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with desired phenotypes are isolated by direct inspection of descendants of muta-
genized or RNAi-treated worms. For example, the very first direct simple screen
using EMS as a mutagen in C. elegans was performed by Sydney Brenner with
particular interests in mutants defective in coordinated movement (
Brenner, 1974
).
He identified mutations in 77 genes affecting movement. Notably, one of these
genes, unc-6, was later shown to be the ortholog of the vertebrate netrin gene,
encoding an important extracellular cue directing axon outgrowth and broadly
conserved across the animal kingdom (
Harris et al., 1996; Hedgecock et al.,
1990; Ishii et al., 1992; Kennedy et al., 1994; Lauderdale et al., 1997; Mitchell
et al., 1996; Serafini et al., 1994
).
3. Forward Screens with the Aid of the Green Fluorescence Protein (GFP)
In contrast to phenotypes observed in direct simple screens, many phenotypical
changes, particularly cellular biological ones, are invisible at the behavioral and light
microscope levels. For some phenotypes, this limitation can be overcome with
fluorescent proteins. Fluorescent markers, in particular, green fluorescence protein
(GFP) from the jellyfish Aequoria victoria (
Chalfie et al., 1994
), have facilitated
screens in many biological processes, including axon guidance (
Zallen et al., 1998
),
vesicle transportation (
Grant and Hirsh, 1999; Grant et al., 2001; Sato et al., 2008
),
and synapse formation (
Liao et al., 2004; Shen and Bargmann, 2003; Zhen et al.,
2000
). In these screens, GFP is utilized as a visual indicator of phenotypic alterations
for identification of mutants, as it allows selective visualization of normally invisible
proteins, subcellular structures, specific cells or tissues, and even gene transcription
status by placing the GFP open reading frame downstream of genes
0
cis-regulatory
regions. In these types of screens, worms that are engineered to transgenically
express GFP are mutagenized and examined for changes in GFP expression levels
or patterns. For instance, to investigate the mechanisms underlying left-right func-
tional asymmetry of chemoreceptor gene expression between two morphologically
symmetrical neurons, ASE left (ASEL) and ASE right (ASER),
Chang et al. (2003)
performed a screen on transgenic worms with GFP expression in either ASEL or
ASER under the control of the cell-specific cis-regulatory regions. They identified
mutations that lost left-right functional asymmetry by isolating mutants that sym-
metrically expressed GFP in both cells or neither. From this screen, they uncovered
several microRNAs and transcription factors that formed a complex regulatory
cascade directing left-right asymmetrical chemoreceptor gene expression, thus
shedding light on chemosensory neuron differentiation (
Chang
et al., 2004;
Johnston and Hobert, 2003
).
The manual isolation of mutants using fluorescence markers is often laborious, as
it requires visual inspection of a large number of mutagenized worms at the micro-
scopic level. Recently, an automated worm sorter (Complex Object Parametric
Analysis and Sorter, COPAS), which is a flow cytometry machine used to sort worms
based on their optical sizes, density, changes in color, and fluorescence intensity
(
Doitsidou et al., 2008
), has been developed to facilitate isolation of mutants. For
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