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Fig. 2. Apical attachment of the dorsal forerunner cell cluster to the
enveloping layer and subsequent detachment to form a cellular rosette during
gastrulation. (A-B') Confocal images of
Tg(β-actin:HRAS-EGFP) zebrafish embryos immunolabelled
with an anti-aPKC-
antibody. GFP (green) and aPKC- (red) channels
are merged. Single dorsal focal planes (A,B) and sagittal sections at the
position of the dotted line (A',B'; embryo surface to the right)
are shown. Animal pole is to the top. A surface plane through an embryo at 50%
epiboly shows that presumptive DFCs (asterisks) are partially positioned
underneath the EVL (A). Clear enrichment of aPKC- is found at the
contact point between one of the DFCs and the overlying EVL (A,A';
arrowheads). At 75% epiboly, the EVL completely covers the DFCs (B). A
sagittal section shows bottle-shaped DFCs with strong aPKC- enrichment
at the contact points with the EVL (B'; arrowheads). (C-D')
Confocal images of Tg(sox17:GFP)-expressing embryos immunolabelled
with an anti-ZO-1 antibody. GFP (green) and anti-ZO-1 (red) channels are
merged. At 80% epiboly, DFCs form a compact cluster that is in contact with
the interior surface of the EVL close to the margin. Anti-ZO-1 signal is
enriched at contact points (arrowheads) between DFCs and the overlying EVL
(dorsal z-projection is shown in C; sagittal section at the position
of the dotted line in C'). At the bud stage, a ZO-1-rich focal point
(arrowhead) is seen at the centre of the DFC cluster (single focal plane is
shown in D; sagittal section at the position of the dotted line in D').
The cluster is disconnected from the overlying EVL. (E-F') Images
of a time-lapse multi-photon confocal movie of a
Tg(β-actin:HRAS-EGFP) embryo, showing DFC cluster
arrangement and tracing of two DFCs (green and yellow dots). Single dorsal
focal planes (E,F) and sagittal sections at the position of the dotted line
(E',F') are shown. At 90% epiboly, several bottle-shaped DFCs are
attached to the overlying EVL via a single focal point (asterisk), including
the two marked cells (E,E'; arrow indicates the position of the EVL). At
+102 minutes, the marked cells have internalised and become part of a DFC
rosette that is arranged around an interior focal point (F,F'; focal
point marked by asterisk). Scale bars: 30 µm.
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