We have identified two members of a novel class of genes in Drosophila that encode putative transmembrane proteins with six leucine-rich repeats and a single immunoglobulin loop. These two molecules, Kek1 and Kek2, show striking conservation in their extracellular domains and have large and more divergent intracellular regions. Both genes are expressed in neurons as they differentiate in the embryonic central nervous system (CNS). kek1 is also expressed in other patterned epithelia, such as the follicle cells of the developing egg chamber, where it is found in a dorsal-ventral gradient around the oocyte. The homology of the kek genes to other known adhesion and signaling molecules, together with their expression patterns, suggests that both genes are involved in interactions at the cell surface. Genetic analysis reveals that deletion of the kek1 gene causes no obvious developmental defects. The coexpression of kek2 in the CNS leads us to suggest that Kek1 is part of a family of cell surface proteins with redundant function.
Specification of cell fates in the nonsegmented terminal regions of developing Drosophila embryos is under the control of a signal transduction pathway mediated by the receptor tyrosine kinase Torso (Tor). Here, we identify tyrosines (Y) 630 and 918 as the major sites of Tor autophosphorylation. We demonstrate that mutation of Y630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of Tor signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gammal, increases Tor signaling. Interestingly, when receptors contain mutations in both the Y630 and Y918 sites, Tor signaling is restored to wild-type levels. These results identify a novel mechanism whereby Tor function is regulated using compensatory signals generated from distinct autophosphorylation sites and reveal an underlying signaling pathway for terminal development.
In Drosophila, the Wingless and Notch signaling pathways function in m any of the same developmental patterning events. Genetic analysis demonstrates that the dishevelled gene, which encodes a molecule previously implicated in implementation of the Winglass signal, interacts antagonistically with Notch and one of its known ligands, Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways.
We have identified a putative Drosophila STAT protein named Marelle that exhibits mutant phenotypes identical to mutations in the Hopscotch/JAK kinase. We show that a reduction in the amount of marelle gene activity suppresses the phenotype associated with a gain-of-function mutation in hopscotch and enhances the phenotype associated with a weak hopscotch mutation. We propose that Hopscotch activates Marelle to regulate transcription of target genes such as the pair rule gene even-skipped. Our results demonstrate the existence of an invertebrate JAK/STAT system.
Notch (N) and other neurogenic genes have been implicated in two fundamental processes, lateral specification of cell fates, and epithelial development. Previous studies have suggested that the neurogenic gene brainiac (brn) is specifically required for epithelial development (Goode, S., Morgan, M., Liang, Y-P. and Mahowald, A. P. (1996). Dev. Biol. 178, 35-50). In this report we show that egghead (egh), a gene with phenotypes identical to brn, encodes for a novel, putative secreted or transmembrane protein. We describe the role of egh and brn germline function in the morphogenesis of the follicular epithelium from the time it is born through the time that it migrates towards the oocyte late in oogenesis. By comparing the function of germline egh and brn to N during oogenesis, we have obtained direct evidence for the involvement of Notch in maintenance of the follicle cell epithelium, and the specificity of brn and egh in epithelial development during oogenesis. The most striking phenotype observed for all three genes is a loss of apical-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte. The spatiotemporal onset of this adenoma-like phenotype correlates with the differential accumulation of egh transcripts in the oocyte at stage 4 of oogenesis. In contrast to N, we find that brn and egh are essential for the organization, but not specification, of stalk and polar cells. The expression patterns and functional requirements of brn, egh, and N lead us to propose that these genes mediate follicular morphogenesis by regulating germline-follicle cell adhesion. This proposal offers explanations for (1) the involvement of egh and brn in N-mediated epithelial development, but not lateral specification, (2) why brn and egh embryonic neurogenic phenotypes are not as severe as N phenotypes, and (3) how egh and brn influence Egfr-mediated processes. The correlation between the differential expression of egh in the oocyte and the differential requirement for brn, egh, and N in maintaining the follicular epithelium around the oocyte, suggests that Egghead is a critical component of a differential oocyte-follicle cell adhesive system.
Corkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.
The Wnt protein Wingless (Wg) functions as a signal in patterning of both the Drosophila embryo and imaginal discs. Lack of porcupine (porc) activity is associated with mutant phenotypes similar to those of wg mutations. In porc mutant embryos, Wg protein is confined to the cells that produce it, suggesting that Porc plays a role in processing or secretion of Wg. porc encodes a novel transmembrane protein that appears to be concentrated at the endoplasmic reticulum. We present both genetic and in vitro evidence demonstrating that porc is involved specifically in the processing of Wg. We identified a human sequence related to Porc suggesting the existence of a family of proteins involved in processing of Wnts.
The imaginal discs of Drosophila, which give rise to the adult appendages, are patterned during a period of intense cell proliferation. The specification of differing regions occurs in some cases by subdividing the disc epithelium into lineage compartments. However, in most cases precise boundaries are formed between different cell types without early compartmentalization. One such boundary occurs between the wingless (wg)-expressing cells of the wing margin and the adjacent proneural cells, which give rise to margin sensory bristles. Here we show that this boundary arises in part by a mechanism of 'self-refinement', by which wingless protein (Wg) represses wg expression in adjacent cells. Cells unable to receive the Wg signal do not resolve the boundary between wg-expressing and proneural cells.
Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal "FLP-DFS" technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.
One major challenge in the fields of signal transduction and pattern formation is to understand how multiple signals are integrated to determine cell fates. Two developmental systems, vulval development in Caenorhabditis elegans and axis formation during Drosophila melanogaster oogenesis, require the epidermal growth factor receptor tyrosine kinase and the NOTCH signaling pathways to specify cell fates. Current work in both systems has provided new opportunities to investigate the potential for the cross-talk between these different signaling pathways.
In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.
The process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, which are controlled by asymmetrically distributed maternal determinants. The anterior determinant bicoid, a homeodomain transcription factor, forms an anterior-to-posterior concentration gradient. It interacts with the maternal transcription factor hunchback to activate the anterior zygotic patterning genes, including the central gap gene Krüppel (Kr). In contrast, the posterior maternal system does not provide such a decisive transcription factor, but rather prevents the repressor hunchback from acting in the posterior half so that the gap genes giant (gt) and knirps (kni) are activated by an as yet unknown transcription factor. Here we show that caudal, a conserved homeodomain protein that forms a posterior-to-anterior concentration gradient, and the anterior determinant bicoid cooperate to form a partly redundant activator system in the posterior region of the embryo.
Signaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled gene product is required for wingless (Wnt1 homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh product in dorsal axis formation and in neural induction.
We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.
The Drosophila gene wingless is a member of the Wnt gene family, a group of genes that are involved in embryonic development and the regulation of cell proliferation. wingless encodes a secreted glycoprotein that plays a role in embryogenesis as well as in the development of adult structures. In the primordia of the adult limbs, the imaginal discs, wingless is expressed in an anterior ventral sector and is required for specification of ventral fate. Ectopic expression of low levels of Wingless in the leg discs leads to partial ventralization and outgrowths of the proximodistal axis. Wingless has thus been proposed to specify ventral fate in a concentration dependent manner (i.e., as a morphogen) and to organize the proximodistal axis. We have extended the analysis of Wingless function in the leg primordium through targeted ectopic expression. We find that Wingless has two functions in the leg disc. In the specification of ventral fate, our data indicate that Wingless does not function as a morphogen but instead appears to collaborate with other factors. In addition to its role in ventral fate specification, Wingless inhibits the commitment of dorsal cells toward a determined state and influences the regulation of proliferation. We propose a model in which Wingless achieves separate functions via spatially regulated mechanisms and discuss the significance of these functions during axial patterning and organization.
Proper spatial expression of the wingless (wg) gene in the Drosophila embryonic epidermis is crucial to intrasegmental patterning. Single cell wide wg expression is initiated at the blastoderm stage in response to combinatorial regulation by the pair rule genes. Later, during gastrulation, when the epidermal expression of the pair rule genes has disappeared, wg becomes regulated by the activity of the segment polarity genes. The segment polarity gene engrailed (en) is expressed in cells adjacent to the wg-expressing cells and is required to maintain wg transcription. Since wg is in turn required to maintain en expression, wg appears to autoregulate its own expression through an endependent paracrine feedback loop. In this paper, we demonstrate that wild-type wg expression requires wg activity during stage 9, prior to its requirement for en maintenance, indicating that wg has an autoregulatory role that is distinct from its paracrine feedback loop through en. In addition, by misexpressing Wg and En in distinct spatial patterns in the epidermis, we find that En is capable of inducing expression from the endogenous wg gene only in immediate adjacent cells which have been exposed to Wg. Furthermore, exogenous Wg expression enables maintenance of endogenous wg transcription in both wg and en mutant embryos. Our results support the model that in the wild-type embryo, wg has an autoregulatory function which is distinct and separable from paracrine regulation via en. We also provide evidence that late, localized Wg expression is crucial for the asymmetric patterning of epidermal cell types as reflected in the larval cuticle.
The Drosophila segment polarity gene wingless (wg) is required in the regulation of engrailed (en) expression and the determination of cell fates in neighboring cells. This paracrine wg activity also regulates transcription of wg itself, through a positive feedback loop including en activity. In addition, wg has a second, more direct autoregulatory requirement that is distinct from the en-dependent feedback loop. Four gene products, encoded by armadillo (arm), dishevelled (dsh), porcupine (porc) and zeste-white 3 (zw3), have been previously implicated as components of wg paracrine signaling. Here we have used three different assays to assess the requirements of these genes in the more direct wg autoregulatory pathway. While the activities of dsh, zw3 and arm appear to be specific to the paracrine feedback pathway, the more direct autoregulatory pathway requires porc.
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.
Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso.