1 Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA
2 Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA
*Author for correspondence (e-mail: jl1{at}astro.ocis.temple.edu)
Accepted March 26, 2001
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SUMMARY |
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Key words: Fate map, Heart-forming region, Bmp2, Nkx2.5, Chick
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INTRODUCTION |
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Recently, Erhman and Yutzey (Erhman and Yutzey, 1999) placed the lateral border of the HFR (stage 5) at the lateral most region of the mesoderm, adjacent to the extra-embryonic tissues, more lateral than that proposed by earlier studies (Rawles, 1936; Rawles, 1943; Rudnick, 1948; DeHaan and Urspurng, 1965; Rosenquist and DeHaan, 1966; Rosenquist, 1966; Rosenquist, 1970); and the posterior border at the level of HN. At stage 8, the posterior boundary of the HFR was placed at the level of the first condensing somite (Erhman and Yutzey, 1999), which is more anterior than that previously defined by others (Rawles, 1936; Rawles, 1943; Rudnick, 1948; DeHaan and Urspurng, 1965; Rosenquist and DeHaan, 1966; Rosenquist, 1966; Rosenquist, 1970). In addition, recent findings by Colas et al. (Colas et al., 2000) describe the heart as arising from paired and separated regions rather than a single crescent, which fuses at the midline between stages 9- and 9. Thus, the boundaries of the HFR (between stages 4 and 8) and the origin of the cells that contribute to the various regions of the developing heart remain unclear. In order to clarify this issue, we have generated cardiac fate maps from stages 4-8 of avian development.
The newly established boundaries generated by us accurately represent the HFR. The cardiogenic fate map of the stage 4 embryo presented in this paper is the first one ever to be generated and reported. Our fate maps were generated by labeling a small group of cells/embryo with DiI (1,1'-dioctadecyl-3, 3,3', 3'-tetramethyl-indocarbocyanine perchlorate), which, although simple, is more direct and technically accurate than the earlier experiments (Rawles, 1936; Rawles, 1943; Rosenquist and DeHaan, 1966; Erhman and Yutzey, 1999). We also discuss the boundaries of our fate maps in relation to the expression of putative cardiogenic molecular markers Bmp2 (Wozney et al., 1988) and Nkx2.5 (Bodmer, 1993), which have been proposed to govern events during early heart development. Our data suggest that neither of these molecules accurately mark the entire cardiogenic region during early embryogenesis. These newly generated fate maps accurately represent the in vivo cardiogenic region and will serve as a standard for studies on heart development.
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MATERIALS AND METHODS |
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In all cases, embryos were imaged identically to ensure that the original proportions of each embryo was maintained. Fig. 1 shows an example of the imaging process. The fluorescent and bright field images at each time point, 0 and 20 hours post-injection, were superimposed in Adobe PhotoShop 5.5 and the difference option in the show layers function was used to determine the exact position of DiI label at the two time points separately. The embryo from which the data was derived is identified by a number, which appears on the grids.
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DiI localization using the grid system
Representative images of embryos at stages 4-8 were used as templates to record injection sites at 0 hour. A grid was superimposed on each template. The axis of the embryo was placed centrally on the grid at column location 0. Equidistant columns to the left and right of the PS were designated from 1 to 7 and +1 to +8, respectively. Equidistant rows (from A-S) intersected the vertical columns to create the grid. At stage 4, the rostralmost point of HN was positioned on the line between rows F and G, and the area opaca/area pellucida (AO/AP) boundary in the anterior region (above HN) was in the middle of row A. At stage 5, the line between rows E and F was positioned in the region of HN, where an asymmetric deflection is seen to either the left or right. The AO/AP boundary was above row A. At stage 6, the beginnings of the head fold was immediately above the line between rows B and C, and the regressed HN in row J. The AO/AP boundary was in row A. At stage 7, the first somite was placed in row G, the regressed HN in row L and the head fold in the row immediately above row A. At stage 8, the first (anteriormost) somite was placed in row H, and the arch above the anterior intestinal portal (AIP) in the row above row A. An identical grid, as used on the template image, was placed over the composite bright field and fluorescence image of each embryo at 0 hours in Adobe PhotoShop 5.5.
Owing to small variations in the size of embryos at comparable developmental stages, an absolute measure of position was not used to mark the location of the labeled cells. Instead, we used the Cartesian coordinate grid system in which the coordinate points in the grid were fixed, but the dimensions of the grid were adjusted proportionately to fit the size of each embryo (Tam and Schoenwolf, 1999). In addition, to correct for the minor variations in size of the embryos, relative distances from the site of injection were measured (1) to HN and the PS for stage 4, (2) from the regressed HN and the PS at stage 5, (3) from HN, the PS and the head fold at stage 6, and (4) from the newly forming somites, the central axis and the head fold at stages 7 and 8. The AO/AP were also used as guides in marking the injection site on templates. This allowed for accurate transposition of injection site location onto the respective stage-specific template. The accurate transposition of the site of injection onto the templates was also preserved because all embryos were carefully imaged using identical microscope and imaging parameters. The movement of the DiI dye 20 hours post-injection was documented and correlated (in terms of location), to the initial site of injection.
In situ hybridization
Immediately after labeling, embryos (stages 5-8) were imaged as described above and processed for in situ hybridization according to Wilkinson (Wilkinson, 1992) with modifications as recommended by Schultheiss (Schultheiss et al., 1995). The digoxigenin-labeled Nkx2.5 antisense mRNA probe was generated by in vitro transcription (Boheringer Mannheim). The Nkx2.5 cDNA was generously provided by T. Schultheiss (Harvard Medical School, Boston, MA). As a positive control, stage 11 embryos were stained for Nkx2.5 expression using the probe described above. The alkaline phosphatase reaction was developed for 24 hours for stages 5 and 6, and for 2 hours for stages 7-8 and 11 embryos. These reaction times were similar to those used by T. Schultheiss (personal communication).
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RESULTS |
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HFR at stage 6
The boundaries of the HFR were deduced from the sum of the data presented in Fig. 2F (n=28). Table 2 provides detailed information on the final location of all DiI-labeled cells for each embryo injected at stage 6. Cells located in the anterior region of the embryo, adjacent to the embryonic axis (columns 1 and +1), and those located to the far lateral regions (columns -4 and +4) of the AP, adjacent to the AO, did not incorporate into the heart. Labeled cells anterior and lateral to the regressing HN in columns 2 and 3 on the left side of the PS and +2 and +3 on the right side of the PS contributed to the heart. The anterior boundary of the HFR was at row E, and the posterior boundary was just posterior to the regressed HN in row J. At this stage, the HFR (black rectangles) overlaps the region stained for Bmp2 mRNA (Fig. 2G) to a greater extent than at stages 4 and 5, but is not completely coincident. At stage 6, the expression of Nkx2.5 mRNA encompasses only the anterior half of the HFR (Fig. 2H).
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Specificity of labeling cells in the mesodermal layer
To ensure that cells in the mesoderm were labeled, stages 4 (Fig. 4) and 5 (data not shown) embryos (n=10) injected with DiI or DiO were immediately fixed post-injection, sectioned using a vibratome and viewed using fluorescence microscopy to locate DiI- or DiO-labeled cells. In eight out of the ten cases, labeled cells were detected solely in the mesoderm. In one case, labeled cells were detected in the mesoderm and ectoderm and in another embryo they were detected in the mesoderm and endoderm.
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DISCUSSION |
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Small groups of cells labeled with DiI were followed and their collective fate was observed 20 hours post-injection. The data showed that in some cases, all labeled cells in a group were confined to only one region of the embryo (e.g. stage 8, embryo number 107; Fig. 3D), whereas in other instances, the labeled descendants were visualized in multiple regions of the embryo (e.g. stage 8, embryo number 570; Fig. 3D).
Our studies clearly showed that, at stage 4, the anterior border is just above the level of HN and extends to one quarter the distance of the PS, below HN. The medial border was
0.3 mm from the primitive groove (PG), and the lateral border extended almost to the AO/AP boundary. This is the only published fate map of the HFR generated at stage 4 in the chicken embryo. The cardiogenic specification marker; Nkx2.5 (Lough and Sugi, 2000), has not been reported to be detected by in situ hybridization until stage 5 (Schultheiss et al., 1995), and therefore cannot be used to ascertain the location of cardiogenic cells prior to stage 5. However, Bmp2 expression can be detected at stage 4, but only in the lateral endoderm at the level of the anterior PS (Schultheiss et al., 1997) and, conversely, at low levels in the mesodermal layer (Andree et al., 1998). The discrepancy in location of Bmp2 at stage 4 in these two studies remains unresolved. In addition, the expression of Bmp2 was not restricted to the HFR, but was also detected in the head fold and posterior PS (Schultheiss et al., 1997; Andree et al., 1998). Our data clearly show that Bmp2 expression is not coincident with the HFR, thereby suggesting that Nkx2.5 and Bmp2 cannot be used to define the HFR.
Another observation shown in Fig. 1 is that labeled cells on the right of the PS (ventral side up) appear to contribute to the C shaped wall of the ventricle. Our data are consistent with the findings of Stalsberg (Stalsberg, 1969). A few sentences from her paper are relevant: She stained the presumptive heart regions of amphibian embryos with vital dyes and stated that the ventricle was later seen to be formed predominantly by material from the left side. She concluded that the primary loop of the heart is caused by an over growth of the left-sided material in the ventricular region (Stalsbergs description of the work of Wilens, 1955). At stage 10-, significantly more cells have been contributed to the epimyocardium from the right side than from the left side. This relation is maintained in the cephalic part of the bulboventricular loop at later stages. In the caudal part of the bulboventricular loop, the relation is reversed, with significantly more cells originating from the left side. The asymmetry in cell numbers may be the expression of a primary difference between the developmental potencies of right and left heart primordia through which the laterality of the heart loop formation is determined (Stalsberg, 1969). Therefore, our data are consistent with those of Wilens and Stalsberg, such that most of the ventricular loop labeled with DiI originated from cells on the left side of the embryo (Fig. 1A is the ventral view, therefore the right side).
The prospective HFR at stage 5 as defined by Rawles (Rawles, 1936; Rawles, 1943), by donor/host chorio-allantoic membrane transplant experiments, showed that the HFR was localized on either side of the PS extending from the anterior most point of the head process down to half the length of the PS (Fig. 8 from Rawles, 1936) (Fig. 6 from Rawles, 1943). The medial boundary was 0.13 mm from the primitive groove (PG) and extended almost to the AO/AP boundary. Studies using labeled grafts (Rosenquist and DeHaan, 1966) identified the HFR as being similar to that defined by Rawles, although the medial border was more lateral,
0.3 mm from the PG. More recently (Erhman and Yutzey, 1999), molecular markers (Nkx2.5 and Bmp2; ventricular myosin heavy chain 1 (VMHC1) (Bisaha and Bader, 1991); and antibody against sarcomeric myosin (Bader et al., 1982)) were used to identify the HFR at stages 5 and 7. In Erhman and Yutzeys study, the lateral boundary of the HFR was placed adjacent to the extra embryonic tissue and the medial boundary at the lateral border of the mesoderm overlying the prospective neural plate, a more laterally located HFR than previously described (Rawles, 1936; Rawles, 1943; Rudnick, 1948; DeHaan and Urspurng, 1965; Rosenquist and DeHaan, 1966; Rosenquist, 1966; Rosenquist, 1970). The posterior boundary was determined to be at the level of HN (Erhman and Yutzey, 1999). These experiments (Erhman and Yutzey, 1999), because of the techniques and marker (VMHC1) used, more appropriately define the ventricle-forming region rather than the entire HFR. Our in vivo studies at stage 5 mapped the anterior border of the HFR anterior to HN, and the posterior border
one quarter the distance of the PS below HN. The medial border was
0.3 mm from the PG, similar to that identified by Rosenquist (Rosenquist and DeHaan, 1966) but inconsistent with that of Rawles (Rawles, 1936; Rawles, 1943) and the lateral border was close to the AO/AP border. Our data do not agree with the recent mapping study (Erhman and Yutzey, 1999), in that the anterior border of the HFR in our study was coincident with the posterior border of Nkx2.5 with minimal, if any, overlap. To confirm that cells within our HFR but outside of the Nkx2.5 expression domain contribute to the heart, we injected stage 5 embryos (outside of the Nkx2.5 expression domain but within the HFR shown in Fig. 2C) and either processed them for in situ hybridization at 0 hours or sectioning 20 hours post-injection (Fig. 6). Very low levels of Nkx2.5 mRNA were detected at stage 5 (confirmed by T. Schultheiss, personal communication). These data confirmed that the HFR extended posterior to the region of Nkx2.5 expression (Fig. 6A,B) and that the labeled cells outside the Nkx2.5 expression domain contributed to the heart (Fig. 6D-G).
According to the classical mapping studies, cardiac cells at stage 6 should lie in a crescent-shaped region on either side of a central embryonic axis, anterior and lateral to the regressed HN, in the lateral mesoderm (DeHaan and Urspurng, 1965; Rosenquist and DeHaan, 1966). At stage 7, the cells move closer towards the head fold region and fuse above the AIP between stages 6 and 7 (DeHaan, 1963a). We identified HFR cells above the AIP at stage 8, but not at stages 6 and 7, which suggests that the heart primordia fuse above the AIP between stages 7 and 8. The posterior border of the HFR at stage 8 extended to the fourth somite, which is more caudal than previously described (Erhman and Yutzey, 1999; DeHaan and Urspurng, 1965). This location of the HFR is further corroborated by our data on chamber specification (Patwardhan et al., 2000). The discrepancy in the location of the posterior boundary of the HFR at stage 8, can be explained by the fact, that we looked at cells contributing to all regions of the heart, while others (Erhman and Yutzey, 1999) have focused on ventricle-forming cells alone. We would place the posterior border of the ventricle-forming region at the same level as Erhman and Yutzey (Erhman and Yutzey, 1999), and this boundary is coincident with the posterior boundary of Nkx2.5 expression domain (Fig. 3D,E), suggesting that Nkx2.5 may better define the ventricle-forming region than the HFR.
Our data address another important aspect, which is rostrocaudal patterning during heart development. There is no direct evidence in the literature to suggest that cardiogenic cells continue to be ordered in a rostral-to-caudal direction at stages 4 and 5, as they are in the PS at stage 3 (Garcia-Martinez and Schoenwolf, 1993). Schoenwolf created quail-chick chimeras at stage 3 and observed the location of cells in the heart at stage 10, although this does not tell one exactly what happened at stages 4-9. The findings of Garcia-Martinez and Schoenwolf (Garcia-Martinez and Schoenwolf, 1993) and those of DeHaan (DeHaan, 1963a; DeHaan, 1963b) have been used by others to conclude that the cardiogenic cells, after gastrulation, remain in the same organized pattern between stages 3 (Garcia-Martinez and Schoenwolf, 1993) and 12 (DeHaan, 1963a; DeHaan, 1963b, reviewed by Fischman and Chein, 1997).
On the contrary, we found that between stages 4 and 6, the cardiogenic cells are not organized in a rostral to caudal pattern. Similar findings of DeHaan (DeHaan, 1963a) where iron oxide particles attached to the endoderm were used to track the movement of migrating cells between stages 5 and 12 were described as At stage 5 the regions of the thickened mesoderm (LHFR,RHFR) are broad and rather diffuse; however, they match nicely the areas shown to have heart forming capacity as explants. If the movements of individual clusters are traced on film through subsequent stages, it is noted that for the first few hours during stages 5-6, the direction of migration of a given cluster may bear no relation to its later "goal" at the AIP. Clusters move at different speeds and in different directions from their neighbors...With development through stages 6 and 7, the anterior medial border of each mass of heart-forming cells extends forward and mesiad forming the crescent of cardiogenic material which arcs rostral to the precordal plate. At this time a pattern of organization of the clusters within the crescent emerges, relating their position with their later differentiation (DeHaan, 1965). DeHaan also detected clumps of cells that started in rostral positions but ended up in a caudal position (DeHaan, 1963a).
Our data are similar to those of DeHaans, except that we would say that this pattern begins to form at stage 7 and a definitive pattern re-established by stage 8. We confirmed this trend by labeling four groups of cells (two on either side of the embryo) in the HFR with DiI and DiO, and followed them at intervals over 20 hours. The rationale for using multiple dyes was that in our experience it is misleading to inject a single embryo in multiple sites with the same tag. This process introduces error as it is difficult to monitor and identify each tagged group of cells over time. We observed that an overall rostrocaudal pattern prevailed but there was considerable overlap of the proliferating cell populations within that pattern. This can be seen in Fig. 5 where the general rostrocaudal relationship of the red and green labeled cells is maintained though a considerable overlap of the caudal cells (green) into the rostral cells (red) was observed.
In summary, our studies have established accurate boundaries for the HFR. Moreover, our data present evidence that the proposed putative cardiogenic markers Bmp2 and Nkx2.5 are not coincident with the HFR between stages 4 and 8. Therefore, even though these well characterized early cardiac regulators may have a functional role in cardiac development (Erhman and Yutzey, 1999; Schultheiss et al., 1995; Schultheiss et al., 1997), they fall short of demarcating the entire HFR during early developmental stages. Furthermore, neither of these molecules has been shown to be sufficient for cardiac commitment or differentiation, making Nkx2.5 and Bmp2 erroneous candidates as HFR markers. Therefore, we believe that it is inaccurate to use these markers to identify the HFR. Finally, our highly accurate fate maps, including the stage 4 HFR fate map (reported for the first time), can now be used as a benchmark for studies involving cellular and molecular events during cardiac development.
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ACKNOWLEDGMENTS |
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