Molecular cloning of fused, a gene required for normal segmentation in the Drosophila melanogaster embryo.

Using the chromosomal walk technique, we isolated recombinant lambda bacteriophage and cosmid clones spanning 250 kilobases (kb) in the 17C-D region of the X chromosome of Drosophila melanogaster. This region was known to contain the segment polarity gene fused. Several lethal fused mutations were used to define more precisely the localization of this locus. Southern analysis of genomic DNA revealed that all of them were relatively large deficiencies, the smallest one being 40 kb long. None of the 12 viable fused mutations examined possessed detectable alterations. We isolated a cosmid containing an insertion covering the entire smallest fused deletion (40 kb). We injected this DNA into fused mutant embryos and obtained a partial phenotypic rescue of the embryonic pattern, indicating that this region contained all the sequences necessary for the embryonic expression of the fu+ gene. Within this DNA, a subclone of 14 kb codes for poly(A)+ RNAs of 3.5, 2.5, 1.6, and 1.3 kb detected in embryos from various developmental stages as well as in adults. All these transcripts showed the same developmental expression. This transcribed region was injected into fused mutant embryos, and once again we obtained a partial rescue of the embryonic phenotype, confirming that this region contained at least the fused gene.

Segmental polarity in Drosophila melanogaster: genetic dissection of fused in a Suppressor of fused background reveals interaction with costal-2.

fused (fu) is a segment polarity gene that encodes a putative serine/threonine kinase. A complete suppressor of the embryonic and adult phenotypes of fu mutants, Suppressor of fused (Su(fu)), was previously described. The amorphic Su(fu) mutation is viable and displays no phenotype by itself. We have used this suppressor as a tool to perform a genetic dissection of the fu gene. Analysis of the interaction between Su(fu) and 33 fu alleles shows that they belong to three different classes. Defects due to class I fu alleles are fully suppressed by Su(fu). Class II fu alleles lead to a new segment polarity phenotype in interaction with Su(fu). This phenotype corresponds to embryonic and adult anomalies similar to those displayed by the segment polarity mutant costal-2 (cos-2). Class II alleles are recessive to class I alleles in a fu[I]/fu[II];Su(fu)/Su(fu) combination. Class 0 alleles, like class I alleles, confer a normal segmentation phenotype in interaction with Su(fu). However class II alleles are dominant over class 0 alleles in a fu[0]/fu[II];Su(fu)/Su(fu) combination. Alleles of class I and II correspond to small molecular events, which may leave part of the Fu protein intact. On the contrary, class 0 alleles correspond to large deletions. Several class I and class II fu mutations have been mapped, and three mutant alleles were sequenced. These data suggest that class I mutations affect the catalytic domain of the putative Fu kinase and leave the carboxy terminal domain intact, whereas predicted class II proteins have an abnormal carboxy terminal domain. Su(fu) enhances the cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). All together these results suggest that a close relationship might exist between fu, Su(fu) and cos-2 throughout development. We thus propose a model where the Fu+ kinase is a posterior inhibitor of Costal-2+ while Su(fu)+ is an activator of Costal-2+. The expression pattern of wingless and engrailed in fu and fu;Su(fu) embryos is in accordance with this interpretation.

Functional domains of fused, a serine-threonine kinase required for signaling in Drosophila.

fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fuO, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment.

Drosophila null slimb clones transiently deregulate Hedgehog-independent transcription of wingless in all limb discs, and induce decapentaplegic transcription linked to imaginal disc regeneration.

Drosophila Slimb (Slmb) is a F-box/WD40 protein which potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless (wg) and decapentaplegic (dpp), as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional mechanisms in which slmb is required, such as leg dorsal-ventral restriction. We have isolated new alleles of the slmb gene in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, we have determined that regulation of Dpp and Wg morphogens by Slmb could be different from what has already been published. We show here that in leg discs, lack of slmb function derepresses the transcription of wg independently of Hh signaling. We present evidence that ectopic legs resulting from slmb(-) clone induction only come from wg misexpression in the normal dpp domain, as ectopic proximo-distal axis are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. We also describe, in discs bearing amorphic slmb clones and in discs of two other slmb(-) contexts, a novel pattern of dpp expression consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb(-) cell elimination. We have investigated the fate of slmb(-) clones, which disappear before adulthood, and found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out which excludes all slmb(-) clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, as in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc.

Production of dominant female sterility in Drosophila melanogaster by insertion of the ovoD1 allele on autosomes: use of transformed strains to generate germline mosaics.

We have cloned a 7 kb genomic fragment containing the dominant female-sterile mutation ovoD1. This fragment confers to transgenic females a sterility phenotype, the severity of which depends both on the genetic background and the ratio of ovoD1 product to ovo+ product. Females containing two copies of the ovoD1 transgene, or those containing one recessive null allele at the ovo locus, are about as sterile as ovoD1 females. Twenty transformed strains were obtained and five of them were tested and shown to be excellent tools for identifying a germline clone of cells sustaining mitotic recombination on the autosomes. One of the tested strains carries an insert on chromosome 4, which enabled us to show that mitotic recombination on that chromosome is not a rare event: it is in fact frequent enough for the maternal effects of the zygotic lethal mutations cubitus interruptus Dominant (ciD) and l(4)29 to be studied.

Modulation of Hedgehog target gene expression by the Fused serine-threonine kinase in wing imaginal discs.

The Fused (Fu) serine-threonine kinase and the Suppressor of fused (Su(fu)) product are part of the Hedgehog (Hh) signaling pathway both in embryos and in imaginal discs. In wing imaginal discs, the Hh signal induces Cubitus interruptus (Ci) accumulation and activates patched (ptc) and decapentaplegic (dpp) expression along the anterior/posterior (A/P) boundary. In this paper, we have examined the role of the Fu and Su(fu) proteins in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. We show that, at the A/P boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed (en) expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. In contrast, in more anterior cells which do not receive Hh signal, we show that Fu plays a role independent of its kinase function in the regulation of Ci accumulation. In these cells, Fu may be involved in the stabilization of a large protein complex which is probably responsible for the regulation of Ci cleavage and/or targeting to nucleus. We propose that the Fused function is necessary for the activation of full-length Ci and counteracts the negative Su(fu) effect on the pathway, leading to en, ptc and dpp expression.

Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster.

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5' part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed.

[Injection of DNA from wild-type strains into the eggs of mutant Drosophila melanogaster]. / Injection d'ADN extrait de souche sauvage dans des oeufs mutants de Drosophila melanogaster

DNA was extracted from wild-type Drosophila Melanogaster or from v; bw mutant and injected into the eggs of v; bw strain. Progeny of flies obtained from injected eggs was examined for several generations. Colored eyes appeared occasionally in the progeny of flies obtained after injection of either DNA. A recovery of colored strains was possible in progeny of flies obtained after injection of wild type DNA (4 out of 15), and not in progeny of 34 flies obtained after injection of v; bw DNA. Genetic alteration inducing color was localized in three of the recovered strains at Su (s) site and in one strain at v site.

Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes.

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.

A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.

Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.

A putative serine/threonine protein kinase encoded by the segment-polarity fused gene of Drosophila.

The segmented pattern of the Drosophila embryo depends on a regulatory cascade involving three main classes of genes. An early regulatory programme, set up before cellularization, involves direct transcriptional regulation mediated by gap and pair-rule genes. In a second phase occurring after cellularization, interactions between segment-polarity genes are involved in cell communication. Segment-polarity genes are required for pattern formation in different domains of each metamere and act to define and maintain positional information in each segment. The segment-polarity gene fused is maternally required for correct patterning in the posterior part of each embryonic metamere. It is also necessary later in development, as fused mutations lead to anomalies of adult cuticular structures and tumorous ovaries. Here we provide molecular evidence that this gene encodes a putative serine/threonine protein kinase, a new function for the product of a segmentation gene. This result provides further insight into segment-polarity interactions and their role in pattern formation.

Differential requirements of the fused kinase for hedgehog signalling in the Drosophila embryo.

The two signalling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper we show that one component of the Hedgehog pathway, the Fused kinase accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signalling. We present direct evidence that Fused is required autonomously in anterior cells neighbouring Hedgehog in order to maintain patched and wingless expression while Wingless is in turn maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells we could show that the Hedgehog signalling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, we show that the morphogen Hedgehog can pattern the dorsal cuticle independently of Fused. In order to account for these differences in Fused requirements, we propose the existence of position-specific modulators of the Hedgehog response.