We are studying the morphogenesis and differentiation of two adjacent, single-cell tubes in the digestive tract called
pm8 and
vpi1. These cells are born in the dorsal sector of the digestive tact primordium.
pm8 and
vpi1 partially delaminate from their neighbors, and invade ventral cells to wrap around the midline of the primordium to form tubes. These cells are born in identical sublineages, but show several differences. For example, each cell uses a distinct fusogen to self-fuse into a tube (thereby avoiding cross-fusion), and
pm8 becomes a pharyngeal muscle while
vpi1 is a non-muscular valve cell. We previously found that many, if not all, of these differences result from Notch signaling in
pm8, and we here investigate how Notch controls the fate of
pm8. While Notch interactions can involve only a few direct targets, such as genes encoding the REF-1/Hairy/Enhancer of Split proteins, we have thus far found at least six different Notch targets in the
pm8 interaction.
pm8 expression of the Notch target
pax-1 appears to require synergy between Forkhead box-containing transcription factors and the Notch transcriptional effector, LAG-1/Su(H). Predicted binding sites for both proteins are essential for
pm8 expression, and are conserved in
pax-1 orthologs in the five sequenced Caenorhabditis genomes. Using comparative genomics, we identified additional C. elegans genes with similar, conserved sites. Thus far, three of the candidate genes tested are expressed in
pm8, but not
vpi1. We found that the predicted enhancer from one of these,
inx-11, is necessary and sufficient for
pm8 expression, and LAG-1/Su(H) binds the
inx-11 enhancer in vitro.
inx-11 encodes an innexin, a component of invertebrate gap junctions, which may function to coordinate the contraction of
pm8 with other pharyngeal muscles. Gap junctions may be particularly important for
pm8 function because, unlike the other pharyngeal muscle groups,
pm8 is not directly connected to the nervous system. These results suggest that "Notch + Forkhead" is a combinatorial code that can drive
pm8-specific expression, in contrast to the previously described "Notch + GATA" code that can drive intestine-specific expression. There appear to be at least two additional codes that regulate expression of the Notch targets
ref-1 and
ceh-24 in
pm8, as enhancers for these genes contain critical sequences that do not resemble the Forkhead consensus motif. We are using a similar, comparative genomics approach to decipher how these additional networks may regulate
pm8 morphogenesis and/or myogenesis.