Asymmetric Fate of the Posterior Part of the Second Heart Field Results in Unexpected Left/Right Contributions to Both Poles of the HeartNovelty and Significance
Rationale: The second heart field (SHF) contains progenitors of all heart chambers, excluding the left ventricle. The SHF is patterned, and the anterior region is known to be destined to form the outflow tract and right ventricle.
Objective: The aim of this study was to map the fate of the posterior SHF (pSHF).
Methods and Results: We examined the contribution of pSHF cells, labeled by lipophilic dye at the 4- to 6-somite stage, to regions of the heart at 20 to 25 somites, using mouse embryo culture. Cells more cranial in the pSHF contribute to the atrioventricular canal (AVC) and atria, whereas those more caudal generate the sinus venosus, but there is intermixing of fate throughout the pSHF. Caudal pSHF contributes symmetrically to the sinus venosus, but the fate of cranial pSHF is left/right asymmetrical. Left pSHF moves to dorsal left atrium and superior AVC, whereas right pSHF contributes to right atrium, ventral left atrium, and inferior AVC. Retrospective clonal analysis shows the relationships between AVC and atria to be clonal and that right and left progenitors diverge before first and second heart lineage separation. Cranial pSHF cells also contribute to the outflow tract: proximal and distal at 4 somites, and distal only at 6 somites. All outflow tract–destined cells are intermingled with those that will contribute to inflow and AVC.
Conclusions: These observations show asymmetric fate of the pSHF, resulting in unexpected left/right contributions to both poles of the heart and can be integrated into a model of the morphogenetic movement of cells during cardiac looping.
- cardiac progenitor cells
- cell fate clonal analysis
- embryonic development
- left/right asymmetry
- mouse heart development
- second heart field
Cardiac precursor cells are located bilaterally as 2 symmetrical regions of the lateral plate splanchnic mesoderm at vertebrate primitive streak stages.1,2 These cardiac precursors fuse at the ventral midline to form the cardiac crescent and subsequently the initial heart tube.3 The classic view assumed that all cardiac progenitors reside in the heart tube,4 but the idea that arterial pole cells may be added later, after looping, was suggested long ago5 and has been definitively demonstrated in chick6,7 and mouse.8 It is now clear that, in all vertebrates, there is a population of cells, termed the second heart field (SHF), lying dorsal to the heart tube in pharyngeal mesoderm, which is a major source for all chambers of the heart, except the crescent-derived left ventricle.9
The extent of the SHF in mouse has been defined by the strong expression of islet-1, and the contribution to the heart has been revealed using Islet1-Cre genetic tracing.10,11 Retrospective clonal analyses have provided complementary linage data, revealing the early segregation of the first and second myocardial lineages, which correspond to the first heart field and SHF. Specific patterns of clones contributing to the looped heart tube have been identified, such as those colonizing both the inflow and outflow poles.12,13 However, the embryological origin and genetic characteristics of the precursors have remained unknown. The boundaries of, and within, the heart-forming regions have been examined by a variety of approaches,9,14 from which it appears that the anteroposterior (craniocaudal) axis of the SHF is patterned and varies in fate.
The anterior portion of the SHF (anterior heart field [AHF]) expresses Fgf8, Fgf10, and Tbx1, and genetic tracing using these genes, and an enhancer of Mef2c, shows that most of the right ventricle and all the outflow tract (OFT) myocardium are AHF-derived.8,15,16 There is no equivalent expression marker of posterior SHF (pSHF), although podoplanin has been suggested,17 but this is not specific for heart precursor cells. Controlling signals for the SHF include a Wnt2 pathway required for pSHF expansion18 and a retinoic acid–mediated mechanism that restricts AHF growth.19 Recently, it was shown that 3′ Hox a1 and b1 genes are regulated by retinoic acid and that these transcription factors act as effectors of SHF patterning.20
Previously, using the Mlc1v-lacZ-24 transgenic reporter of Fgf10, we provided some evidence for an atrial fate of the Islet1+/Fgf10− pSHF cell population.21 In this study, we have analyzed in detail the fate of small groups of pSHF cells using lipophilic dye labeling and mouse embryo culture to form a fate map of the Islet1+/Fgf10− subdomain. This shows that pSHF is a source of cells for both inflow and outflow cardiac regions, with left-right, craniocaudal, and stage-dependent patterns of contribution.
Outbred MF1 mice were used, except for the clonal analyses. The pSHF was defined by in situ hybridization for Islet1 and Mlc2a expression10 and by X-gal staining of the Fgf10 transgenic reporter line,8 Mlc1v-nLacZ-24. Cells within the Mlc2a−/Islet1+/Fgf10− pSHF region (4- and 6-somite stages; Online Figures IH and II) or the most caudal Mlc2a−/Islet1+/Fgf10+ region (2 somites; Online Figure IG) were labeled by injection of a lipophilic carbocyanine, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) or DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide), as described previously.22 Initial studies showed that injections label a contiguous group of 20 to 30 cells within the splanchnic mesoderm layer (see Online Data Supplement, Online Figure IVA–IVC).
Injected embryos were photographed immediately (t0), and the exact localization of dye was systematically recorded using a grid overlay (Online Figure VA–VD). Embryos were cultured for 40 hours (t40) in rolling bottles and then hearts were isolated, fixed, and dye distribution examined by confocal microscopy. The locations of label were charted onto systematically defined cardiac regions (Online Figure VE–VH), each of which is identified here by a color/symbol code (Online Figure VI) when related back to the site of injection.
Clonal analyses were made at E8.5 in embryos from the α-cardiac actinnlaacZ1.1/+ transgenic line that spontaneously generates random clones of β-galactosidase (β-gal)–positive myocardial cells, as previously described.12,13 Statistical methods are given in the Online Data Supplement.
Establishing the Extent of pSHF With Molecular Markers
By definition, we consider the pSHF as that part of the Islet1-expressing SHF that does not express AHF markers, such as Fgf10.21 We charted this domain precisely in embryos of 2 to 6 somites, using Islet1, Fgf10 (Mlc1v-nLacZ-24), and Mlc2a (myocardial marker for crescent) expression (Online Figures IB, IC, IE, IF, IH, and II). At 2 somites, even the most caudal region of the Islet1-expressing domain also showed Mlc1v-nLacZ-24 staining (Online Figure IA, ID, and IG), so no clear pSHF can be defined at this stage using these techniques.
pSHF Cells Contribute to the Venous Pole of the Heart With Craniocaudal, Left-Right, and Stage-Dependent Patterns
Previously, we showed that pSHF contributes to the ipsilateral atrium.21 Here, we find that the left pSHF at 4 to 6 somites contributes to the dorsal wall of the left (common) atrium (LA), whereas the right pSHF contributes to both the dorsal and ventral regions of the right (common) atrium (RA; Figure 1A–1D). Interestingly, the right pSHF also contributes contralaterally to the ventral side of the LA (Figure 1C–1G). Observation of labeled cells in the ventral LA after left pSHF injection was rare (2 cases, ≈2% injections; Figure 1C–1G for details).
Considering the whole pSHF, about two thirds of all injections resulted in labeling of the atrium, and this proportion did not differ between the 4- and 6-somite stages (65% and 63% of injections, respectively; Online Figure IIA). However, the cranial and caudal halves of the pSHF show clear spatial differences in contribution, again similarly at 4 and 6 somites. The caudal portion of the pSHF was less likely than the cranial to contribute to the atria (Figure 1C and 1D; Online Figure IIIC and IIID). Furthermore, the caudal portion also contributes more distally to the atria and to the sinuatrial region (Figure 2A and 2B and Figure 3C and 3D), whereas more cranial injections yielded labeled cells in proximal portions of the atria, closer to the atrioventricular junction.
There seems to be a difference in the allocation process between the dorsal and ventral RA. Of all injections that labeled the RA, 46% contributed to the dorsal portion only, 51% to dorsal and ventral, and 3% to ventral only. So, 97% of the ventral label also displayed dorsal label, which could be accounted for by cells contributing first dorsally, then a subpopulation moving ventrally. In support of this, contiguous regions of labeled cells were observed in the lateral dorsal region through the right lateral wall and into the ventral portion (Figure 2A–2C).
The right cells that contribute contralaterally to the ventral LA lie in the cranial region of the pSHF: 10% cranial injections compared with 2% caudal. Furthermore, the vast majority (81%) of injections that resulted in contralateral contribution also resulted in ipsilateral labeling in the ventral and dorsal side RA. Again, this may reflect a progressive contribution from the cranial right pSHF to dorsal right, ventral right, and then ventral left regions of the atrium.
Injections in the pSHF also produced labeled cells in the sinus venosus region, which we define at this stage as that region caudal to the visible sulcus at the atrium–body wall junction (Figures 1A, 1B, 2A, 2B, 2D, and 2E). Of all cases of sinus venosus contribution, 70% showed contribution to the atrium also, with a contiguous region of labeled cells. However, in contrast to the observations in the atria, contralateral labeling from one side of the pSHF to the other sinus venosus was very rare (1 and 2 cases from left and right [L&R], respectively). The overall area occupied by sinus venosus precursors in the pSHF is somewhat greater than that for the atria, extending more caudally and medially (Figure 2D and 2E and Online Figure IIIC and IIID). In accordance with observations in the atria, sinus venous–destined cells were more likely to reside in the caudal half of the pSHF at the 4-somite stage (Figure 2D and 2E and Online Figure IIC). However, contrary to the atria, there was a clear stage difference between the 4- and 6-somite stages, with consistently more sinus venosus contribution at the older stage (Online Figure IIC). This indicates that sinus venosus precursors are added to the domain of Isl1 expression between the 4- and 6-somite stages.
We did not systematically analyze contribution to the endocardium (indeed all observations described here refer to myocardium), but sectioning of hearts shows that, in the inflow region, labeled cells are found in both endocardium and myocardium (Figure 2F and 2G). This is consistent with analyses of Islet1-cre mice, suggesting a common origin from Islet1-expressing precursors for these 2 cell types.10
Left and Right pSHFs Contribute Differently to the AVC Region
Overall, 14% of pSHF injections at 4- and 6-somite stages showed labeled cells in the atrioventricular canal (AVC) region, of which the vast majority (97%) also showed atrial contribution. Strikingly, the contributions to the AVC from the left and right pSHFs were markedly different. Consistently, labeled cells from the right pSHF contributed to the inferior AVC (Figure 3A, 3B, and 3E–3G), whereas cells from the left pSHF contributed to the superior and left lateral AVC (hereafter superior AVC), corresponding with the outer curvature of the AVC (Figure 3C, 3D, and 3E–3G). Curiously, no labeled cells from the pSHF were observed in the right lateral side of the AVC, corresponding with the inner curve, closer to the OFT region. Furthermore, there were no cases of an individual injection resulting in labeling of both the inferior and the superior AVC.
Although the AVC-destined region of the pSHF overlapped with the atria/sinus venosus precursors (Online Figure IIIE and IIIF), AVC-contributing cells were localized almost exclusively in the most cranial portion of the pSHF (Figure 3E–3G and Online Figure IID). In contrast to the sinus venosus, the proportion of injections that resulted in AVC-labeled cells decreased between 4 and 6 somites (Online Figure IIA). This indicates that AVC precursor cells have left the SHF by the 6-somite stage and have been fully recruited to the heart tube.
There is a close relationship between contributions to regions of the atria and regions of the AVC. Thus, in 100% of cases showing labeled cells in the superior AVC region, there were also labeled cells in the dorsal LA. Conversely, 88% of inferior AVC-labeled hearts also showed contribution to the ventral LA. There were no examples of superior AVC plus ventral LA nor of inferior AVC plus dorsal LA patterns of labeling in this study. L&R pSHF contributions to atria and AVC are summarized in Figure 7B and 7C.
We aimed to gain insight into the clonal relationships between the inferior and superior AVC, as well as between the AVC and inflow regions. To investigate these relationships by a complementary approach, we studied α-cardiac actinnlaacZ1.1/+ E8.5 embryos, which allows retrospective clonal analysis of myocardial cells in the mouse heart.12 Thus, from a collection of 4967 α-cardiac actinnlaacZ1.1/+ embryos of 8 to 21 somites, we selected all those that had β-gal–positive groups of cells in the AVC or atria (Online Table I). Among these 96 embryos, 33% showed β-gal–positive cells in the superior AVC (Figure 4C, 4F, and 4H), whereas only 10% displayed positive cells in the inferior AVC (Figure 4E, 4G, and 4I). This difference, in a random collection of clones, probably reflects the difference in size, and therefore numbers of progenitor cells, between these 2 regions, the superior AVC being larger. The exclusive participation in either region of the AVC in most embryos suggests that these 2 regions have different clonal origins and that up to the stage of 21 somites there is no intermixing of these populations. We observed only 2 embryos (Figure 4A and 4B), with β-gal expressing cells that occupied both the inferior and the superior AVC. Statistical analysis (Online Table I) suggests that these clones arise from common precursors of the 2 AVC regions. Indeed, the clone in Figure 4A, which contains 1370 β-gal–positive cells colonizing all regions of the heart, is the biggest clone of the collection, indicative of a very early event of recombination. The other clone colonizing both AVC regions (Figure 4B) contains 42 β-gal–positive cells in the right and left ventricles and the AVC. It has been classified as a large clone of the first myocardial lineage.13 However, most large clones of the first (n=13) or second (n=13) myocardial lineage, as well as very large clones arising from common precursors of the first and second myocardial lineages (n=4; Figure 4C and 4E), have an exclusive contribution to either the superolateral or the inferior AVC. This means that the segregation between the 2 AVC regions precedes the segregation of the 2 lineages (Figure 4D).
β-gal–positive labeling restricted to a region of the AVC was frequently associated with positive cells in the atria (80%). The majority (63%) of embryonic hearts with LacZ-labeled cells in the superior AVC only also showed β-gal–positive cells in the dorsal LA (Figure 4H), and less frequently (20%) in the ventral LA (Figure 4C). There was a single example of LacZ labeling on both the superior AVC and right atrium. Statistical analysis confirms the clonal relationship between the superior AVC and the LA, but not with the RA. In contrast, all hearts that showed β-gal–positive cells in the inferior-only AVC also contained LacZ-labeled cells in the ventral LA or the RA or both (Figure 4E, 4G, and 4I). Statistical analysis indicates that the inferior AVC is clonally related to the ventral LA and the RA, but not to the dorsal LA (Online Table II).
Taken together, these results suggest that the inferior and superior regions of the AVC have different origins, and the inferior part is clonally related to the ventral portion of the atria, whereas the superior portion is clonally related to the LA (Figure 4D). This is wholly consistent with our dye labeling data, and given our conclusion that left pSHF contributes to the dorsal wall of the LA, we conclude that the bulk of AVC (the superolateral portion) is also of left pSHF origin.
pSHF Cells Colonize the Arterial Pole of the Heart
Interestingly, labeling of the most cranial portions of the pSHF in embryos from 4 to 6 somites resulted in labeled cells at the arterial pole of the developing heart (Figure 5). At the 25-somite stage examined, our data show an ipsilateral cell contribution from the pSHF to the OFT (summarized in Figure 7D). We found OFT precursor cells in an extensive area of the pSHF, overlapping with those obtained for the inflow tract (IFT)/AVC cardiac regions (Figure 5H–5J and Online Figure IIIG and IIIH). Almost all OFT-labeled embryos also presented staining of the IFT (94%), suggesting that IFT- and OFT-derived cells may share a common precursor pool in the pSHF and the pSHF may be composed of an intermingling of IFT-destined plus OFT-destined cells.
As previously described for IFT and AVC-destined cells, OFT precursor cells followed a craniocaudal pattern of contribution, that is, the OFT contribution was higher in the cranial portions of the pSHF (Figure 5H–5J; Online Figure IIE). In addition, at 4 somites, labeled cells contributed to both distal (aortic sac/OFT boundary) and proximal (OFT/right ventricle boundary) portions of the arterial pole, whereas only distal contribution to the OFT was observed at 6 somites (Figure 5H–5J). This suggests a sequential process of pSHF recruitment to the OFT (Figure 5H–5J; Online Figure IIE).
pSHF Supplies Fgf10-Expressing and Nonexpressing Cells to the OFT
Previous studies, including those using the Mlc1v-nLacZ-24 reporter, have demonstrated that much of the OFT is derived from Fgf10-expressing cells.8 Our results, showing a contribution of the Fgf10-negative pSHF to the OFT, led us to ask whether or not these cells switch on Fgf10 when contributing to the arterial pole. Sections of OFT from Mlc1v-nLacZ-24 embryos show a mix of β-gal–positive and β-gal–negative cells (Figure 6A–6D). We dye-injected the pSHF of Mlc1v-nLacZ-24 embryos producing dye labeling of the dorsal pericardial wall at t40, overlapping with the β-gal domain of Mlc1v-nLacZ-24 staining (Figure 6E–6F). Isolation of this region and further examination demonstrated that the dye was localized in β-gal–negative and β-gal–positive cells (Figure 6G–6I). However, we cannot estimate what fraction of all OFT contributing pSHF cells do not express Fgf10. Also, we cannot rule out the possibility that there is slow or incomplete activation of the Mlc1v-nLacZ-24 transgene in this population.
The Posterior-Most SHF at 2 Somites Is Predominantly of OFT Fate
We find that the posterior-most portion of Islet1+ splanchnic mesoderm at 2 somites also expresses Fgf10, showing that there is no pSHF, by our molecular definition, at this stage. Nevertheless, we labeled the posterior-most Islet1+ region of 2 somite embryos and found that 95% contributed to the OFT with only 48% showing IFT labeling (n=10; Online Figure IIF). This contrasts with the pSHF at 4 and 6 somites, which is mainly destined to form the inflow region. At 2 somites, inflow progenitors lie slightly more medial than outflow progenitors (Figure 7A). The contribution to the inflow cardiac regions displayed, overall, similar left-right and craniocaudal patterns to those observed at 4 to 6 somites (data not shown). These observations suggest that from the 2-somite stage there is recruitment of cells to the pSHF, with progressive anterior-ward movement of cells within the SHF.
Craniocaudal Patterning of the pSHF
Several lines of evidence have shown that the fate of cells from the SHF in the mouse varies according to the anteroposterior (craniocaudal) position within this region. An anterior domain is marked by the expression of genes, including Fgf8, Fgf10, Tbx1, and an Mef2c enhancer, and forms the OFT and right ventricle.8,10,15,16 A posterior domain contributes to the atrial chambers.10,21 We now show that there is further craniocaudal patterning within the pSHF domain, although without sharp boundaries.
The most cranial pSHF contributes to the atrioventricular canal, the most caudal part contributes to the sinus venosus, and atrial progenitors lie predominantly in between. This suggests that cells within the pSHF add sequentially to the heart tube, with anteroposterior position predicting regional contribution to the heart, from outflow to inflow. Little is known of the molecular mechanisms that may encode craniocaudal identity within the pSHF. Retinoic acid signaling limits the extent of the SHF,19 and recently it has been shown to be required for the correct deployment of Hox-expressing SHF cells.20 Furthermore, regionalized expression of Hoxb1 and Hoxa1 seems to be required for anteroposterior patterning of the SHF.20
Importantly, at the stages examined here, the cranial pSHF contributes asymmetrically to the left and right sides of the heart, whereas derivatives from the caudal pSHF contribute symmetrically. We have looked at pSHF contributions to the heart at the 20- to 25-somite stage, and the region we have identified as the sinus venosus will develop further into inflow regions of the atria, the coronary sinus, and proximal parts of the caval veins, so it is possible that some asymmetric contributions might be revealed at later stages. However, a companion article23 to this demonstrates clonal relationships between the LA and the left superior caval vein, and between the RA and the right superior caval vein at E14.5, thus strongly supporting the progressive ipsilateral development that we have observed from the pSHF to the future atria and systemic veins.
The sequential craniocaudal arrangement of precursors within the mouse pSHF is stage-dependent and is overlapped by progenitors of the arterial pole (see below). In apparent contrast, in the chick the medial and lateral cardiogenic mesoderm contribute to the most cranial and caudal portions of the heart, respectively.24 We see no evidence of mediolateral patterning at 4 to 6 somites in the mouse, but this is probably explained by the earlier stage examined in the chick before splanchnic mesoderm rotation.25 At 2 somites, we do observe a more medial and lateral disposition of outflow and IFT progenitor cells, respectively, comparable with the arrangement in the chick heart-forming regions.24
pSHF Also Contributes to the Arterial Pole
We find a robust contribution of the more cranial part of the pSHF to the arterial pole, as well as to the inflow regions. This may seem surprising, given that genetic tracing studies suggested that the OFT is entirely derived from the AHF.8,10,15,16 However, some previous studies in the chick26 and recent genetic tracing and clonal studies in the mouse support this notion. Cre tracing shows that pSHF cells that have expressed Hoxb1, Hoxa1, and Hoxa3 contribute not only to the inflow but also to the arterial pole, specifically to the OFT myocardium of the pulmonary trunk.20 In addition, clonal analysis in the E8.5 heart13 and E14.5 heart23 shows a clonal relationship between the atria and the OFT myocardium.
From our current studies, we are not able to relate left and right pSHF progenitors to the pulmonary or aortic parts of the OFT. At the 20- to 25-somite stage at which we assessed contribution, the left pSHF contributed to the left side of the OFT and the right side similarly. However, we and others have shown a functional rotation of the OFT at later stages of development, which moves cells from the left side to populate the subpulmonary myocardium,27 and there is a clonal relationship between pulmonary myocardium and left head muscles derived from the SHF.28 As is the case for the inflow, we found evidence for sequential addition of cells from the pSHF to the OFT, with progenitors labeled at 4 somites contributing proximally and at 6 somites distally. A similar sequential relationship is seen in descendants of Hoxb1-expressing cells, which populate the proximal OFT, and Hoxa1 and Hoxa3 descendants that appear more distally.20
As discussed, there are clonal and genetic data that support the existence of SHF cells that are progenitors of both outflow and inflow myocardium. Because we labeled groups of cells, we cannot draw any conclusions about lineage relationships, but, nevertheless, it is very clear from our observations that adjacent pSHF cells contribute to both poles of the heart, which raises questions about their paths of movement and how they are allocated to one pole or the other. The majority of pSHF cells move into the atrioventricular canal, atria, and sinus venosus, which seems to be a contiguous route to the heart tube via the sinus horns. In contrast, those pSHF cells that contribute to the OFT are likely to move via the dorsal pericardial wall, which is the location of the AHF cells that form the majority of the OFT. This is compatible with the caudal movement of the outflow region of the heart, relative to the pharyngeal region, over this time period, and we have observed labeled cells in the dorsal pericardial wall at 20 somites after labeling of pSHF at 4 to 6 somites. It is possible that this pathway of migration is controlled by Tbx1. It has been shown that, in Tbx1−/− mice, AHF cells marked by the Mlc1v-nLacZ-24 transgene are found ectopically in the dorsal RA, rather than in their normal location in the OFT.29
If pSHF cells move to the OFT via the dorsal pericardial wall, which is populated by AHF cells, it may be expected that the originally pSHF cells switch on AHF molecular markers during their relocation. Again using the Mlc1v-nLacZ-24 transgene marker, we found that this is the case for some cells labeled in the pSHF, but apparently not all. It is possible that some cells switch on AHF markers slowly, so are not yet expressing at the 20- to 25-somite stage we assessed, or that the transgene is not fully reflecting endogenous Fgf10 expression or that Fgf10 does not mark all AHF cells. Alternatively, the OFT at this stage may be a molecular mosaic, which has not been previously reported.
Left-Right Asymmetric Contributions From the pSHF
We observed 2 marked left-right asymmetries in pSHF contributions, in relation to the atrioventricular canal and to regions of the atria. The left pSHF gives rise to the myocardium overlying the superior atrioventricular region, whereas the right pSHF contributed to the inferior region. Labeling much earlier, at the 4-cell stage, has shown this same left-sided origin of the superior AVC region in Xenopus30 but has not been reported previously in mammals. Left-right asymmetries in heart development, excepting looping, are controlled by the left-sided expression of Pitx2c, which is known to pattern the SHF.21 Pitx2 is strongly expressed in superior, but not inferior, atrioventricular myocardium.31 Thus, Pitx2 is expressed in cells at the origin and destination of the movement from left pSHF to superior region of the atrioventricular canal, but it is not clear whether Pitx2 is required for this relocation. Pitx2c mutant hearts have AVC abnormalities,32 but any relationship to movement of cells from the left pSHF is untested.
Although the pSHF predominantly contributes to the ipsilateral atria, as previously reported in mouse21 and chick,25 we found that the right pSHF contributes to a subportion of the left ventral atrium. It is not clear what region of the fully formed atrium is represented in the left ventral atrium at the 20- to 25-somite stage, but again there is a correlation with Pitx2 expression. Recent detailed analysis of Pitx2 expression, using a reporter transgene, shows that at E9.5 the ventral LA and inferior AVC are Pitx2-negative, whereas the dorsal region is positive, with a domain extending over the superior AVC.31
Our clonal analyses show that the 2 lineages which give rise to left and right progenitor populations segregate from common precursors before the separation of the first and second heart lineages (Figure 4D). This is compatible with the common precursors being located in the primitive streak and the progenitor populations in the initial bilateral heart fields, each of which then contributes to the first and second heart lineages (fields). Thus, cells of the superior AVC are already allocated in the left heart field.
The cell movements we describe may be part of the tissue-level forces that drive early heart morphogenesis. The progressive movement of cells of the atrial/atrioventricular region from dorsal right to ventral left (Figure 7E and 7F) may be related to rightward looping, the initial leftward jog of the AVC, and the clockwise (viewed from the aortic sac) torsion of the heart tube.33,34 It is possible that abnormality in this process could lead to congenital looping defects such as tetralogy of Fallot. That left pSHF cells do not move rightward, contributing instead to the superior AVC (Figure 7E and 7F), may explain the expansion of this region, relative to the inferior AVC. Human laterality syndromes include a wide range of congenital heart defects, including the atrioventricular connections such as double outlet right ventricle, which may be explained by defects in the left-right asymmetrical contributions of the pSHF.
We thank E. Pecnard for his participation in collecting α-cardiac actinnlaacZ1.1/+ embryos and J-F. Le Garrec for help with the statistical analyses. S.M.M. is an INSERM research scientist.
Sources of Funding
This work was supported by the European Community’s Sixth Framework Programme contract (Heart Repair) SHM-CT-2005–018630 and by a British Heart Foundation Programme grant RG/03/012.
In July 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.48 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.271247/-/DC1.
Non-standard Abbreviations and Acronyms
- anterior heart field
- atrioventricular canal
- inflow tract
- left (common) atrium
- outflow tract
- posterior SHF
- right (common) atrium
- second heart field
- Received April 12, 2012.
- Revision received August 29, 2012.
- Accepted September 4, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
The embryonic mammalian heart is initially a simple inverted Y-shaped tube, most of which will eventually become the left ventricle, whereas the rest of the heart is formed by the addition of cells to the single arterial pole and the bilateral (left and right) venous poles.
The cells added to the heart tube are from the second heart field (SHF), which lies bilaterally in the pharyngeal and splanchnic mesoderm and which differs in fate and molecular properties in its cranial and caudal parts.
The cranial SHF (also called anterior heart field) adds to the arterial pole, forming the right ventricle and the outflow tract, whereas the exact contributions to the heart made by the caudal, or posterior, SHF (pSHF) are unclear.
What New Information Does This Article Contribute?
Cells in the pSHF contribute to all regions of the heart, including the outflow tract but except the ventricles, with differing frequency depending on the position within the pSHF; cells that lie more cranially tend to contribute to the atrioventricular canal (AVC) and atria, whereas those more caudally generate the sinus venosus, but there is considerable intermixing of fate throughout the pSHF.
Although the left and right pSHF contribute symmetrically to the outflow tract and the sinus venosus, they add asymmetrically and contralaterally to the AVC and atria, with the left pSHF moving to dorsal left atrium and superior AVC, whereas right pSHF contributes to right atrium, ventral left atrium, and inferior AVC.
This same, unexpected, contralateral development is also demonstrated by clonal analyses, which show that the left atrium is clonally related to the superior AVC and the right atrium to the inferior AVC.
The second heart field, part of the embryonic splanchnic mesoderm, contains the progenitors of all regions of the heart excluding the left ventricle. Knowing how these progenitors are arranged within this field is required to understand heart development and may help explain some congenital defects. We examined the contributions of the posterior region of the second heart field, which has 2 separate halves, on the left and right of the embryo. Unexpectedly, these halves contributed similarly to the poles of the heart, the outflow tract, and sinus venosus, but quite differently to the atrioventricular canal and the atria. The dorsal left atrium and superior atrioventricular canal are derived from left pSHF, whereas the ventral left atrium, inferior atrioventricular canal, and right atrium form from the right pSHF. Charting these movements of cells adds a novel part to the understanding of the tissue morphogenesis of cardiac looping and chamber formation. Future studies of these processes, and related congenital defects, should include further searching for signaling pathways and their cellular responses that are left-right asymmetrical.