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 link between oncogenesis and normal development is well illustrated by the study of the Wnt family of proteins. The first Wnt gene (int-1) was identified over a decade ago as a proto-oncogene, activated in response to proviral insertion of a mouse mammary tumor virus. Subsequently, the discovery that Drosophila wingless, a developmentally important gene, is homologous to int-1 supported the notion that int-1 may have a role in normal development. In the last few years it has been recognized that int-1 and Wingless belong to a large family of related glyco-proteins found in vertebrates and invertebrates. In recognition of this, members of this family have been renamed Wnts, an amalgam of int and Wingless. Investigation of Wnt genes in Xenopus and mouse indicates that Wnts have a role in cell proliferation, differentiation and body axis formation. Further analysis in Drosophila has revealed that Wingless function is required in several developmental processes in the embryo and imaginal discs. In addition, a genetic approach has identified some of the molecules required for the transmission and reception of the Wingless signal. We will review recent data which have contributed to our growing understanding of the function and mechanism of Drosophila Wingless signaling in cell fate determination, growth and specification of pattern.
An elegant combination of genetic and biochemical approaches has been used to investigate a variety of signal transduction pathways in developmental processes. Here, we describe the 'terminal' signaling system in the Drosophila embryo, which is responsible for pattern formation in the polar regions of the embryo. This pathway involves a membrane-bound receptor tyrosine kinase (RTK) that is similar to other Drosophila RTKs, such as sevenless, and the mammalian RTKs, such as the epidermal growth factor or platelet-derived growth factor receptors.
The isolation and characterization of Drosophila mutations in receptor protein tyrosine kinases (RPTKs) have allowed a detailed analysis of the cellular processes regulated by these proteins. Recent investigations have identified a number of putative ligands involved in the activation of the receptors, and have demonstrated that these RPTKs trigger an evolutionarily conserved biochemical pathway. In addition to molecules previously identified from vertebrate studies, i.e. Grb2, Sos, Ras-Gap, p21ras, Raf, MEK and MAPK, genetic studies have suggested that two novel proteins, the protein tyrosine phosphatase (PTPase) Csw and the transmembrane protein Rho, are involved in RPTK signalling.
Pattern formation at the anterior and posterior termini of the Drosophila embryo involves intercellular communication via the Torso receptor tyrosine kinase (RTK). Recent advances in the understanding of Torso signaling has provided further support for the conservation of a signal transduction cassette downstream of RTKs. In addition, the analysis of the Torso pathway has begun to reveal general molecular mechanisms by which cells may impart patterning information to their neighbors through the use of RTKs.
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.