discrete cell states along the epithelial-mesenchymal spectrum of
phenotypes has been raised [4, 5]. Therefore, observing EMT
progression of living animals is a powerful tool to understand
what the intermediate cell states are, and how cells reach these
intermediate states.
One of the central steps during EMT is the loss of cell-cell
junctions, so that the cells restricted within the epithelial sheet can
dissociate from each other [6]. As the basic structure to connect
cells and support cell shapes, cell-cell junctions not only respond to
EMT signaling, but also are regulated by many other intrinsic and
extrinsic cues, including other signals or physical tension. Therefore, cell-cell junctions such as adherens junctions can act as a
platform where other inputs modulate EMT progression. Indeed,
EMT during development often happens simultaneously with
other morphogenic events. During gastrulation in Drosophila melanogaster (Drosophila), the mesoderm primordium undergoes both
EMT and mechanical tension-driven epithelial folding. While the
EMT program aims to disassemble junctions, epithelial folding
requires strong junctions to maintain tissue integrity and effect
tissue-wide shape changes. It was shown that mechanical tension
not only drives epithelial folding but also overrides the EMT program to strengthen adherens junctions [7, 8]. This provides an
excellent system to study EMT in the context of epithelial
morphogenesis.
Drosophila embryos have several characteristics that facilitate
high-quality live imaging and in order to understand early embryo
morphogenesis, it is important to do live imaging properly. The
adherens junctions in early Drosophila embryos are very close to the
embryo surface and therefore can be steadily imaged with laser
scanning confocal microscopy. Before gastrulation, Drosophila
embryos consist of a single layer of epithelial cells surrounding
the yolk with apical surfaces facing outside (Fig. 1a). Cells are
about 30 μm tall and 6–7 μm wide, with spot adherens junctions
localized in a relatively narrow zone within 6–7 μm below the apical
surface (Fig. 1b). This single layer of epithelial cells is generated
during a process called cellularization, where cell membranes of the
syncytial embryo gradually ingress between nuclei and grow deeper
to eventually enclose the nuclei and cytoplasm surrounding the
yolk. Cellularization happens 2 h after egg laying and it takes
50 min to 1 h for the cellularization membrane front to reach the
end of the cytoplasm. Therefore, the depth of cellularization front
from the embryo surfaces can be used to estimate the embryo’s age
for live imaging (Fig. 2). Spot adherens junctions are formed in all
the cells during early cellularization and appear as clusters of
cadherin-catenin complexes around the subapical region and only
become more belt-like much later in the development. No other
junctions, such as septate junctions and focal adhesions, have
formed during this early stage of development [9]. Immediately
66
Lingkun Gu and Mo Weng
phenotypes has been raised [4, 5]. Therefore, observing EMT
progression of living animals is a powerful tool to understand
what the intermediate cell states are, and how cells reach these
intermediate states.
One of the central steps during EMT is the loss of cell-cell
junctions, so that the cells restricted within the epithelial sheet can
dissociate from each other [6]. As the basic structure to connect
cells and support cell shapes, cell-cell junctions not only respond to
EMT signaling, but also are regulated by many other intrinsic and
extrinsic cues, including other signals or physical tension. Therefore, cell-cell junctions such as adherens junctions can act as a
platform where other inputs modulate EMT progression. Indeed,
EMT during development often happens simultaneously with
other morphogenic events. During gastrulation in Drosophila melanogaster (Drosophila), the mesoderm primordium undergoes both
EMT and mechanical tension-driven epithelial folding. While the
EMT program aims to disassemble junctions, epithelial folding
requires strong junctions to maintain tissue integrity and effect
tissue-wide shape changes. It was shown that mechanical tension
not only drives epithelial folding but also overrides the EMT program to strengthen adherens junctions [7, 8]. This provides an
excellent system to study EMT in the context of epithelial
morphogenesis.
Drosophila embryos have several characteristics that facilitate
high-quality live imaging and in order to understand early embryo
morphogenesis, it is important to do live imaging properly. The
adherens junctions in early Drosophila embryos are very close to the
embryo surface and therefore can be steadily imaged with laser
scanning confocal microscopy. Before gastrulation, Drosophila
embryos consist of a single layer of epithelial cells surrounding
the yolk with apical surfaces facing outside (Fig. 1a). Cells are
about 30 μm tall and 6–7 μm wide, with spot adherens junctions
localized in a relatively narrow zone within 6–7 μm below the apical
surface (Fig. 1b). This single layer of epithelial cells is generated
during a process called cellularization, where cell membranes of the
syncytial embryo gradually ingress between nuclei and grow deeper
to eventually enclose the nuclei and cytoplasm surrounding the
yolk. Cellularization happens 2 h after egg laying and it takes
50 min to 1 h for the cellularization membrane front to reach the
end of the cytoplasm. Therefore, the depth of cellularization front
from the embryo surfaces can be used to estimate the embryo’s age
for live imaging (Fig. 2). Spot adherens junctions are formed in all
the cells during early cellularization and appear as clusters of
cadherin-catenin complexes around the subapical region and only
become more belt-like much later in the development. No other
junctions, such as septate junctions and focal adhesions, have
formed during this early stage of development [9]. Immediately
66
Lingkun Gu and Mo Weng