Activation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. By using a recently developed technique of germline mosaics, we find that D-Raf can be activated by torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. Our study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway.

The determination of specific cell fates and polarity within each segmental unit of the Drosophila embryo involves the products of the segment polarity genes. One of these, wingless (wg), encodes a secreted protein that is homologous to the mammalian proto-oncogene Wnt-1 (refs 4, 5). In the embryonic epidermis, wg is expressed in a single row of cells within each segmental unit, although its activity is required for the correct patterning of most of the epidermis. Initially Wg signals to adjacent posterior cells, maintaining engrailed (en) expression. Later during embryogenesis, wg specifies the differentiation of naked cuticle. Wg signalling functions by inactivating or antagonizing the activity of zestewhite 3 (zw3). We have investigated the requirement in the Wg signal transduction pathway for the three genes armadillo (arm), dishevelled (dsh) and porcupine (porc), all of which have embryonic mutant phenotypes similar to wg. Our results indicate that dsh and porc act upstream of zw3, and arm acts downstream of zw3.

The Wnt genes encode conserved secreted proteins that play a role in normal development and tumorigenesis. Little is known about the signal transduction pathways of Wnt gene products. One of the best characterized Wnt family members is the Drosophila segment polarity gene wingless. We have investigated whether segment polarity genes with a wingless-like phenotype mediate the wingless signal. We used a wingless transgene controlled by a heat-shock promoter for genetic epistasis experiments. We show that wingless acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed and cuticle differentiation.

The Drosophila Wnt-1 homolog, wingless (wg), is involved in the signaling of patterning information in several contexts. In the embryonic epidermis, Wg protein is secreted and taken up by neighboring cells, in which it is required for maintenance of engrailed transcription and accumulation of Armadillo protein. The dishevelled (dsh) gene mediates these signaling events as well as wg-dependent induction across tissue layers in the embryonic midgut. dsh is also required for the development processes in which wg functions in adult development. Overall, cells lacking dsh are unable to adopt fates specified by Wg. dsh functions cell autonomously, indicating that it is involved in the response of target cells to the Wg signal. dsh is expressed uniformly in the embryo and encodes a novel protein with no known catalytic motifs, although it shares a domain of homology with several junction-associated proteins. Our results demonstrate that dsh encodes a specific component of Wg signaling and illustrate that Wnt proteins may utilize a novel mechanism of extracellular signal transduction.

We have identified dominant mutations that suppress the lethality associated with an R217-->L mutation in the GTP.Ras binding region (CR1) of the Drosophila raf (D-raf) serine/threonine kinase. Four intragenic and seven extragenic suppressors were recovered. Each of the four intragenic mutations contains one compensatory amino acid change located in either the CR1 or the kinase domain of D-raf. The seven extragenic suppressors represent at least four genetic loci whose effects strongly suggest that they participate in both the sevenless and Drosophila EGF receptor (DER) signaling pathways. One of these mutations, Su(D-raf)34B, is an allele of D-mek which encodes the known signaling molecule MAPK kinase (MEK). A D83V mutation in D-MEK is identified and shown to be sufficient to confer the dominant activity of Su(D-raf)34B.

In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2). Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.

In Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. We describe a new role for Raf in Drosophila development, showing that Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. We show that Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, in that either can induce ventral follicle cells to assume a dorsal fate.

We describe the characterization of the Drosophila gene, hopscotch (hop), which is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. We demonstrate that the effect of hop on the expression of these genes is stripe-specific. The hop gene encodes a putative nonreceptor tyrosine kinase of the Janus kinase family, based on an internal duplication of the catalytic domain. We present a model in which the Hop tyrosine kinase is involved in the control of pair-rule gene transcription in a stripe-specific manner. Our results provide the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase.

MEK, a dual specificity threonine/tyrosine kinase, has been postulated to be a convergent point for signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors. In contrast to yeast and mammalian cells where several MEKs have been isolated, only one Drosophila MEK (D-Mek) has been characterized to date. Previous studies have shown that D-Mek acts in the Torso RTK signaling pathway. To demonstrate that D-Mek also operates downstream of other RTKs, we generated a temperature-sensitive allele of D-mek (D-mekts) by site-directed mutagenesis based on the amino acid change of a yeast cdc2ts mutation. Using D-mekts, we show that in addition to its role in Torso signaling, D-Mek operates in the Sevenless and in the Drosophila epidermal growth factor RTK pathways. Because loss-of-function mutations in D-mek and the upstream receptors give rise to similar phenotypes, it suggests that D-mek is the only MEK activated by Drosophila RTKs. In addition, we demonstrate that different RTK pathways respond differently to alteration in D-Mek activity.

The 'dominant female-sterile' technique used to generate germ-line mosaics in Drosophila is a powerful tool to determine the tissue specificity (germ line versus somatic) of recessive female-sterile mutations as well as to analyze the maternal effect of recessive zygotic lethal mutations. This technique requires the availability of germ-line-dependent, dominant female-sterile (DFS) mutations that block egg laying but do not affect viability. To date only one X-linked mutation, ovoD1 has been isolated that completely fulfills these criteria. Thus the 'DFS technique' has been largely limited to the X-chromosome. To extend this technique to the autosomes, we have cloned the ovoD1 mutation into a P-element vector and recovered fully expressed P[ovoD1] insertions on each autosomal arm. We describe the generation of these P[ovoD1] strains as well as demonstrate their use in generating germ-line chimeras. Specifically, we show that the Gap1 gene, which encodes a Drosophila homologue of mammalian GTPase-activating protein, is required in somatic follicle cells for embryonic dorsoventral polarity determination.

Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the torso (tor) receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew (csw). We found that expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. To demonstrate that p21ras/Ras1 mediates tor signaling, we injected mammalian p21ras variants into early Drosophila embryos. We found that the injection of activated p21v-ras rescued the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. In addition, wild-type embryos show a terminal-class phenotype resembling csw when injected with p21rasN17, a dominant-negative form of p21ras. Furthermore, we have analyzed the maternal-effect phenotype of Son of sevenless (Sos), a positive regulator of Ras1, and showed that embryos derived from germ cells lacking Sos+ activity exhibit a terminal-class phenotype. Our study demonstrates that the Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway.

Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the D-raf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein.

We describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.

We have characterized the molecular nature of mutations in wingless (wg), a segment polarity gene acting during various stages of Drosophila development. Embryo-lethal alleles have undergone mutations in the protein-encoding domain of the gene, including deletions and point mutations of conserved residues. In a temperature sensitive mutation, a conserved cysteine residue is replaced by a serine. In embryo-viable alleles, the wg transcriptional unit is not affected. Immunostaining of mutant embryos shows that the embryo-lethal alleles produce either no wg antigen or a form of the protein that is retained within cells. Interestingly, embryos mutant for the segment polarity gene porcupine show a similar retention of the wg antigen. We have also transfected wild type wg alleles into Drosophila tissue culture cells, which then display wg protein on the cell surface and in the extracellular matrix. In similar experiments with mutant alleles, the proteins are retained in intracellular compartments and appear not to be secreted. These data provide further evidence that wg acts as a secreted factor and suggest that porcupine provides an accessory function for wg protein secretion or transport.

BACKGROUND: Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms. Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive. In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays. Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death. Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types. RESULTS: A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker. This method can be applied to cell lineage and mosaic analysis in Drosophila. The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT). In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker. Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase. Of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue. CONCLUSIONS: We demonstrate that, by intermolecular recombination, we can use FLIP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues. This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals.

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

We describe the characterization of the Drosophila gene, corkscrew (csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the torso signal via tailless, to downstream terminal genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.

We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.

Lethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.

An efficient method for generating embryonic mosaics using a yeast site-specific recombinase (FLP), under the control of a heat shock promoter, is described. FLP-recombinase can promote mitotic exchange between homologous chromosomes that contain FRT (FLP Recombination Target) sequences. To demonstrate the efficiency of FLP-recombinase to generate embryonic mosaics, clones of the recessive and cell autonomous mutation armadillo (arm), detected by their ability to differentiate ectopic denticles in the naked cuticle of each abdominal segment, have been induced. We have analyzed the parameters of FLP-recombinase induced embryonic mitotic recombination and have demonstrated that clones can be efficiently induced during the postblastoderm mitotic divisions. We discuss applications of this technique for the analyses of the roles of various mutations during embryonic patterning.