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.

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.

The segment-polarity gene fused is highly conserved in Drosophila.

The segment polarity gene fused (fu) is involved in specification of positional information inside embryonic segments in Drosophila melanogaster (Dm). The predicted Fused (Fu) protein contains a serine/threonine kinase domain and a second domain with unknown function. We cloned and sequenced the fu homologous gene from Drosophila virilis (Dv) and made an interspecific DNA sequence comparison to identify regions that have been conserved during evolution. Comparison of the predicted amino acid (aa) sequences reveals two regions of strong homology, one corresponding to the kinase domain (268 aa), the other located in the third exon of the Dm fu gene, suggesting a functional importance for this region. Stretches of significantly conserved sequences are also observed in the 5' and 3' untranslated regions. Weak homology is seen in the intronic sequences although the adjacent exonic sequences are mostly conserved. These findings indicate a high conservation of the predicted Fu protein during the evolution of Drosophila.

The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment.

Suppressor of fused, Su(fu), was identified as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused in Drosophila melanogaster. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, we have cloned the Su(fu) region and identified chromosomal rearrangements associated with Su(fu) mutations. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assigned the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the kar RNA mapped within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttraductionnal modification of a common target in the hedgehog signaling pathway.

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.

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.

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 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.

[Histopathological studies in young Lymnae glabra Müller of live or of abortive infestation]. / Etudes histopathologiques chez de jeunes Lymnae glabra Müller avec une infestation évolutive ou abortive.

Very young snails had been individually exposed to a single miracidium of Fasciola hepatica; they were bred at 23 degrees C during 49 days and killed at days 42 and 49 postexposure for histological studies. Tissue lesions were observed in six organs of infected snails and none in uninfected and control snails. All snails with living parthenitae showed a necrotized and atrophic gonad; a reconstitution with epithelial hyperplasia was noted in the most digestive glands, kidneys and genital annex glands of these snails. Variable tissue lesions occurred in organs of snails with abortive infections: the lesions - i.e. necrosis of epithelial cells followed by a reconstitution with cell hyperplasia - were often seen in the kidneys, more rarely in the other organs. No lesions in ventricle and no amoebocytic reaction were found in all these snails. Cells of nerve ganglia showed a dense and homogeneous coloration in 53% of infected snails and a normal colored structure in the others. Epithelial reconstitution of organs was only seen in infected snails with a higher shell at the end of experiment, epithelial necrosis in infected ones with a variable shell height.

Ecdysteroids during the third larval instar in 1(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster.

The temperature-sensitive 1(3)ecd-1ts mutation (A. Garen, L. Kauvar, and J.A. Lepesant (1977). Proc. Natl. Acad. Sci USA 74, 5099-5103.) has been used in several laboratories to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20 degrees C) and restrictive temperatures (29 degrees C) was compared. Pupariation was inhibited when larvae were shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remained active, did not leave the food, and reached a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29 degrees C). Ecdysone was then the predominant moulting hormone, whereas 20-hydroxyecdysone was the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29 degrees C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5 degrees C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER.

Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovo( D1)) or few eggs (ovo(D2)); females heterozygous for the third mutation, ovo(D3), lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovo(D3) and ovo(D2), but females carrying ovo(D1) and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.

[Effect of the number of miracidia and age of the mollusc on the survival and degree of infestation of Lymnaea glabra Müller by Fasciola hepatica L]. / Influence du nombre de miracidiums et de l'âge du mollusque sur la survie et le degré d'infestation de Lymnaea glabra Müller par Fasciola hepatica L.

This report summarizes studies on the infection of Lymnaea glabra by Fasciola hepatica in connection with the following: 1) the number of miracidia for a single exposure; 2) the snail height (0,5 mm, 1 mm or 2 mm) at miracidial exposure. Snails with evolutive infection were only observed in 0,5- and 1-mm-high groups at day post-exposure. In 0,5-mm-high groups, the percentage of these infected snails showed a peak for 5 miracidia per snail. In 1-mm-high groups, there was no infected snails until 5 miracidia per snail; then the percentage of infected snails increased in connection with the number of miracidia used per snail. L. glabra is an accidental snail host for F. hepatica.

[Experimental data on the infestation of the young of 5 species of Lymnaea by Fasciola hepatica L]. / Données expérimentales sur l'infestation des jeunes de cinq espèces de limnées par Fasciola hepatica L.

The ability of five lymnaeid species (Lymnaea glabra, L. palustris, L. peregra, L. stagnalis, L. truncatula) to act as intermediate hosts of Fasciola hepatica was investigated under controlled conditions. Each snail born in the laboratory (at 23 degrees C) was infected when 1-24-hours-old, using 3 miracidia of F. hepatica. The percentage of infected snails ranged from 15.8% to 64.5% on day 30 post-exposure. The shell height of snails with evolutive infection was significantly reduced than the shell height of snails with abortive infection or controls. The mean number of cercariae produced by the snails with evolutive infection was low: from 12.2 to 18.4 cercariae per snail. The length of the shedding period was one day for 114 snails, two days for 41 snails and 3-31 days for the 54 other snails. We did not observe a distinct rythm in cercarial production of these snails. All infections of guinea-pigs with these metacercariae were positive. The significance of these observations is discussed.