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First published online 2 June 2004
doi: 10.1242/dev.01185

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Organization of cardiac chamber progenitors in the zebrafish blastula

¹ Developmental Genetics Program and Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA
² Department of Developmental Biology, Biology I, University of Freiburg, Freiburg, Germany

View larger version (85K): [in a new window]   Fig. 1. Labeling chamber progenitors by activating caged fluorescein. (A) Lateral view of zebrafish blastula at 40% epiboly, animal pole to the top, dorsal to the right. Red arrowheads indicate the embryonic margin, where the blastoderm meets the yolk. Nuclei of the yolk syncytial layer are visible below the margin. White arrows demarcate blastomere tiers 1, 2, 3 and 4, counting up from the margin. (B) In this example, caged fluorescein is activated in 2 neighboring blastomeres. (C) Overlay of DIC image and fluorescent image, pseudocolored in green, demonstrates that the labeled blastomeres are in tier 1. (D) Animal view, dorsal towards the right. After incubation at 20°C for 1 hour, the longitudinal position of labeled cells (arrowhead) is recorded as an angular measurement around the circumference of the embryo, with the center of Tg(gsc:gfp) expression (arrow) defined as 0°. In this embryo, the labeled cells occupy positions 90-95° from dorsal. (E-L) Assessment of cardiac contributions at 44 hpf. Lateral views of the heart, anterior towards the left. Labeled myocardial (E,F,I,J) and endocardial (G,H,K,L) progeny are marked with a blue precipitate, and myocardium is counterstained with a pink precipitate (see Materials and methods). Identities of labeled cells are determined by morphology and location, either in whole mount (E,G,I-L) or in longitudinal sections (F,H). We show a series of typical results. (E) After labeling three blastomeres in tier 1, 105-115° from dorsal, we found six labeled cells in the ventricular myo cardium (arrowhead). (F) Sectioning confirms the myocardial identity of the labeled cells in E (arrowhead). (G) After labeling two blastomeres in tier 4, 125-130°, we found multiple labeled cells in the atrial endocardium (arrow). (H) After labeling two blastomeres in tier 2, 88-9 3°, we found multiple clusters of labeled cells in the atrial endocardium, shown via sectioning (arrows). (I) After labeling 2 blastomeres in tier 1, 87-93°, we found six myocardial cells arranged along the long axis of the ventricle (arrowhead). (J) After labeling two blastomeres in tier 2, 122-128°, we found five myocardial cells arranged along the long axis of the atrium (arrowhead). (K) After labeling two blastomeres in tier 1, 110-118°, we found labeled cells in the ventricular endocardium (arrow). (L) After labeling two blastomeres in tier 2, 92-98°, we found a ch ain of endocardial cells extending from the atrium to the ventricle (arrows). Open arrowheads indicate the plane of the atrioventricular boundary.

View larger version (106K): [in a new window]   Fig. 5. Progenitor location prior to gastrulation correlates with location of progeny in LPM. (A-D) Lateral views at tailbud stage, rostral towards the top. Blue precipitate indicates locations of cells derived from labeled blastomeres. (E-H) Higher magnification views of embryos shown in (A-D). Blastomeres located 62-86° (A,E) or 67-95° (B,F) from dorsal in tier 1 contribute to rostral and medial regions of the LPM. By contrast, blastomeres located 94-120° (C,G) or 120-144° (D,H) from dorsal in tier 3 contribute to relatively caudal and lateral regions of the LPM. (I-M) Dorsal views of embryos at 22 somites (I-K,M) and 20 somites stages (L), anterior towards the top. In situ hybridization indicates expression of cmlc2 (I,L,M), vmhc (J),and amhc (K) with a blue (I-K) or pink (L,M) precipitate. Progeny of labeled blastomeres are indicated with a blue precipitate (L,M). cmlc2 is expressed in all cells of the myocardial cone (I), with more robust expression in the central region that also expresses vmhc (J) than in the peripheral portion that also expresses amhc (K) (Yelon et al., 1999; Berdougo et al., 2003). (L) Blastomeres located 101-107° from dorsal in tier 1 contribute to the central region of the myocardial cone. (M) By contrast, blastomeres located 110-116° from dorsal in tier 3 contribute to the peripheral region of the myocardial cone.

View larger version (45K): [in a new window]   Fig. 6. Antagonism of Nodal signaling alters myocardial fate assignment in tier 1 blastomeres. (A-D) Whole-mount immunofluorescence with the anti-myosin heavy chain monoclonal antibodies MF20 (TRITC) and S46 (FITC) at 36 hpf. (A,B,D) Lateral views, anterior towards the left. (C) Dorsal view, anterior towards the top. (A) In wild-type (wt) embryos, double exposure indicates MF20 staining in the ventricle (red, V), and overlap of MF20 and S46 staining in the atrium (yellow, A). In embryos injected with small amounts of lefty1 mRNA (B-D), we observe variable reduction of myocardium, ranging from a moderately reduced ventricle and a subtly affected atrium (B) to severe reductions of ventricular tissue together with significant reductions of atrial tissue (C,D). In addition, ectopic expression of lefty1 can disrupt endoderm formation, causing cardia bifida as shown in C (Yelon, 2001). (E,F) Examples of myocardial contributions by tier 1 blastomeres in embryos injected with lefty1. Labeled myocardial cells (blue precipitate) are examined with reference to an atrium-specific marker, amhc (pink precipitate) (Berdougo et al., 2003). (E) Frontal view at 36 hpf, dorsal towards the top. The amount of myocardium present resembles that in the embryo depicted in B. In this embryo, blastomeres located 65-150° from dorsal in tier 1 contributed to both atrial myocardium (arrows) and ventricular myocardium (below eyes and out of focal plane). (F) Dorsal view at 36 hpf, anterior towards the top. The amount of myocardium present resembles that in the embryo depicted in C. In this embryo, blastomeres located 90-135° from dorsal in tier 1 contributed to atrial myocardium (arrow). (G) Fate map of myocardial chamber progenitors in embryos injected with lefty1. Data are depicted as described for the myocardial fate map (Fig. 2B), except that experiments were performed only in tier 1. In this map, bar colors correspond to the myocardial contribution of labeled cells: red, contribution to ventricular myocardium; yellow, contribution to atrial myocardium; cyan, contribution to both ventricular and atrial myocardium; black, no myocardial progeny. When Nodal signaling is antagonized, both ventricular and atrial myocardial progenitors reside in tier 1. The dorsoventral organization of these progenitors roughly resembles that observed in tiers 2 and 3 of the wild-type fate map (Fig. 2), in that blastomeres located more dorsally than 125° give rise to ventricular myocardium and blastomeres located more ventrally than 90° give rise to atrial myocardium.

View larger version (42K): [in a new window]   Fig. 4. Multiple lineages arise from the lateral marginal zone. Although we did not score non-cardiac lineages thoroughly, we noted the origins of some frequently detected cell types. (A,C,E,G) Lateral views at 44 hpf, anterior towards the left, showing examples of labeled cells (arrows). (B,D,F,H) Fate maps indicating experiments in which labeled blastomeres gave rise to head vessels, circulating blood cells in cranial locations, pharyngeal pouches or pectoral fin mesenchyme. Progenitors for head endothelium (A,B) and presumed myeloid cells (C,D) are found 45-100° from dorsal in tiers 1 and 2. Progenitors for pharyngeal pouches (E,F) and pectoral fin mesenchyme (G,H) are found 50-150° from dorsal in tiers 1-4.

View larger version (25K): [in a new window]   Fig. 2. Ventricular and atrial myocardial progenitors are spatially organized at 40% epiboly. (A) The coordinates of our fate map projected onto a lateral view at 40% epiboly; background image adapted, with permission, from Karlstrom and Kane (Karlstrom and Kane, 1996). Latitude, on the vertical axis, is expressed in tiers, or cell diameters, from the margin. Longitude, on the horizontal axis, is expressed in degrees around the circumference of the embryo, with the center of Tg(gsc:gfp) expression defined as 0°. (B) Fate map of myocardial chamber progenitors. Each horizontal bar represents an individual experimental embryo. The position of a bar on the map corresponds with the location of labeled blastomeres. Bar color indicates myocardial contribution of labeled cells: red, contribution to ventricular myocardium; yellow, contribution to atrial myocardium; black, no myocardial progeny. All data from Table 1, except for experiments labeling five blastomeres, are included. As progenitor distribution, chamber contribution and density appear equivalent on both the left and right sides of the embryo (Table 1), the data are summarized together in the fate map. Compiling all data, ventricular myocardial progenitors are found 60-125° from dorsal in tie rs 1, 2 and 3. By contrast, atrial myocardial progenitors are found 90-140° from dorsal in tiers 2, 3 and 4.

View larger version (14K): [in a new window]   Fig. 3. Ventricular and atrial endocardial progenitors are intermingled at 40% epiboly. The fate map of endocardial chamber progenitors is depicted as described for the myocardial fate map (Fig. 2B). In this map, bar colors correspond to the endocardial contribution of labeled cells: red, contribution to ventricular endocardium; yellow, contribution to atrial endocardium; cyan, contribution to both ventricular and atrial endocardium; black, no endocardial progeny. All data from Table 2, except for the experiment labeling five blastomeres, are included. Endocardial progenitors are found 55-140° from dorsal in all four tiers.

View larger version (81K): [in a new window]   Fig. 7. Model of myocardial morphogenesis. The spatial organization of myocardial chamber progenitors at 40% epiboly and the locations of their progeny at tailbud and mid-somitogenesis stages suggest orderly migration of these populations. (A-I) Schematic representations of zones containing ventricular myocardial progenitors (red) or atrial myocardial progenitors (yellow). Zones containing both ventricular and atrial progenitors are depicted in red and yellow stripes. (A) Representation of myocardial fate map at 40% epiboly; primary data shown in Fig. 2B. (B-F) Frames from an animated model of myocardial morphogenesis (see Movie 1 at http://dev.biologists.org/supplemental); lateral views, dorsal towards the right, at (B) 40% epiboly, (C) shield, (D) 70% epiboly, (E) 85% epiboly and (F) tailbud stages. Background images adapted, with permission, from Karlstrom and Kane (Karlstrom and Kane, 1996). (G-I) Dorsal views, anterior towards the top, depicting myocardial progenitor zones within the LPM at (G) 5 somites, (H) 15 somites and (I) 22 somites.