Irmgard Wech

The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes.

Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su(H)) mediated transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex (E(spl)-C), encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. Here we show that the E(spl)-C contains three additional Notch responsive, non-bHLH genes: m4 and ma are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide.

Distinct expression patterns of different enhancer of split bHLH genes during embryogenesis of Drosophila melanogaster.

Abstract E(spl) bHLH genes are targets of the Notch pathway: they are transcriptionally activated in response to the Notch signal. Yet, during imaginal development, additional regulatory factors appear to modulate transcription resulting in different expression patterns. During early embryogenesis all E(spl) bHLH genes are expressed in roughly the same domain, namely the neurogenic ectoderm. Within this region these seven genes show a highly dynamic, yet distinct transcriptional activity. Our analysis further detected tissue specific expression of some E(spl) genes at later embryonic stages. Prominent differences were observed in the dorsolateral and procephalic neuroectodermal regions as well as in the mesoderm. These observations indicate that other factors in addition to the Notch signal participate in the regulation of the individual E(spl) genes not only in imaginal tissues but also during neuroblast specification and other cell fate determination events in the embryo.

Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an ‘Orange’ protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.

Mutations in rugose promote cell type-specific apoptosis in the Drosophila eye

rugose (rg) encodes an A kinase anchor protein and was isolated as a genetic interactor of the Notch and epidermal growth factor receptor (EGFR) pathways during eye development in Drosophila. rg mutants display a small, rough eye phenotype primarily caused by the loss of cone cells. Here we show that the basis of this phenotype is cell type-specific apoptosis rather than transformation and hence can be rescued by reduction of proapoptotic signals. Moreover, a nearly complete rescue is observed by an increased Notch signal suggesting an antiapoptotic function of Notch in this developmental context. Cone cell loss in rg mutants is accompanied by enhanced Jun N-terminal kinase activity and, concomitantly, by a reduction of EGFR signalling activity. Together, these findings support the idea that rg plays an important role in the integration of different signals required for the exact regulation of cone cell development and survival.

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.

Protein Kinase D regulates several aspects of development in Drosophila melanogaster

BackgroundProtein Kinase D (PKD) is an effector of diacylglycerol-regulated signaling pathways. Three isoforms are known in mammals that have been linked to diverse cellular functions including regulation of cell proliferation, differentiation, motility and secretory transport from the trans-Golgi network to the plasma membrane. In Drosophila, there is a single PKD orthologue, whose broad expression implicates a more general role in development.ResultsWe have employed tissue specific overexpression of various PKD variants as well as tissue specific RNAi, in order to investigate the function of the PKD gene in Drosophila. Apart from a wild type (WT), a kinase dead (kd) and constitutively active (SE) Drosophila PKD variant, we also analyzed two human isoforms hPKD2 and hPKD3 for their capacity to substitute PKD activity in the fly. Overexpression of either WT or kd-PKD variants affected primarily wing vein development. However, overexpression of SE-PKD and PKD RNAi was deleterious. We observed tissue loss, wing defects and degeneration of the retina. The latter phenotype conforms to a role of PKD in the regulation of cytoskeletal dynamics. Strongest phenotypes were larval to pupal lethality. RNAi induced phenotypes could be rescued by a concurrent overexpression of Drosophila wild type PKD or either human isoform hPKD2 and hPKD3.ConclusionOur data confirm the hypothesis that Drosophila PKD is a multifunctional kinase involved in diverse processes such as regulation of the cytoskeleton, cell proliferation and death as well as differentiation of various fly tissues.

ISOLATION AND CHROMOSOMAL LOCALIZATION OF A GERM LINE-SPECIFIC HIGHLY REPETITIVE DNA FAMILY IN ACRICOTOPUS LUCIDUS (DIPTERA, CHIRONOMIDAE)

Abstract. In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line.

Neural hyperplasia induced by RNA interference with m4/mα gene activity

The E(spl) complex (E(spl)-C) contains three different classes of genes that are downstream of Notch signaling. The bHLH genes mediate the Notch signal by repressing proneural gene activity, for example during the singularization of mechanosensory organ precursor cells (SOPs). Genes of the second class, the E(spl) m4/malpha family, antagonize this process if overexpressed. Here we show that this is based on dominant-negative effects since RNA interference gives neurogenic phenotypes indistinguishable from E(spl)-C mutations. Furthermore, a third member of the m4/malpha gene family, named bbu/tom, behaves differently with respect to RNA expression patterns, its regulation by Notch signaling and loss of function phenotypes.