mesoderm migrate toward apical direction and are strengthened—
eventually become highly concentrated at the apical edge of the
cells [7].
Adherens junction levels during the EMT of Drosophila mesoderm primordium do not change unidirectionally. Instead, there is
a transient upregulation following the initial downregulation
[7]. EMT in those cells is driven by the transcription factor Snail,
a conserved master regulator of EMT in many other systems
[10]. In the early Drosophila embryo, Snail is zygotically expressed
specifically in the mesoderm and gradually accumulates to high
levels from before cellularization to gastrulation [7, 11]. This
high level of Snail eventually drives the mesoderm to undergo
EMT [12]. Although the disassociation of cells only occurs after
the folding of the mesoderm, it was shown that the adherens
junctions start to be broken down before the epithelial folding
event [7]. This is consistent with many of Snail’s transcriptional
targets being activated or repressed before ventral furrow formation
[13–15]. However, during the epithelial folding, the contractile
myosin, which drives the apical constriction and eventually the
folding of the mesodermal epithelium, relocates and strengthens
adherens junctions despite the continued expression of Snail
(Figs. 2 and 3). Such a reverse in junction levels is possible because
junctions are downregulated by Snail at the step of assembly and
disassembly [8]. Only after the mesoderm is completely internalized and myosin is inactivated, does Snail-dependent junction
disassembly resume, and mesodermal cells undergo a full EMT.
The approach described here relies on mounting the embryos
directly on the cover glass taking advantage of the fact that both the
embryos and the glass are hydrophobic and therefore stick to each
other. This method for live imaging embryos avoids the use of glue
and allows continued readjustment of the orientation of the
embryo. While it creates a small footprint of the embryo on the
glass, within this area light goes through only the vitelline membrane, glass, and emersion oil, thus greatly improving image quality. We will discuss how to identify healthy embryos of the right
stages and orientation, as this is also critical for the efficient imaging. Finally, how fast imaging can be achieved through the reduction of photobleaching will also be discussed.
ä
Fig. 2 (continued) Nuclei on the ventral side appear to be less aligned, indicating the initiation of apical
constriction. Gastrulation begins. (f) Gastrulation proceeds with the ventral furrow already formed and the
posterior midgut reaching the dorsal side
Live Imaging of EMT in Chick Neural Crest
69
eventually become highly concentrated at the apical edge of the
cells [7].
Adherens junction levels during the EMT of Drosophila mesoderm primordium do not change unidirectionally. Instead, there is
a transient upregulation following the initial downregulation
[7]. EMT in those cells is driven by the transcription factor Snail,
a conserved master regulator of EMT in many other systems
[10]. In the early Drosophila embryo, Snail is zygotically expressed
specifically in the mesoderm and gradually accumulates to high
levels from before cellularization to gastrulation [7, 11]. This
high level of Snail eventually drives the mesoderm to undergo
EMT [12]. Although the disassociation of cells only occurs after
the folding of the mesoderm, it was shown that the adherens
junctions start to be broken down before the epithelial folding
event [7]. This is consistent with many of Snail’s transcriptional
targets being activated or repressed before ventral furrow formation
[13–15]. However, during the epithelial folding, the contractile
myosin, which drives the apical constriction and eventually the
folding of the mesodermal epithelium, relocates and strengthens
adherens junctions despite the continued expression of Snail
(Figs. 2 and 3). Such a reverse in junction levels is possible because
junctions are downregulated by Snail at the step of assembly and
disassembly [8]. Only after the mesoderm is completely internalized and myosin is inactivated, does Snail-dependent junction
disassembly resume, and mesodermal cells undergo a full EMT.
The approach described here relies on mounting the embryos
directly on the cover glass taking advantage of the fact that both the
embryos and the glass are hydrophobic and therefore stick to each
other. This method for live imaging embryos avoids the use of glue
and allows continued readjustment of the orientation of the
embryo. While it creates a small footprint of the embryo on the
glass, within this area light goes through only the vitelline membrane, glass, and emersion oil, thus greatly improving image quality. We will discuss how to identify healthy embryos of the right
stages and orientation, as this is also critical for the efficient imaging. Finally, how fast imaging can be achieved through the reduction of photobleaching will also be discussed.
ä
Fig. 2 (continued) Nuclei on the ventral side appear to be less aligned, indicating the initiation of apical
constriction. Gastrulation begins. (f) Gastrulation proceeds with the ventral furrow already formed and the
posterior midgut reaching the dorsal side
Live Imaging of EMT in Chick Neural Crest
69