The bulk of the C. elegans pharynx consists of eight muscle groups (
pm1-8), each with unique morphology that give the pharynx its distinct bilobed structure. Muscle group
pm6 is unique in that it produces the pharyngeal grinder, a chitinous structure involved in the mechanical breakdown of food. How the different pharyngeal muscle sub-types are specified is largely unclear and little is known about the specification and development of the
pm6 cells. To begin to examine
pm6 development, we first identified a set of
pm6-specific genes by searching available gene expression databases (in particular, the Kohara group''s NEXTDB). From a list of candidate
pm6-specific genes, we selected three genes (C04G6.10, F41G3.3, F45B8.3) for further study, and constructed GFP reporters for all three genes to verify that each was expressed specifically in the
pm6 cells. We next performed a detailed analysis of the C04G6.10 promoter and identified two cis-regulatory elements (PM6-2 and PM6-3) that are critical for
pm6-specific expression. The first element, PM6-2, contains a predicted PHA-4 binding site. Consistent with regulation by PHA-4, extensive mutation of the PM6-2 sequence abolishes C04G6.10 expression. However, more discrete point mutations indicate that PM6-2 contains an element in addition to the PHA-4 site. Interestingly, when placed in multiple copies upstream of a "promoterless" GFP, PM6-3 is sufficient for
pm6-specific expression. Thus, two mechanisms appear to regulate
pm6-specific expression of C04G6.10. The first, acting through PM6-2, appears to be a factor that represses expression in non-
pm6 cells, possibly by interfering with PHA-4 binding and/or function. The second, acting through PM6-3, appears to respond to a
pm6-specific activator. We are currently examining the other two
pm6-specific reporters (F41B3.3 and F45B8.3) to determine whether their expression is regulated by the same cis-elements present in the C04G6.10 promoter. Preliminary data suggest that expression of both is dependent on a PM6-3-like sequence, arguing that expression of many
pm6-specific genes is regulated by a common upstream activator. To identify the putative
pm6-specific transcription factor acting through PM6-3, we will perform a yeast one-hybrid screen, test candidate factors by RNAi and perform a forward genetic screen for mutants that lack expression of a
pm6-specific reporter. This work will ultimately lead to the identification of factors required for specification of the
pm6 muscle group and formation of the pharyngeal grinder.