Molecular and Genetic Approaches for the Analysis of C. elegans Neuronal Development - Caenorhabditis Elegans: Molecular Genetics and Development - page 411

mutants were correlated with behavioral defects, such as locomotion and chemo-
taxis. Ultrastructural examination was particularly instrumental to deciphering ana-
tomical disruptions. These studies provided ground-breaking discoveries in the
development of the nervous system, such as identifying UNC-6/Netrin axon guid-
ance pathway, and discovering regulators of neuron specification.
The introduction of green fluorescent protein (GFP) from Aequorea victoria as a
noninvasive in vivo reporter revolutionized the research in C. elegans ( Chalfie et al.,
1994 ). Nearly all neurons and their molecular components or cellular compartments
can be visualized using a plethora of transgenic fluorescent protein reporters in live
animals. Combined with the awesome power of genetic analysis, C. elegans is a
prime system for elucidating the molecular and functional logic of neuronal circuits.
In this chapter, we summarize the general strategies for transgenically labeling
neurons and cellular compartments, and provide guidelines for genetically dissect-
ing genes and pathways controlling neuronal development.
II. Strategies for In Vivo Labeling of Neurons and
Compartments
Rationale: The transparency of C. elegans and the ease of making transgenic
animals offer great advantages for in vivo labeling of cells, tissues, and cellular
components. The starting point to study the development of neurons of interest is to
learn about the position, morphology, and unique features of the neurons. This
entails the understanding of the cell lineage that generates the neurons and finding
the promoters or regulatory DNA sequences that are selectively active in these
neurons. Nearly every neuron in C. elegans can be labeled using promoter (see
supplemental table in Chelur and Chalfie, 2007 ). Since the initial use of GFP, there
are now over 58 fluorescent protein reporters (XFPs) that can be excited from a wide
range of wavelengths and emit different colors. The most commonly used XFPs in
C. elegans are GFP, YFP, CFP, mCherry, and more recently, tdTomato and
mStrawberry. The preference for transgenic labeling is based on the question of
interest, from simple promoter reporters to complex labeling of proteins and orga-
nelles. Below, we summarize the common strategies.
A. Constructing Transgenic Reporters to Visualize Neuronal Morphology
The general design of reporter constructs began with Andy Fire ' s toolkit in the
1990s ( Fire et al., 1990 ). XFP coding sequences are optimized for the starting ATG
and codon usage and inclusion of synthetic introns. Multiple cloning sites at 5 0 and 3 0
allow for insertion of promoters, making fusion proteins, or attaching 3 0 untranslated
regions (3 0 UTR) ( Fig. 3 A). Most Fire vectors contain the 3 0 UTR of the unc-54 gene
for stable mRNA production. These basic features have recently been transposed
into the recombinase-based Gateway cloning system ( www.invitrogen.com ) .
Practical issues to consider when designing a reporter construct are: strength and