Development of the C. elegans pharynx requires the activity of the gene
pha-4. Animals that lack
pha-4 activity fail to make a pharynx, while ectopic expression of
pha-4 leads to the ectopic production of pharyngeal markers at the expense of other cell types. These data indicate that
pha-4 specifies pharyngeal organ identity. The PHA-4 product is a member of the family of winged helix transcription factor and is expressed in all pharyngeal cells. Several genes are known to be specifically expressed in the pharynx, and at least two of these (
ceh-22 and
myo-2) require PHA-4 for their expression. Therefore,
pha-4 probably functions by activating pharynx-specific gene expression. For
pha-4 to direct development of the entire pharynx, it must act either directly or indirectly on all pharyngeal genes. One possibility is that
pha-4 directly activates a small subset of pharyngeal genes, which then activate other downstream targets. This model proposes a regulatory hierarchy with PHA-4 directly activating those genes immediately downstream of it. Another hypothesis is that
pha-4 directly regulates all pharyngeal genes, whether they act early or late in pharyngeal development. A third possibility is that
pha-4 directly activates a subset of genes at multiple levels in the hierarchy and indirectly activates others. To understand how
pha-4 directs organogenesis we have identified potential targets of PHA-4 using microarray experiments. Probes were made from RNA from
skn-1 embryos, which produce no pharyngeal cells, and from
par-1 embryos, which produce excess pharyngeal cells. The results of these experiments have identified ~500 genes whose transcripts are more abundant in
par-1 versus
skn-1 embryos. Among these genes are known targets of PHA-4 (such as
myo-2) as well as other genes that are involved in pharyngeal development, including
pha-4 itself and
ceh-22. We are currently analyzing the other positives to determine whether they are expressed in the pharynx, and whether they are direct or indirect targets of PHA-4.