During development, cells acquire distinct cell fates in response to a host of regulators, many of which encode transcription factors. A longstanding issue has been the nature of the target genes controlled by these transcriptional regulators: do they activate a few genes at the top of a hierarchy, or control many genes that function at multiple stages throughout development? This question has been difficult to resolve because few direct targets have been identified. To address how transcription factors influence cell fate decisions, we are studying the role of the fork head box protein PHA-4 during pharynx development. In embryos that lack
pha-4 activity, cells that would normally become part of the pharynx develop as ectoderm instead. Conversely, ectopic expression of
pha-4 produces excess pharyngeal cells at the expense of other cell types. Because
pha-4 normally functions in pharyngeal cells irrespective of the cells' lineage or cell type, we propose that
pha-4 endows cells with pharyngeal organ identity. We have used a microarray chip approach to identify genes selectively expressed in the pharynx and have analyzed those genes to determine which are direct PHA-4 targets. We took advantage of maternal effect mutants that produce embryos with excess (
par-1) or no (
skn-1) pharyngeal cells to identify candidate pharyngeal genes. Included among our positive clones were most (>70%) genes known to be expressed in the pharynx, indicating that our approach worked well. Three lines of evidence suggest that PHA-4 directly activates most or all pharyngeally-expressed genes. First, we examined the pharynx-specific expression of a random set of microarray positives and found that the expression of each depended on one or more predicted PHA-4 binding sites. Second, PHA-4 is able to bind these sites in vitro. Third, temperature-shift studies indicate that PHA-4 is required throughout development, consistent with a model in which PHA-4 regulates both early and late acting genes. Our analysis also showed that the affinity of PHA-4 for different binding sites regulates the relative time of onset of the different target genes. When a PHA-4 binding site is converted by a single base-pair to a higher affinity site, expression initiates earlier. Conversely, a single base-pair mutation of a PHA-4 recognition sequence to one with lower affinity leads to a delay in the onset of expression. The affinity of PHA-4 binding in vitro matches the behavior of these reporter constructs in vivo. These findings suggest that modulating the affinity of PHA-4 binding sites within a promoter provides the embryo with a subtle mechanism to coordinate expression temporally throughout a developing organ. Since direct target genes of mammalian FOXA proteins include genes expressed early (e.g. endoderm) or late (e.g. liver) during vertebrate development, we suggest that global regulation of transcription within the foregut may be a common feature of this family of developmental regulators. We are indebted to Stuart Kim, Rebecca Begley, Carrie van Doren, Kyle Duke, and Min Jiang, who performed the microarray hybridization experiments.