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Roles of the E(spl) Genes in the Notch pathway

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Petros Ligoxygakis1, Sarah Bray2, Anette Preiss3 and Christos Delidakis1.

1 Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece.
2 Department of Anatomy, University of Cambridge, UK.
3 Institute für Allgemeine Genetik, Universität Hohenheim, Stuttgard, Germany.

Introduction:

Cell fate acquisition in multicellular systems is based on cell lineage and cell communication. In processes of cell-cell signalling the transmembrane Notch protein appears to play a central role. Notch functions as a receptor in a signal transduction pathway whose other components include the ligands Delta and Serrate , the intacellular transducer Suppressor of Hairless and the nuclear proteins encoded by the genes of the Enhancer of split complex. Highly concerved Notch homologues as well as other elements of the pathway have now been identified in vertebrates, amphibians and nematodes.

The Enhancer of split genes are considered to be the last step in the reception of the Notch signal and are likely to modulate transcription of downstream genes. The locus includes seven genes encoding related basic-helix-loop-helix proteins (m‰, mÁ, m‚, m3, m5, m7, m8). The presence of the bHLH motif and their expression patterns in the embryo and wing imaginal discs are consistent with these proteins suppressing the neural fate in proneural cluster cells, by repressing genes of the achaete-scute complex (AS-C). The effects of E(spl)bHLH proteins on transcription may involve interaction with the product of the neighbouring gene groucho (gro) (Figure 1). Gro has sequence similarity to the yeast co-repressor TUP1 and complexes formed between Gro and E(spl)bHLH proteins repress transcription of target genes (Fisher et al, 1996).

However although in some processes Notch represses specific downstream genes (e.g. achaete, scute) it appears to transcriptionaly activate others (e.g. wingless, cut, vestigial) during wing margin development. We want to determine the extent to which E(spl) proteins might participate in these different responses to Notch activity. Because mutants deleting individual E(spl) bHLHs are viable we used the GAL4/UAS system to characterise the function of individual E(spl) bHLH genes (m‰, mÁ, m5) in different developmental contexts. Here we report results using the E(spl) m‰

Fig 1: Scematic representation of the Notch signal trasduction pathway (see introduction for details)

Fig 2a / Fig 2b: Loss of scutellar bristles when UASm‰ expression is driven by the ptc-GAL4 activator. Suppression of neural development is not only shown by lack of sensory organs but also by lack of expression in early neural markers like asense.

Fig 3: Loss of sensory organs in the abdomen when UASm‰ expression is driven by the sca-GAL4 activator. Partial rescue of the phenotype is observed when m‰ is ectopicaly expressed in a boss14 /+ background. boss14 is a deficiency of the E(spl) locus that deletes almost all the genes of the locus except m8 and groucho.

Fig 4: Loss of wing veins when UASm‰ expression is driven by 32BGAL4 activator.

Fig 5a / Fig 5b: Complete rescue of the wing vein loss phenotype when m‰ ectopic expression is driven in a boss14 /+ or in a E73/+ genetic background. E73 is a null point mutant of groucho.

Fig 6a / Fig 6b / Fig 6c / Fig 6d : Complete rescue of wing notching when UASm‰ expression is driven by 32BGAL4 activator.

Discussion

The above results show the effects of E(spl)m‰ ectopic expression in three different developmental contexts. In the development of bristles, wing veins and wing blade. Targeted expression of E(spl)m‰ in imaginal discs inhibits formation of bristles. This is accompanied by abolition for staining of the early neural marker asense in cells corresponding to the SMCs. Ectopic expression of E(spl)m‰ mimics the Notch gain of function phenotype in the notum where clones of cells expressing activated Notch fail to produce sensory bristles (Struhl et al, 1993).

Drosophila wing vein morphogenesis involves various forms of cell-cell communication in which Notch plays a lateral inhibitory role. During later stages of wing development Notch has been implicated in restricting the breadth of veins and is doing so in earlier stages by restricting the number of cells initiating vein development (Sturtevant and Bier, 1995). The loss of vein phenotype from ectopic expression of E(spl)m‰ resemble theNAX gain of function phenotype.

With the help of clonal analysis and molecular markers Notch is found to be locally activated in dorsal and ventral cells at the D/V boundary through the action of two ligands Delta and Serrate (de Celis et al, 1996). Su(H) is required to mediate actions of Notch in these cells. Notch activity at the D/V boundary is critical for wing growth and margin development and it is necessary to restrict and maintain the expression of genes such as wingless and cut to the cells of this boundary (de Celis et al, 1996, Diaz-Benjumea and Cohen, 1995). Ectopic activation of Notch through ectopic expression of its ligands using the GAL4/UAS system causes ectopic wing margin formation and ectopic wg expression accompanied by overgrowth of the disc (Thomas et al, 1995, Kim et al, 1995, Doherty et al, 1996).

Flies heterozygous for loss of function alleles of Notch or hemizygous for hypomorphs (like nd3) show wing scalloping phenotypes. This loss involves loss of sensory organs and adjacent wing tissue. Ectopic expression of m‰ driven by the 32BGAL4 activator restores the wing margin and additionally suppresses the thick veins phenotype caused by these alleles. Clones of cells lacking all the E(spl) bHLH genes that abut the wing margin do not produce wing nicks. Also ectopic expression of four E(spl) bHLH genes, two of which are normally detected in the cells at the dorsoventral boundary, fails to mimic the above mentioned effect of ectopic Notch activation (de Celis et al, in press). Although this is evidence that the E(spl) bHLH genes are not essential for Notch function at the D/V boundary ectopic expression of m‰ suggests a potential involvement of E(spl) in this process perhaps at a stage after the initial boundary setting.

The suppression of both loss of bristles and loss of vein phenotypes in a boss14/+ genetic background show that in order for the ectopic expression of m‰ to have an effect the rest of the E(spl) bHLHs are required. There is evidence that E(spl)m‚ has evolved to participate inthe Notch pathway during vein development. Its expression in intervein regions during wing imaginal disc development and its effect after ectopic expression are such evidence (Jose de Celis pers. commun.). When UASm is driven by 32BGAL4 the result is an extensive loss of vein. When this ectopic expression takes place in a boss14/+ genetic background using the same GAL4 activator no suppression of the phenotype is observed (data not shown). It is therefore possible that there is some kind of cross-regulation (or interdepedency) between m and the other E(spl) bHLHs (namely m‚ in the context of wing vein development), because ectopic expression of m and m‚ place the former upstream of the latter during vein differentiation.

Finally the suppression of the wing vein phenotype in a gro E73 background correlates well with evidence suggesting gro as a partner of the E(spl) bHLH genes in transcriptional repression (Fisher et al, 1996).

References

de Celis, J.F., de Celis, J., Ligoxygakis, P., Preiss, A., Delidakis, C.& Bray, S.J. Development (in press).

de Celis, J.F., Garcia-Bellido, A.& Bray, S.J. (1996) Development 122, 359-369.

Diaz-Benjumea, F.& Cohen, S.M. (1995) Development 121, 4125-4225.

Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L.Y., Jan, Y.N. (1996) Genes & Dev. 10, 421-434.

Fisher, A.L., Oshako, S.& Caudy, M. (1996) Mol. Cell. Biol. 16, 2670-2677.

Kim J., Irvine K.D.& Carroll S.B. (1995) Cell 82, 795-802.

Struhl, G., Fitzgerald, K. & Greenwald, I. (1993) Cell 74, 331-345.

Sturtevant, M.A.& Bier, E. (1995) Development 121, 785-801.

Thomas, U., Johnson, F., Speicher, S.A.& Knust, E. (1995) Genetics 139, 203-213.

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