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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.
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