363been identified. Even less is known about how Nodal signals are controlled in
chicken and mammals.
Significant questions remain about how a morphogen gradient of Nodal signals
could specify cell fates when the positions of the responding cells are not fixed. The
primitive streak in birds and mammals, and the embryonic margin of teleosts are
characterized by dramatic cell movements at the time they respond to Nodal signals.
In these cases, germ layer identities may be determined by the total cumulative dose
of Nodal signals to which cells are exposed, as a function of absolute dosage and the
length of time to which the cells are exposed to Nodal. Cells in a moving population
could be exposed to Nodal signals for different lengths of time by virtue of their
different movement paths. Those cells that remain near a Nodal source for longer
times would be exposed to a higher Nodal dosage than cells which move away—or
are pushed away—more quickly.
The molecular mechanism by which cells could interpret such a temporal gradi-
ent is not understood. Elucidating this mechanism will certainly involve the devel-
opment of more sophisticated mathematical models, variations on Turing’s
Reaction-Diffusion Model that take into account subtle changes in Nodal concen-
tration over time and the movements of cells in the blastoderm stage. Our success
depends upon the development of new techniques and strategies to image the
response to Nodal signaling in vivo and in vitro. One area of progress has been the
invention of techniques to culture single embryonic stem cells in microfluidic cham-
bers (Warmflash et al. 2014 ). In these chambers, the Nodal dosage can be controlled
over time with great precision, and the response to Nodal can be assessed at single
cell resolution in real time. These studies can be used to generate models for the
time-dependent response to Nodal signaling, which can be tested in whole wild type
embryos as well as in various mutant backgrounds. In combination with more estab-
lished embryological techniques, these new computational and imaging tools will
provide an increasingly granular view of the molecular and cellular basis of germ
layer formation in vertebrates.
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7 Establishment of the Vertebrate Germ Layers