Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 372

I. Introduction
Caenorhabditis elegans has obvious advantages that make it well suited for
analyzing morphogenesis of living embryos. Its organizational simplicity, transpar-
ency, and essentially invariant development enabled the determination of the com-
plete embryonic cell lineage ( Sulston et al., 1983 ). Such invariant development
allows the assessment of mutant phenotypes at the level of single cells in C. elegans.
The wild-type embryonic lineage was originally determined by direct observation
using Nomarski microscopy. This was a very slow process, since only one or two of
the > 500 total embryonic cells could be followed per embryo. More recently, the use
of histone::GFP and other technologies, along with automated analysis, has stream-
lined lineaging even further. These developments have been discussed elsewhere
( Murray et al., 2008 ; see the chapter by Cowan and Chisholm, this volume). The
analysis of postmitotic movements of cells in embryos has likewise benefitted from
technological advances. The advent of four-dimensional (4D) microscopy made
analysis of morphogenesis much more practical by using computer-controlled equip-
ment to record development of embryos in three dimensions over time. Simultaneous
software advances made analysis of 4D movies practical ( Thomas et al., 1996 ).
The fundamental concepts of microscopy that apply to any context in C. elegans
are covered elsewhere in this volume (see the chapter by Maddox and Maddox, this
volume). In this chapter, we focus on the uses of modern microscopy specifically for
imaging later morphogenesis in C. elegans embryos, after many embryonic cells
have undergone their terminal divisions. We describe preparation of standard agar
mounts and other approaches for immobilizing embryos and treating embryos prior
to performing routine 4D microscopy, discuss simple methods for capturing 4D
movies, and discuss various probes for imaging fluorescently tagged cells or struc-
tures in living embryos. We also describe one way of performing correlative fluo-
rescence and transmission electron microscopy (F-TEM) during embryonic
morphogenesis.
Since the most dramatic movements occur among hypodermal cells during this
period of embryonic development, much of this chapter focuses on techniques that
are particularly useful for analysis of hypodermal cells. Because the terms ''hypo-
dermal'' and ''epidermal'' are used interchangeably, this chapter will use the latter
for better consistency with standard usage in other organisms (see Chisholm and
Hardin, 2005 for discussion).
II. Methods
A. Mounting Embryos for Imaging Morphogenesis
1. Agar Mount (Modified from Heid and Hardin, 2000 )
The agar mount is a simple way to prepare C. elegans embryos for microscopy.
The basic technique is presented elsewhere in this volume (see the chapter by