neurulation (SN) [4, 5]. During PN, the lateral ends of the anterior
neural plate elevate and the bilateral neural folds fuse with each
other to form the anterior NT [6–8]. By contrast, in SN mesenchymal neuromesodermal progenitors are recruited to elongate the
caudal body axis and drive the caudal elongation of the NT. The
MET of neuromesodermal progenitors (NMPs) is the central event
of SN, along with the formation of a compact nerve cord and its
subsequent cavitation to form the caudal NT. In human embryos,
the transition from the primary to the secondary NT occurs at the
lumbosacral level; therefore the development of the lumbar, sacral,
coccygeal and equinal cord largely involves SN [9–12].
Here, we propose that the chick embryo is an ideal model to
understand the MET events that occur during amniotic SN. While
manipulating and imaging mammalian embryos is still problematic,
the chick embryo provides an easily accessible model in which the
stages of development can be readily identified [13]. At early stages
of development the avian body plan is very similar to that of
mammals, and these embryos have excellent optical properties as
they are thin and planar [14]. Furthermore, the SN region in the
chick embryo extends up to the lumbar region [15, 16], closely
resembling human development, whereas SN only occurs at the tail
level in mice [17–19].
In the chick, SN starts when undifferentiated NMPs converge
onto the dorsal midline, adopt a neural cell identity and undergo
MET. The complete MET process can be followed in stage HH15
chick embryos, as different degrees of polarization exist along the
anteroposterior axis (Fig. 1). The first cells to undergo MET are
located in the periphery of the medullary cord, while the central
cells remain mesenchymal until the very end of the process. It is
between these two cell populations that small cavities of varied size
and shape form, later coalescing to form a single central lumen
(Fig. 1c–g) [20, 21].
However, the analysis of SN in vivo has always been technically
difficult. Cell tracing studies have identified the epiblast region
occupied by cells that undergo SN in the future (the preSN region),
located caudo-medially to Hensen’s Node at stage HH9 chick
embryos [22–26]. However, the NT is still open in the posterior
domains of stage HH9 chick embryos, so for electroporation the
DNA must be injected on top of the epiblast and the electrodes
positioned above and below the embryo (Fig. 2a, b). Earlier embryonic stages have always been electroporated ex ovo in this way
[27, 28], which facilitates the electroporation of the flat epiblast,
although cultured embryos do not grow to stage HH15 with
normal body elongation [29].
Here we propose a new method for the in vivo analysis of MET
during SN that overcomes all these technical difficulties. The
method involves combining early in ovo chick embryo electroporation with time-lapse imaging in a culture setup specifically adapted
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Elena Gonzalez-Gobartt et al.
neural plate elevate and the bilateral neural folds fuse with each
other to form the anterior NT [6–8]. By contrast, in SN mesenchymal neuromesodermal progenitors are recruited to elongate the
caudal body axis and drive the caudal elongation of the NT. The
MET of neuromesodermal progenitors (NMPs) is the central event
of SN, along with the formation of a compact nerve cord and its
subsequent cavitation to form the caudal NT. In human embryos,
the transition from the primary to the secondary NT occurs at the
lumbosacral level; therefore the development of the lumbar, sacral,
coccygeal and equinal cord largely involves SN [9–12].
Here, we propose that the chick embryo is an ideal model to
understand the MET events that occur during amniotic SN. While
manipulating and imaging mammalian embryos is still problematic,
the chick embryo provides an easily accessible model in which the
stages of development can be readily identified [13]. At early stages
of development the avian body plan is very similar to that of
mammals, and these embryos have excellent optical properties as
they are thin and planar [14]. Furthermore, the SN region in the
chick embryo extends up to the lumbar region [15, 16], closely
resembling human development, whereas SN only occurs at the tail
level in mice [17–19].
In the chick, SN starts when undifferentiated NMPs converge
onto the dorsal midline, adopt a neural cell identity and undergo
MET. The complete MET process can be followed in stage HH15
chick embryos, as different degrees of polarization exist along the
anteroposterior axis (Fig. 1). The first cells to undergo MET are
located in the periphery of the medullary cord, while the central
cells remain mesenchymal until the very end of the process. It is
between these two cell populations that small cavities of varied size
and shape form, later coalescing to form a single central lumen
(Fig. 1c–g) [20, 21].
However, the analysis of SN in vivo has always been technically
difficult. Cell tracing studies have identified the epiblast region
occupied by cells that undergo SN in the future (the preSN region),
located caudo-medially to Hensen’s Node at stage HH9 chick
embryos [22–26]. However, the NT is still open in the posterior
domains of stage HH9 chick embryos, so for electroporation the
DNA must be injected on top of the epiblast and the electrodes
positioned above and below the embryo (Fig. 2a, b). Earlier embryonic stages have always been electroporated ex ovo in this way
[27, 28], which facilitates the electroporation of the flat epiblast,
although cultured embryos do not grow to stage HH15 with
normal body elongation [29].
Here we propose a new method for the in vivo analysis of MET
during SN that overcomes all these technical difficulties. The
method involves combining early in ovo chick embryo electroporation with time-lapse imaging in a culture setup specifically adapted
184
Elena Gonzalez-Gobartt et al.