C. elegans
nfi-1 belongs to the highly conserved Nuclear Factor I (NFI) family of site-specific DNA-binding proteins known to function in the regulation of gene expression and development in metazoans. We showed previously that loss of
nfi-1 in C. elegans results in multiple behavioral defects; slower pharyngeal pumping, impaired egg laying, defective motility and a shortened life span. It is important to determine the specific cell types in which
nfi-1 functions to alleviate the phenotypes observed in
nfi-1 null worms. Since
nfi-1 is normally expressed in both muscle cells and selected neurons in worms, and defects in either cell type could affect pharyngeal pumping rates, we generated transgenic worms to determine in which cells
nfi-1 must be expressed to rescue the pharyngeal pumping phenotype. In one transgenic construct an
nfi-1-GFP fusion cassette was driven by the well-characterized pharyngeal muscle-specific
myo-2 promoter. In a second construct the
nfi-1-GFP cassette was expressed from the neuronal-specific F25B3.3 promoter. This
nfi-1-GFP fusion cassette contains the 4.2 kb
nfi-1 genomic region with all the coding exons of
nfi-1 and the GFP cassette as a translational fusion to the last residue of
nfi-1 and the 1.4 kb 3''UTR of
nfi-1. Transgenic expression of
nfi-1 under the
myo-2 promoter, but not under F25B3.3, rescued the pharyngeal pumping defect of
nfi-1 null worms. Surprisingly, transgenic expression of
nfi-1 under the
myo-2 promoter also rescued the shortened lifespan of
nfi-1 null worms, indicating that cell-autonomous expression of
nfi-1 in pharyngeal muscles is sufficient for this role of
nfi-1. These data suggest that as pharyngeal pumping rates decrease in
nfi-1 null animals, the animals starve, leading to progressive muscle wasting, paralysis and death. Thus two of the major phenotypes of
nfi-1 null worms are rescued by
nfi-1 expression solely in pharyngeal muscles. We used QPCR to examine expression levels of some candidate genes known to be expressed in pharyngeal muscles or involved in the regulation of pharyngeal pumping. We found that levels of both
myo-1 and
myo-2 transcripts were reduced by ~3 fold in the
nfi-1-null worms compared to N2 controls. The observed
nfi-1 effect on
myo-1 and
myo-2 expression is very likely indirect, since there is an absence of NFI binding sites within the genes, their promoters and 3UTRs. Currently we are using chromatin immunoprecipitation (ChIP) to identify direct NFI targets in pharyngeal muscles.