Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

Four mouse POU domain genomic DNA clones-Brain-i, Brain-2, Brain-4, and Scip-and Bran-2 cDNA, which are expressed in adult brain, were cloned and the coding and noncoding regions ofthe genes were sequenced. The amino acid sequences of the four PoU domains are highly conserved; sequences in other regions of the proteins also are conserved but to a lesser extent. The absence of introns from the coding regions of the four POU domain genes and the similarity of amino acid sequences of the corresponding proteins suggest that the coding region of the ancestral class HI POU domain gene lacked introns and therefore may have originated by reverse transcription of a molecule of POU domain mRNA followed by insertion ofthe cDNA into germ cell genomic DNA. Additional duplications ofthe ancestral class HI POU domain gene (or mRNA) would create the Brain-i, Brain-2, Brain-4, and Scip genes.POU domain proteins bind to specific nucleotide sequences in DNA and regulate gene expression (for reviews, see refs. 1 and 2). The POU domain is a conserved amino acid sequence =150 amino acid residues long. The initial region of 69-72 amino acid residues is termed the POU-specific domain, which is followed by a 15-to 25-amino acid residue linker region and a 60-amino acid residue POU homeodomain. Both the POU-specific domain and the homeodomain are required for specific high-affinity binding to DNA (3, 4).Rosenfeld and his colleagues have sorted POU domains into different groups on the basis of POU domain amino acid sequence similarity (2). Three mammalian class III POU domain cDNAs have been described-human (5) and rat (2) Brain-i and Brain-2 and rat (5-8) and mouse (9-11) Scip (also termed Oct-6 and Tst-J)-that have closely related POU domains and are expressed in embryonic and adult brain. Only the POU domain regions of human and rat Brain-i and Brain-2 cDNA have been sequenced thus far (2, 5), whereas the complete coding sequences of rat (6-8) and mouse (9-11) Scip cDNA have been reported. Scip RNA is expressed in a subset of neurons, oligodendroglia, Schwann cells, and in the testis (5-11). The expression of the Scip gene is promoted by cAMP (6, 7).In this report, a fourth class III mouse POU domain gene, Brain4, is described, which is also expressed in adult mouse brain. Brain4 is similar to the recently reported XLPOU 2 POU domain partial cDNA of Xenopus laevis (12). Three additional mouse class III POU domain genomic clonesBrain-i, Brain-2, and Scip-and Brain-2 cDNA were obtained and the nucleotide sequences of the coding and noncoding regions were determined. Comparison of the deduced amino acid sequences of the four POU domain proteins shows that the structure of the genes and the amino Fig. 4). DNA inserts were subcloned in pBluescript II SK+ and KS+. The nucleotide sequences of both strands of DNA were determined with universal or specific oligodeoxynucleotide primers and single-stranded DNA templates by the dideoxynucleotide chain-termination method (13).OlUgdeoxynucleotide Probes. Four oligodeoxynucleotides (48 bases...

Four novel murine homeobox genes, Uncx-4.1, OG-2, OG-9, and OG-12, were cloned and partially sequenced. The amino acid sequence of the mouse Uncx-4.1 homeodomain is closely related to the sequence of the unc-4 homeodomain of Caenorhabditis elegans. However, the OG-2, OG-9, and OG-12 homeodomains are relatively diverged and are not closely related to any previously described homeodomain. Northern blot analyses revealed multiple bands of Uncx-4.1, OG-2, OG-9, and OG-12 poly(A)+ RNA in RNA from mouse embryos and adults that change during development and showed that each gene is expressed in a tissue-specific manner. OG-12 cDNAs were cloned that correspond to two alternatively spliced species of OG-12 mRNA. Three major bands of Uncx-4.1 poly(A)+ RNA were found only in RNA from adult mouse brain, but an additional band was observed in RNA from all of the other tissues tested. Major bands of OG-9 and OG-2 poly(A)+ RNA were found only in RNA from striated muscle; however, trace bands were detected in RNA from other tissues.