Although the techniques for cell culture developed in this section are
not fully reproducible, the observation that axons can be grown from
cultured C. elegans neurons is encouraging and suggests that further
optimization should be attempted. Techniques developed for mass culture
of all embryonic cells could form the basis for culture of specific
neuronal types. Experiments in Drosophila suggest that the isolation
and culture of specific cell types from a population of dissociated
embryonic cells might be possible. Krasnow and colleagues (1991) used
a beta-galactosidase substrate, FDG, that can be introduced into living
cells and releases a fluorescein molecule when cleaved. They
dissociated populations of transgenic Drosophila embryos carrying lacZ
fusions, including a neuron-specific enhancer trap line, stained with
FDG, and isolated a population of lacZ-expressing cells by
fluorescence-activated cell sorting. Neurons isolated by this technique
survived and sent out axons. This technique could probably be used in
C. elegans in conjunction with neuron-specific lacZ or GFP fusions to isolate
specific neuronal populations for culture. If neurons can grow at low
density (perhaps with conditioning factors released into the medium by
cells in denser cultures), the interactions of particular cells might be
studied both in culture and intact worms. In particular, unc-30/lacZ
fusions might be used to isolate DD neurons, which in embryos send out
processes that change direction or stop when they contact the cell body
of the adjacent DB or process of the adjacent DD neuron (Durbin, 1987)
.
Another potential use for C. elegans cell culture is in the study of
the electrical properties of neurons and muscle cells. The
electrophysiology of cultured Drosophila neurons, which are similar in
size to the C. elegans cultured cells described above, has been studied
by intracellular and patch recording techniques (Wu et al., 1983) . An
attempt to establish patch clamps with C. elegans cells was unsuccessful
(D. Raizen, E. Jorgensen, B. Sawin, and L. Bloom, unpublished results),
due largely to the poor adhesion of the cells to the coverslip.
Improvements in the culture technique could permit electrophysiological
analysis of a variety of mutants with defects in the regulation of
muscle contraction (Levin and Horvitz, 1992) , calcium channels (L.
Lobel, personal communication), anaesthetic sensitivity (P. Morgan,
personal communication), etc.
Finally, vertebrate cell culture techniques have been used to quantify
the elongation of axons on other axonal substrates (for example, see
Chang et al., 1987 ), and it is possible that the C. elegans culture
techniques can be applied to the study of fasciculation. In particular,
it would be interesting to determine whether axons from mutants
defective in fasciculation in vivo--unc-34, unc-71, and unc-76--are
unusual in their fasciculation behavior in vitro. Genetic mosaic
experiments for the study of fasciculation are difficult in vivo because
neighboring neurons are often closely related by lineage, but an in
vitro fasciculation assay would offer an opportunity to test the cell
autonomy of the fasciculation defects observed in these mutants.
While the important parameters for successful C. elegans cell culture
are not well understood, it is encouraging that cells can be isolated
and cultured in the short term with a reasonable degree of
reproducibility. The observation of neurons growing in defined
conditions required further refinement of these techniques to allow
lower-density cell culture (perhaps with the addition of conditioned
medium from C. elegans cultures). The requirement for a TESPA substrate
suggests that the isolated cells were poorly adhesive, and
investigations of fasciculation or of the effects of specific substrates
on axonal growth (e.g., the laminin-related protein Unc-6 (Ishii et al.,
1992) require isolation techniques that yield healthier cells.
Nevertheless, the initial success of these culture experiments suggest
that further investigation can yield a useful technique.
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