loosely connected to the other tissues. The best stage is 18. At
this stage the neural folds nearly finished closing (Movie 2).
Only the most anterior part of the neural plate is still open and
has the shape of a tear drop. The NC region appears as a bump
lateral to the anterior neural plate and extends roughly to the
middle of the anteroposterior axis. Before that stage, the NC
region is still attached to the neural plate and one would need
to arbitrarily cut the explant out of the embryo without any
clear landmark to use as reference. After stage 18, cells initiate
migration and start adhering strongly to the mesoderm and the
ectoderm. First, many cells are lost while peeling the superficial
ectoderm. Second, it increases the probability of mesoderm
contamination (Figs. 3 and 5, last part of Movie 3). Dissections
at early (prior to stage 18) or late stages (after stage 18) are still
technically possible, but they require an extensive amount of
training. If such stages are absolutely needed for a given project, one should train dissecting by placing embryos at similar
stages that have been stained with a neural crest marker (e.g.,
slug, twist) next to the embryos to be dissected, for reference.
7. Embryos should not be put completely inside the clay. It is
important that roughly a third of the embryo still pops out of
the modelling clay after the embryo has been immobilized.
Otherwise, the region of interest will be difficult to access for
dissection with the hair knife.
8. Once the embryo has been immobilized by applying pressure
with the modelling clay, all previous morphological landmarks
are gone. Thus, it is critical that the orientation of the embryo
to be dissected is correct before applying pressure.
9. At the end of neurulation (stage 18), NC cells adhere to each
other more than they adhere to their surrounding tissues (see
Note 6) making dissection relatively easy. Yet, contamination
from adjacent cell types can sometimes occur. Mesoderm and
endoderm are richer in vitellus and appear whiter than NC
cells, which are comparatively translucent and gray. The last
part of Movie 3 shows an example of a rough dissection during
which a bit of mesoderm is taken out together with the neural
crest. See also Fig. 3 and its legend to see how to proceed when
mesoderm is mistakenly dissected alongside NC cells. Superficial ectoderm may be detected by the presence of dark spots
corresponding to the pigments. Explants should always be
screened for contamination at the end of the culture and
explants with contamination from other cell types should be
excluded. In 2D-culture, ectoderm, neural plate, neural crest,
mesoderm and endoderm can be easily recognized morphologically using several criteria such as the shape of the cells and
nuclei or the size of vitellus platelets (Fig. 5). Nonneural ectodermal cells flatten extensively onto the substrate and form a
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this stage the neural folds nearly finished closing (Movie 2).
Only the most anterior part of the neural plate is still open and
has the shape of a tear drop. The NC region appears as a bump
lateral to the anterior neural plate and extends roughly to the
middle of the anteroposterior axis. Before that stage, the NC
region is still attached to the neural plate and one would need
to arbitrarily cut the explant out of the embryo without any
clear landmark to use as reference. After stage 18, cells initiate
migration and start adhering strongly to the mesoderm and the
ectoderm. First, many cells are lost while peeling the superficial
ectoderm. Second, it increases the probability of mesoderm
contamination (Figs. 3 and 5, last part of Movie 3). Dissections
at early (prior to stage 18) or late stages (after stage 18) are still
technically possible, but they require an extensive amount of
training. If such stages are absolutely needed for a given project, one should train dissecting by placing embryos at similar
stages that have been stained with a neural crest marker (e.g.,
slug, twist) next to the embryos to be dissected, for reference.
7. Embryos should not be put completely inside the clay. It is
important that roughly a third of the embryo still pops out of
the modelling clay after the embryo has been immobilized.
Otherwise, the region of interest will be difficult to access for
dissection with the hair knife.
8. Once the embryo has been immobilized by applying pressure
with the modelling clay, all previous morphological landmarks
are gone. Thus, it is critical that the orientation of the embryo
to be dissected is correct before applying pressure.
9. At the end of neurulation (stage 18), NC cells adhere to each
other more than they adhere to their surrounding tissues (see
Note 6) making dissection relatively easy. Yet, contamination
from adjacent cell types can sometimes occur. Mesoderm and
endoderm are richer in vitellus and appear whiter than NC
cells, which are comparatively translucent and gray. The last
part of Movie 3 shows an example of a rough dissection during
which a bit of mesoderm is taken out together with the neural
crest. See also Fig. 3 and its legend to see how to proceed when
mesoderm is mistakenly dissected alongside NC cells. Superficial ectoderm may be detected by the presence of dark spots
corresponding to the pigments. Explants should always be
screened for contamination at the end of the culture and
explants with contamination from other cell types should be
excluded. In 2D-culture, ectoderm, neural plate, neural crest,
mesoderm and endoderm can be easily recognized morphologically using several criteria such as the shape of the cells and
nuclei or the size of vitellus platelets (Fig. 5). Nonneural ectodermal cells flatten extensively onto the substrate and form a
Using Xenopus Neural Crest to Study EMT
269