Ligand-Induced Cis-Inhibition of Notch Signaling: The Role of an Extracellular Region of Serrate.

Cellular development can be controlled by communication between adjacent cells mediated by the highly conserved Notch signaling system. A cell expressing the Notch receptor on one cell can be activated in trans by ligands on an adjacent cell leading to alteration of transcription and cellular fate. Ligands also have the ability to inhibit Notch signaling, and this can be accomplished when both receptor and ligands are coexpressed in cis on the same cell. The manner in which cis-inhibition is accomplished is not entirely clear but it is known to involve several different protein domains of the ligands and the receptor. Some of the protein domains involved in trans-activation are also used for cis-inhibition, but some are used uniquely for each process. In this work, the involvement of various ligand regions and the receptor are discussed in relation to their contributions to Notch signaling.

An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling.

Cell-to-cell communication via the Notch pathway is mediated between the membrane-bound Notch receptor and either of its canonical membrane-bound ligands Delta or Serrate. Notch ligands mediate receptor transactivation between cells and also mediate receptor cis-inhibition when Notch and ligand are co-expressed on the same cell. We demonstrate in Drosophila that removal of any of the EGF-like repeats (ELRs) 4, 5 or 6 results in a Serrate molecule capable of transactivating Notch but exhibiting little or no Notch cis-inhibition capacity. These forms of Serrate require Epsin (Liquid facets) to transduce a signal, suggesting that ELR 4-6-deficient ligands still require endocytosis for Notch activation. We also demonstrate that ELRs 4-6 are responsible for the dominant-negative effects of Serrate ligand forms that lack the intracellular domain and are therefore incapable of endocytosis in the ligand-expressing cell. We find that ELRs 4-6 of Serrate are conserved across species but do not appear to be conserved in Delta homologs.

The Ser(+r83k) mutation is a second site mutation of SerD affecting the N-terminus of serrate.

The Serrate gene encodes an essential ligand for Notch signaling used during development of the adult wing and other systems in Drosophila melanogaster. Animals heterozygous or homozygous for the Ser(D) allele of this gene display characteristic defects in wing margin formation. We have characterized a spontaneously arising intragenic suppressor of Ser(D) named Ser(+r83k). Homozygous double mutant Ser(+r83k), Ser(D) animals are viable, with normal wing margin formation, but display an aberrant outspread wing posture. The two mutations can be separated by meiotic recombination which restores the Ser(D) mutant phenotype and demonstrates that in the absence of Ser(D) the Ser(+r83k) mutation is homozygous lethal. These two mutations therefore display allelic compensation. Molecular analysis reveals a single C-T transition mutation within the 5' (protein encoding region) of the Ser(+r83k) transcript. This mutation is predicted to change Arginine(176) to Cysteine, possibly leading to altered interactions with the Notch receptor.

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development.

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.

The Drosophila melanogaster importin alpha3 locus encodes an essential gene required for the development of both larval and adult tissues.

The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression.

Drosophila melanogaster importin alpha1 and alpha3 can replace importin alpha2 during spermatogenesis but not oogenesis.

Importin alpha's mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin alpha genes, most of which fall into three conventional phylogenetic clades, here designated alpha1, alpha2, and alpha3. Using degenerate PCR we cloned Drosophila melanogaster importin alpha1, alpha2, and alpha3 genes, demonstrating that the complete conventional importin alpha gene family arose prior to the split between invertebrates and vertebrates. We have begun to analyze the genetic interactions among conventional importin alpha genes by studying their capacity to rescue the male and female sterility of importin alpha2 null flies. The sterility of alpha2 null males was rescued to similar extents by importin alpha1, alpha2, and alpha3 transgenes, suggesting that all three conventional importin alpha's are capable of performing the important role of importin alpha2 during spermatogenesis. In contrast, sterility of alpha2 null females was rescued only by importin alpha2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin alpha2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin alpha2.