Somites are paired segments that form during vertebrate development on either side of the midline structures, the neural tube and
notochord. They are transient embryonic structures which give rise
to progenitors that form the musculoskeletal system—such as cartilage, tendon, muscle, dermis, and endothelial cells. They are
generated in a regular sequence—every 90 min in chick embryos,
from the pre-segmented mesoderm (PSM) at the posterior end of
the embryo [4]. The newest formed somites comprise a sphere of
epithelial cells encapsulating a central cavity, the somitocoel, containing mesenchymal cells. As somites mature and begin to differentiate, the ventral domain dissociates, via epithelial-tomesenchymal transition (EMT), to form the sclerotome, which
gives rise to the axial skeleton. The dorsal domain remains epithelial
to form the dermomyotome and myotome, which give rise to all
trunk and appendicular skeletal muscles [5].
Although much has been learned about the signaling pathways
and key regulatory genes that are important for coordinating the
fate decisions of these cells [6], very little is known about the
cellular dynamics that underlie the morphological transitions during somite differentiation. To observe cellular behaviors and rearrangements that contribute to the morphogenesis of differentiating
somites, we used live multi-photon imaging of transgenic chick
embryos, expressing a membrane-bound GFP. Here, we describe
a method for dissecting somites together with their neighboring
tissues—the neural tube and notochord, lateral plate mesoderm,
surface ectoderm and endoderm, which provide signaling cues
essential for specification of somite derivatives [6]. The tissue slice
can be maintained in culture in order to image somite differentiation at cellular resolution (see Fig. 1). In optimized conditions,
somites are still exposed to their native signaling environment.
They grow over time and individual cells are very active, they
increase in size, divide, change shape, move and extend filopodia
(see examples of observed cell behavior in Figs. 2 and 3) [2]. This
approach can potentially be adapted to study the morphogenesis
and individual cell behavior across a range of tissues within an
embryo.
2 Materials
2.1 Harvesting Avian
Embryos
1. Penicillin/Streptomycin, 100,000 U/mL.
2. Fetal calf serum, FCS.
3. Buffered culture medium such as F12 supplemented with
nutrients. We routinely use Glutamax Ham’s F12 medium
(Invitrogen cat. no. 31765).
4. 30 mm petri dish.
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Gi Fay Mok et al.
notochord. They are transient embryonic structures which give rise
to progenitors that form the musculoskeletal system—such as cartilage, tendon, muscle, dermis, and endothelial cells. They are
generated in a regular sequence—every 90 min in chick embryos,
from the pre-segmented mesoderm (PSM) at the posterior end of
the embryo [4]. The newest formed somites comprise a sphere of
epithelial cells encapsulating a central cavity, the somitocoel, containing mesenchymal cells. As somites mature and begin to differentiate, the ventral domain dissociates, via epithelial-tomesenchymal transition (EMT), to form the sclerotome, which
gives rise to the axial skeleton. The dorsal domain remains epithelial
to form the dermomyotome and myotome, which give rise to all
trunk and appendicular skeletal muscles [5].
Although much has been learned about the signaling pathways
and key regulatory genes that are important for coordinating the
fate decisions of these cells [6], very little is known about the
cellular dynamics that underlie the morphological transitions during somite differentiation. To observe cellular behaviors and rearrangements that contribute to the morphogenesis of differentiating
somites, we used live multi-photon imaging of transgenic chick
embryos, expressing a membrane-bound GFP. Here, we describe
a method for dissecting somites together with their neighboring
tissues—the neural tube and notochord, lateral plate mesoderm,
surface ectoderm and endoderm, which provide signaling cues
essential for specification of somite derivatives [6]. The tissue slice
can be maintained in culture in order to image somite differentiation at cellular resolution (see Fig. 1). In optimized conditions,
somites are still exposed to their native signaling environment.
They grow over time and individual cells are very active, they
increase in size, divide, change shape, move and extend filopodia
(see examples of observed cell behavior in Figs. 2 and 3) [2]. This
approach can potentially be adapted to study the morphogenesis
and individual cell behavior across a range of tissues within an
embryo.
2 Materials
2.1 Harvesting Avian
Embryos
1. Penicillin/Streptomycin, 100,000 U/mL.
2. Fetal calf serum, FCS.
3. Buffered culture medium such as F12 supplemented with
nutrients. We routinely use Glutamax Ham’s F12 medium
(Invitrogen cat. no. 31765).
4. 30 mm petri dish.
174
Gi Fay Mok et al.