fused regulates germline cyst mitosis and differentiation during Drosophila oogenesis.

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.

Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs.

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.

Genetic analysis of viable and lethalfused mutants ofDrosophila melanogaster.

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.

Over-expression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis.

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.