Journal of Neuroscience, Vol 7, 10-22, Copyright © 1987 by Society for Neuroscience
In vitro neuronal differentiation of Drosophila embryo cells
PM Salvaterra, N Bournias-Vardiabasis, T Nair, G Hou and C Lieu
Early gastrula-stage Drosophila embryo cells will differentiate in vitro to
form several cell types, including neurons. We report here the
morphological appearance of cultured embryo cells, the pattern of DNA
synthesis, and the expression of neurotransmitter-metabolizing
macromolecules. The cells initially exhibit no overt morphological
differentiation, and all cells incorporate 3H-thymidine following a 1 hr
pulse-labeling period. As cells undergo morphological differentiation,
fewer total cells as well as qualitatively different cell types incorporate
label. By the time cells are 8 or 9 hr old, no myocytes or myotubes are
labeled. In contrast, some neurons are labeled with a thymidine pulse as
late as 18 hr. We have also stained cultured cells of various developmental
ages with the insect neuron-specific antibody: anti-HRP. Some positive
cells can be detected as early as 5 hr, when no overt morphological
differentiation is apparent. As the cells differentiate, the staining is
limited to the small, round neuronal type and its processes. These findings
suggest that this neuron-specific cell marker is expressed very early in
cultured gastrula-stage cells and may be used to identify neuronal
precursor cells. We have studied the patterns of expression of several
macromolecules involved in acetylcholine metabolism using these cultures.
The appearance of choline acetyltransferase (ChAT), the biosynthetic enzyme
for ACh production, is first detected in 5-hr-old cells. There is an
initial phase of low-level expression, followed by a rapid rise in activity
shortly after the differentiating neuron clusters make contact with one
another. ChAT activity reaches a plateau in 36-48-hr-old cells.
Acetylcholinesterase activity can be detected several hours before ChAT and
also shows a period of low-level expression followed by a rapidly
increasing phase, reaching a plateau at around 36-48 hr.
125I-alpha-bungarotoxin binding appears in cells about 4 hr old and rapidly
approaches maximum levels by about 36 hr. The in vitro expression pattern
for ChAT and AChE is similar to that seen in vivo. AChE activity has been
localized histochemically to the neurons and their processes in vitro. The
normal in vitro expression pattern for ChAT and AChE can be altered by
adding various cholinergic drugs to the culture medium during cell
differentiation. Medium conditioned by older cultures can also result in
lower levels of ChAT and AChE expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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