To identify genes that are primarily expressed in the pharynx, Mango and colleagues [8
] profiled transcripts from the mutant strains par-1
. Worms with par-1
mutations produce an excess of pharyngeal cells following transformation of gut cells to a pharyngeal fate, whereas skn-1
animals produce no pharyngeal cells owing to transformation of pharynx precursors into body muscle and epidermis (Figure ). Comparing expression levels between par-1
animals increased the sensitivity of the analysis, as differences in specific expression levels were much larger than would be seen in a more traditional comparison, such as between wild-type and skn-1
animals. Thus, genes that would have been excluded in a traditional comparison, such as genes that are expressed only in subsets of pharyngeal cells or are expressed at very low levels, were readily detected from the par-1
Figure 1 An outline of the experimental strategy used by Mango and colleagues [8,9] to identify regulatory motifs that specify temporal and spatial patterns of gene expression during pharyngeal development. (a) RNA was isolated from worms with mutations in the (more ...)
The next stage of the analysis was the identification of regulatory elements within the promoters of the identified pharyngeal genes. The pharynx-specific genes were grouped according to their temporal and spatial expression patterns and sequences in the proximal regions of the promoters of grouped genes were analyzed for overrepresented sequence elements (Figure ). One factor that contributed to the success of this stage was the recently completed genome sequence of the related nematode Caenorhabditis briggsae
]; conservation of sequences between the genomes of C. elegans
and the closely related C. briggsae
is often used to make a case for their biological relevance [10
]. When Mango and colleagues [8
] looked at pharyngeal gene promoters, they found that the proximal 500 base-pairs of promoter sequence were the most conserved between C. elegans
and C. briggsae
genes; they therefore decided to limit their analysis to these regions, thus increasing their chances of identifying sequences motifs of biological relevance.
Another important factor contributing to the success of this stage was the use of the Improbizer algorithm [12
], which identifies sequence motifs that occur at significantly high rates within a sample pool and which has the advantage that a priori
knowledge of the cis
-regulatory sequence is not required. Thus, when used on a population of genes associated with a particular biological activity, Improbizer can identify novel sequences involved in gene regulation associated with that particular activity.
The criteria used for subdivision of the pharynx-specific genes into temporal and spatial classes were a critical aspect of the experimental design. In the study by Gaudet et al
], the pharynx-specific genes were subdivided into two temporal classes, depending on whether expression began during mid-embryogenesis ('early' genes) or at the start of terminal differentiation of the pharynx ('late' genes). This grouping was used to identify sequence elements that were enriched in one temporal group compared with the other. In the study by Ao et al
], the total complement of pharyngeal genes was subdivided into five groups on the basis of their spatial expression patterns. Sequence elements that were particularly enriched in the promoters of each group were identified as potential cis
elements involved in regulation of spatial expression patterns. In both studies [8
], the rich resources available to C. elegans
biologists, including databases of expression patterns obtained from in situ
hybridization studies [13
], three-dimensional 'Topo' maps for identifying genes with shared expression patterns [12
] and the wealth of detailed studies on embryogenesis and larval development, were crucial in creating spatial and temporal groupings of genes that were analyzed with the Improbizer algorithm.
The results of these analyses were a set of sequence motifs that were found to be overrepresented in promoters of particular subgroups of pharyngeal genes (Figure ). But are these motifs actually used for gene regulation in the developing worm? Many microarray and bioinformatic approaches flounder when it comes to biological validation of the sequence motifs identified, but Mango and colleagues [8
] took a multipronged approach that not only allowed them to test the identified sequences for biological relevance but also provided information about the function of each promoter element. The initial validation test was for enhancer activity of the identified motif in the context of a minimal exogenous promoter driving a reporter gene. This assay allowed the investigators to evaluate the regulatory element on three different criteria: whether the sequence was sufficient to activate expression and act as an enhancer, whether expression was primarily pharyngeal, and whether it was sufficient to confer a temporal pattern of expression. These tests not only confirmed pharyngeal expression and temporal patterns of expression for candidate sequences, but in one case also showed that an element acted as a repressor. In the second round of validation tests, pharyngeal genes containing each candidate regulatory element were identified, and site-directed mutagenesis of the element was used to evaluate whether loss of function led to loss of the temporal pattern of expression. The native context of the identified temporal elements was further investigated by searching the promoters of the 'early' and 'late' groups of genes for conserved clustering or combinations of temporal elements. The patterns identified were also used in a bioinformatics search to find additional pharyngeal genes that had not been identified from the microarray experiments, further validating the biological relevance of the identified sequences.