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The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, we overexpressed the Su(fu) gene in imaginal discs and observed opposing effects depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. We also show that the Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, we propose a model in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression.

The fused gene encodes a serine-threonine kinase that functions as a positive regulator of Hedgehog signal transduction in Drosophila embryogenesis, wing morphogenesis, and somatic cell development during oogenesis. Here, we have characterized the germline ovarian tumors present in adult ovaries of fused mutant females, a phenotype not observed upon deregulation of any other component of Hedgehog signaling. In the strongest fused mutant contexts, we found that tumorous ovarian follicles accumulate early spectrosome-containing germ cells corresponding to germline stem cells and/or early cystoblasts as evidenced by activated Dpp signal transduction and transcriptional repression of bag-of-marbles, encoding the cystoblast determination factor. These early germ cells are maintained far from their usual position in a specialized niche of somatic cells in the apical part of the germarium, which appears normal in size in fused mutant ovarioles. Therefore, these results indicate a novel function for fused in downregulation of Dpp signaling which is necessary for de-repression of bag-of-marbles and consequent cystoblast determination. The abnormal accumulation of these early germ cells seems to be due primarily to defects in differentiation since we show that germline stem cell proliferation in the germarium is not affected. A later block in germline development, at the 16-cell cyst stage before significant nurse cell and oocyte differentiation, was also observed in tumorous follicles when fused function was only partially lowered. Finally, fused mutant ovaries exhibit some germline cysts having undergone a supernumerary fifth mitotic division. Through clonal analysis, we provide evidence that fused regulates these cystocyte divisions cell autonomously, while the tumorous phenotype probably reflects both a somatic and germline requirement for fused for cyst and follicle development.

Background: In Drosophila and vertebrates, suppressor of fused (Su(fu)) proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling., Results: We sought for novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. We focused on one of these putative partners, dMLF, which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. We demonstrate that dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, we showed, that dMLF is a nuclear, chromosome-associated protein. We over-expressed a dMLF transgene in fly using an inducible expression system and showed that dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF., Conclusion: We propose that dSu(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development.

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.

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.

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 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.

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.

Fused is a segmentation gene belonging to the segment-polarity class. Mutations at thefused locus are known to display pleiotropic effects, causing zygotically determined anomalies of ovaries and of some adult cuticular structures, and maternally determined embryonic segmentation defects. In order to determine the amorphic phenotype offused and to study the genetical basis of its pleiotropy, newfused alleles (18 viable and 11 lethal) were isolated. The phenotype of these mutants and of others already known are described, taking into account zygotic and maternal effects. The main results provided by this analysis are as follows. Firstly, allfused alleles show the whole complex fused phenotype, and a good correlation is observed between the strength of the wing and segmentation defects, suggesting that a single function is involved in both processes. Secondly, all embryonic and larval lethals carry deficiencies which allow us to localizefused between the 17C4 and 17D2 bands of the X-chromosome. Thirdly, the 24 viable and 2 pupal lethals examined behave as point mutants, as shown cytologically or by Southern blot analysis. However, only one of them, the pupal lethalfu mH63 was proven to carry a nullfused allele, since it displays in germ-line clones a strong maternal phenotype and a very low zygotic rescue, similar to those of the small deficiencyDf(1)fu z4 . The phenotype of the amorphic mutant indicates that zygotic ezpression offused is required for normal metamorphosis, while maternal expression is necessary for a normal segmentation pattern, since a complete loss offused expression during oogenesis cannot be compensated zygotically.