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Visualization of C. elegans VD-Motor Neurons using RNAi
Correct
microtubule function is necessary for proper neural development.
Doublecortin (dcx) and doublecortin-like kinase (dclk) are microtubule
associated proteins that stabilize growing microtubules. Mutations in
dcx and dclk have been shown to disrupt cell migration, axon outgrowth
and axon transport in the developing mammalian brain. To study the role
of C. elegans dclk, zyg-8, in neural development, we
constructed temperature sensitive zyg-8 strains with transgenic
GFP markers to visualize motor neuron axons and synapses using
epifluorescence microscopy. Using a GFP marker with a tissue-specific
promoter we can visualize a subset of 19 GABAergic D motor neurons.
Thirteen of these neurons synapse to the ventral body wall muscles of
C. elegans, while 6 synapse to the dorsal side. Unfortunately, the
axons of both dorsal and ventral neurons overlap and the 13 VD’s cannot
be visualized alone with these markers. A tissue specific promoter is
not available for these neurons. However, injection of dsRNA encoding
the gene unc-30 induces a gene knock-down effect that shuts off
GFP expression in the dorsal D neurons born during embryogenesis. This
results in visualization of the 13 VD neurons, which are born later in
the animal’s life cycle. unc-30 dsRNA was synthesized using a
RiboMax Express RNAi System (Promega). The dsRNA was injected into adult
C. elegans at concentrations between 50 ug/ul – 2 ng/ul, and the
progeny were examined for GFP expression. We are using this method to
more accurately characterize the VD neuron synapse and axon phenotypes
associated with mutations in zyg-8. Support provided by: Howard Hughes Medical Institute Undergraduate Science Education Grant |
