Genomic organization, chromosomal assignment, and expression analysis of the mouse suppressor of fused gene (Sufu) coding a Gli protein partner.

Suppressor of fused (Sufu) is a negative regulator of the Hedgehog pathway both in Drosophila and vertebrates. Here, we report the genomic organization of the mouse Sufu gene (mSufu). This gene comprises 11 exons spanning more than 30 kb and encodes a protein with a putative PEST sequence. DNA-consensus sequences recognized by basic helix-loop-helix (bHLH) proteins, referred to as E-box motifs, are found in the 5' flanking region. Analysis by single-strand conformation polymorphism and radiation hybrid positioned the Sufu locus to the distal end of mouse Chr 19 between D19Mit102 and D19Mit9, near the Fgf8 and dactylin genes. Mouse Sufu is expressed in various tissues, particularly in the nervous system, ectoderm, and limbs, throughout the developing embryo. Sufu binds with all three Gli proteins, with different affinities. This report, in conjunction with recent studies, points out the importance of Sufu in mouse embryonic development.

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.

Suppressor of fused links fused and Cubitus interruptus on the hedgehog signalling pathway.

The Hedgehog (Hh) family of signalling proteins [1] mediate inductive interactions either directly or by controlling the transcription of other secreted proteins through the action of Gli transcription factors, such as Cubitus interruptus (Ci) [2]. In Drosophila, the transcription of Hh targets requires the activation of the protein kinase Fused (Fu) and the inactivation of both Suppressor of fused (Su(fu)) and Costal-2 (Cos-2) [3]. Fu is required for Hh signalling in the embryo and in the wing imaginal disc and acts also as an antitumorigen in ovaries [4]. All fu- phenotypes are suppressed by the loss of function of Su(fu) [5]. Fu, Cos-2 and Ci are co-associated in vivo in large complexes that are bound to microtubules in a Hh-dependent manner [6,7]. Here we investigate the role of Su(fu) in the intracellular part of the Hh signalling pathway. Using the yeast two-hybrid method and an in vitro binding assay, we show that Su(fu), Ci and Fu can interact directly to form a trimolecular complex, with Su(fu) binding to both its partners simultaneously. Su(fu) and Ci also co-immunoprecipitate from embryo extracts. We propose that, in the absence of Hh signalling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci.

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.