Biology Reference
In-Depth Information
with excess or missing pharyngeal cells. Most pharynx-
expressed genes identified this way contained binding sites
for the master regulator of pharyngeal development PHA-4
in their promoters, and interestingly, the strength of these
binding sites correlated to some extent with the timing of
transcription induction
[115]
. Further, by searching for
enriched sequence motifs in the promoters of genes activated
either early or late during development (information that was
obtained from the NEXTDB in situ database, see below),
motifs that act in combination with PHA-4 to specify early
or late expression were also identified
[124]
.
As a second example, microarray-based expression
profiling has also been used in combination with genetic
perturbations to map gene regulatory networks during the
development of the posterior of the embryo
[116,125]
. Here,
genetic mutants with fate conversions were used to identify
genes expressed in the C lineage
[116]
,andacombinationof
expression profiling in mutants, yeast one-hybrid analyses
and reporter construct experiments was used to infer regu-
latory interactions among transcription factors
[116,125]
.
Examining the interactions among transcription factors
specifying muscle and epidermal cell fates allowed the
authors to propose that these two fates cross-repress each
other, and also that the two pathways differ in the topology of
the interactions that lead to all-or-none fate conversions
[125]
. Two technical challenges that have possibly discour-
aged the wider adoption of this approach are, first, the need to
obtain pure tissue samples in the perturbed animals and,
second, the redundancy and compensation in transcription
networks that mean inhibiting even master regulators
sometimes only has a subtle effect on gene expression
[126]
.
Genome-Wide Maps of Normal
and Perturbed Gene Expression
The invention of microarrays, and more recently the
implementation of massively parallel sequencing tech-
nology, allows the levels of essentially all mRNAs to be
assayed in parallel (see chapter by Guigo). Initial gene
expression profiling studies used microarrays to quantify
the levels of mRNAs in RNA samples extracted from whole
animals or whole embryos, for example to map how gene
expression changes during development
[103]
, as animals
age
[104
106]
, or in response to environmental perturba-
tions such as heat shock
[107]
. These early experiments,
together with expressed sequence tag (EST) cDNA
sequencing, also served an important role in validating the
existence of many only computationally predicted genes.
More recently, deep sequencing and tiling microarrays
have been used to provide a more comprehensive view of
the transcribed genome, including maps of 5
0
ends
[108]
,
trans-splicing
[109]
, alternative slicing
[110]
,3
0
UTRs
[111,112]
, and non-coding RNAs
[113]
. For example,
hundreds of alternative splicing events were found to be
developmentally regulated
[110]
. Moreover, expression
has been compared between two different nematode
species
[114]
.
A major technical challenge when mapping gene
expression in a rapidly developing multicellular animal
such as C. elegans is the need to map gene expression to
individual cells or tissues. Three main approaches have
addressed this challenge: (1) using mutants with trans-
formed cell fates so that the majority of the mRNA derives
from a single cell lineage or type; (2) using dissection to
isolate tissue samples; and (3) using cell sorting to isolate
cells expressing a reporter gene of interest. Examples of
the first approach include studies that have used well-
characterized mutants that affect the fates of early blas-
tomeres to map genes expressed in particular tissues and
lineages, such as the pharynx
[115]
, the C lineage
[116]
,
and the male and female germlines
[117]
. Examples of the
second approach include the manual dissection of germ-
line tissue
[118]
, and examples of the third approach
include the use of tissue-specifically expressed poly(A)
binding protein to isolate genes expressed in the muscle
[119]
and intestine cells
[120]
, and the use of tissue-
specific GFP reporters and cell sorting to map genes
expressed at particular time points
[121]
or in particular
embryonic cells, such as the touch receptor neurons
[122]
,
and later in many different cell types as part of the mod-
ENCODE project
[123]
.
A few studies have also used genome-wide expression
profiling to understand gene regulation, an elegant example
being studies on the development of the pharynx. Here,
genes expressed in the pharynx were first globally identified
by comparing the expression profiles of mutant embryos
e
Single Cell-Resolution Analysis of Gene
Expression
An alternative approach to using microarrays or RNA
sequencing to globally gene expression is a 'gene-centric'
approach that aims to map all of the cells in which
a particular gene is expressed. Five important contributions
have been made towards this goal in C. elegans.
First, the spatial and temporal expression of many genes
has been studied at low resolution by in situ hybridization,
with the resulting images available in a free-to-access
database (NEXTDB,
http: //nematode.lab.nig.ac.jp/
). These
in situ data provide information on the expression of
thousands of genes, but have the disadvantage that the
expression patterns have not been systematically annotated
in a way that makes them useful for computational analysis.
Obviously, information is only provided on mRNA, not
protein expression.
Second, a computational approach has been developed
that can recognize 357 of the 558 nuclei in the first (L1)
larval stage from their reproducible locations with 86%