that at the last time point, the laser was increased at maximum power in order to reveal more clearly the red labeled cells at the arterial pole.
The contrast had to be enhanced as well. Image at 9h30m is a single optical section. 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. 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. 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 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. We propose that two distinct phases of cardiomyocyte differentiation take place during early heart development. At EHF stage the cc differentiates and starts folding. Cardiomyocytes round up and become contractile while the cardiac progenitors located in the splanchnic mesoderm remain undifferentiated. Subsequently, the cc.
Finally, cardiac differentiation resumes contributing new cardiomyocytes from the splanchnic mesoderm to the arterial pole prospective RV and the dorsal closure of the HT. 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.
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.https://sotaluti.tk
Growth and Morphogenesis during Early Heart Development in Amniotes
This period coincides as well with the onset of cardiac contractility in the embryo Tyser et al. 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.
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. Regarding the specification of FHF and SHF populations, previous prospective clonal analyses showed that these lineages diverge around gastrulation Devine et al. 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.
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. 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. Finally, the endoderm may also play a key role in mediating FHF differentiation.
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