Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 389

excellent results, such as recent vintage Nikon NA 1.45 oil immersion ''TIRF''
lenses.
Ultimately, the choice of a particular imaging modality is largely based on empir-
ical issues, including the type of probe being imaged. We now turn to types of probes
useful for analyzing morphogenesis.
2. Probes for Visualizing Morphogenesis
Introduction
The discovery and widespread use of variants of the green fluorescent protein
(GFP) as a tag for visualizing gene expression and protein localization within living
organisms has revolutionized live embryo imaging, including in C. elegans. Many of
the probes we use routinely for analyzing morphogenetic events are genetically
encoded. However, several other techniques have proven useful for studying mor-
phogenesis in C. elegans.
Junctions
For studying morphogenetic movements in embryos, junction-localized FPs are
extremely useful. Our laboratory used junction-localized FPs to study epithelial
sheet movement in C. elegans early on, and these tools have become standard among
C. elegans researchers. These include AJM-1 ( Mohler et al., 1998 ), DLG-1
( Koppen et al., 2001 ), and HMP-1/ a -catenin ( Raich et al., 1999 ). Others include
JAC-1/p120ctn ( Pettitt et al., 2003 ) and HMR-1/cadherin ( Achilleos et al., 2010 ).
The use of epithelial junctional markers is particularly useful for following cellular
movements at single-cell resolution during events such as dorsal intercalation,
ventral enclosure, and the early steps of elongation. The use of such markers
provides much clearer views of morphogenetic movements than can often be
achieved with Nomarski microscopy ( Fig. 3 ). We have also found that it is possible
to create transgenic lines expressing more than one FP-tagged junctional marker,
such as dlg-1::dsRed and hmp-1::gfp ( Zaidel-Bar et al., 2010 ). In some cases,
however, especially if two junctional proteins physically interact, coexpression
can lead to artifactual aggregation of proteins (C. Lockwood and J. Hardin,
unpublished).
Actin and tubulin
i. Actin
We have employed two basic methods to visualize the F-actin cytoskeleton in
embryos during morphogenesis: (i) phalloidin staining of fixed specimens and
(ii) imaging of the F-actin binding fragment of the spectraplakin, VAB-10, fused