Thinking hard for Scarecrow, the NKX2.1 homolog of Drosophila. Crystal L. Maki, Albert J. Erives. Dept. of Biology, University of Iowa.

   Homeobox-containing genes are important regulators of development and encode tissue- and cell-specific transcription factors that bind DNA via their homeodomains. The Drosophila genes for ventral neurons defective (vnd), bagpipe (bap), tinman (tin), and scarecrow (scro) encode a conserved subfamily of homeobox genes. The genes bap and tin pattern the mesoderm and specify the founder cells that give rise to the musculature. In contrast, vnd and scro are involved in neuroectoderm-specification and CNS development. While much is known about vnd regulation in the early embryo and its role in the patterning, much less is known about scro. Its vertebrate homolog nkx2.1is well studied and is known to function in brain, lung, and thyroid development. Accordingly, in humans nkx2.1 disruption leads to developmental pleiotropic brain-lung-thyroid syndrome. During Drosophila embryogenesis, scro is expressed similarly in the pharyngeal primordia, pharynx, segmental neuronal precursors in the ventral nerve cord, and procephalic neuroblasts that give rise to distinct regions in the brain (Zaffran et al. 2000). We conducted a computational screen for conserved Nkx2.1-like binding sites in D. virilis and D. melanogaster, which represent divergent lineages as old as the genus. We identified all enhancer-size sequences centered on Nkx2.1-like binding sites located in each genome, and used a reciprocal regulatory BLAST method (R-BLAST) to identify conserved modules. We optimized R-BLAST by using the neurogenic ectoderm enhancers (NEEs) of vnd and other loci as positive internal controls. Here, we identified 76 putative enhancer sequences, which we then analyzed for enrichment of Gene Ontology categories in nearby genes. We found over-representation of loci encoding factors involved in cell fate determination and regulation of development. We report on enhancers located at pumilio (pum), neuralized (neur), midline (mid), and other loci. To test scro involvement at these enhancers, we are constructing mutant alleles of scro, which is located in the heterochromatic region of 3L.