Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway.     

Neuralized functions cell autonomously to regulate Drosophila sense organ development

Neurogenic genes, including NOTCH: and DELTA:, are thought to play important roles in regulating cell-cell interactions required for DROSOPHILA: sense organ development. To define the requirement of the neurogenic gene neuralized (neu) in this process, two independent neu alleles were used to generate mutant clones. We find that neu is required for determination of cell fates within the proneural cluster and that cells mutant for neu autonomously adopt neural fates when adjacent to wild-type cells. Furthermore, neu is required within the sense organ lineage to determine the fates of daughter cells and accessory cells. To gain insight into the mechanism by which neu functions, we used the GAL4/UAS system to express wild-type and epitope-tagged neu constructs. We show that Neu protein is localized primarily at the plasma membrane. We propose that the function of neu in sense organ development is to affect the ability of cells to receive Notch-Delta signals and thus modulate neurogenic activity that allows for the specification of non-neuronal cell fates in the sense organ.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

The peripheral nervous system of mutants of early neurogenesis inDrosophila melanogaster

Summary Mutations previously known to affect early neurogenesis inDrosophila melanogaster have been found also to affect the development of the peripheral nervous system. Anti-HRP antibody staining has shown that larval epidermal sensilla of homozygous mutant embryos occur in increased numbers, which depend on the allele considered. This increase is apparently due to the development into sensory organs of cells which in the wild-type would have developed as non-sensory epidermis. Thus, neurogenic genes act whenever developing cells have to decide between neurogenic and epidermogenic fates, both in central and peripheral nervous systems. Different regions of the ectodermal germ layer are distinguished with respect to their neurogenic abilities.     

Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling

The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.     

Multiple roles of the Dcdc42 GTPase during wing development in Drosophila melanogaster

The Rho sub-family of GTPases, comprising Rho, Rac and Cdc42. regulates many biological processes, including morphogenesis, cell polarity, migration, the cell cycle and gene expression. It is important to develop genetic approaches to allow the dissection, in vivo, of the mechanisms of GTPase regulation and signal transmission, and their biological consequences. In this regard, wing development in Drosophila melanogaster is an excellent model system. To investigate the functions of the Drosophila Cdc42 GTPase (Dcdc42), we generated phenotypes during wing development, by expression of the dominant-negative N17 and L89 mutants of Dcdc42. We have identified roles for Dcdc42 in wing growth, and in cell fate choice during the development of the wing veins and the peripheral nervous system. Reduction of Dcdc42 signalling following over-expression of Dcdc42N17 resulted in a broader but more diffuse domain characterised by wing-margin sensory bristles. This was correlated with a broadened stripe of wingless expression along the dorsal-ventral boundary of third-instar wing imaginal discs. Together with genetic interactions with loss- and gain-of-function Notch alleles, these data support a role for wild-type Dcdc42 as a negative regulator of Notch signalling.     

The scabrous protein can act as an extracellular antagonist of notch signaling in the Drosophila wing

Notch (N) is a receptor for signals that inhibit neural precursor specification [1-6]. As N and its ligand Delta (DI) are expressed homogeneously, other molecules may be differentially expressed or active to permit neural precursor cells to arise intermingled with nonneural cells [7,8]. During Drosophila wing development, the glycosyltransferase encoded by the gene fringe (fng) promotes N signaling in response to DI, but inhibits N signaling in response to Serrate (Ser), which encodes a ligand that is structurally similar to DI. Dorsal expression of Fng protein localizes N signaling to the dorsoventral (DV) wing margin [9-11]. The secreted protein Scabrous (Sca) is a candidate for modulation of N in neural cells. Mutations at the scabrous (sca) locus alter the locations where precursor cells form in the peripheral nervous system [12,13]. Unlike fringe, sca mutations act cell non-autonomously [12]. Here, we report that targeted misexpression of Sca during wing development inhibited N signaling, blocking expression of all N target genes. Sca reduced N activation in response to DI more than in response to Ser. Ligand-independent signaling by overexpression of N protein, or by expression of activated truncated N molecules, was not inhibited by Sca. Our results indicate that Sca can act on N to reduce its availability for paracrine and autocrine interactions with DI and Ser, and can act as an antagonist of N signaling.     

Genetic interaction between integrins and moleskin,a gene encoding a Drosophila homolog of importin-7

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. If alphaPS subunits are inappropriately expressed during early pupariation, the epithelia separate, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: wing blisters are not generated efficiently by misexpression of loss-of-function alphaPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant alphaPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen for dominant suppressors of wing blisters generated null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has recently been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK. Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import. These data are discussed with respect to the possible regulation of integrin function by cytoplasmic ERK.     

Notch in the pathway: the roles of Notch signaling in neural crest development

Here, we review recent studies that suggest that Notch signaling has two roles during neural crest development: first in establishing the neural crest domain within the ectoderm via lateral induction and subsequently in diversifying the fates of cells that arise from the neural crest via lateral inhibition. The first of these roles, specification of neural crest via lateral induction, has been explored primarily in the cranial neural folds from which the cranial neural crest arises. Evidence for such a role has thus far only been obtained from chick and frog; results from these two species differ, but share the feature that Notch signaling regulates genes that are expressed by cranial neural crest through effects on expression of Bmp family members. The second of these roles, diversification of neural crest progeny via lateral inhibition, has been identified thus far only in trunk neural crest. Evidence from several species suggests that Notch-mediated lateral inhibition functions in multiple episodes in this context, in each case inhibiting neurogenesis. In the 'standard' mode of lateral inhibition, Notch promotes proliferation and in the 'instructive' mode, it promotes specific secondary fates, including cell death or glial differentiation. We raise the possibility that a single molecular mechanism, inhibition of so-called proneural bHLH genes, underlies both modes of lateral inhibition mediated by Notch signaling.     

rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development.

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila EGF-Receptor, spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid (rho) and Star (S) encode membrane proteins. In this study, we show that rho-mediated hyperactivation of the EGFR/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordium during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. We show that rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independently of spi, to promote vein development through the EGFR/MAPK signaling pathway.     

Phenotypic and Molecular Characterization of Ser(d), a Dominant Allele of the Drosophila Gene Serrate

The Drosophila gene Serrate (Ser) encodes a transmembrane protein with 14 epidermal growth factor--like repeats in its extracellular domain, which is required for the control of cell proliferation and pattern formation during wing development. Flies hetero- or homozygous for the dominant mutation Ser(D) exhibit scalloping of the wing margin due to cell death during pupal stages. Ser(D) is associated with an insertion of the transposable element Tirant in the 3' untranslated region of the gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation signals located further downstream. This leads to increased stability of Ser RNA and higher levels of Serrate protein. In wing discs of wild-type third instar larvae, the Serrate protein exhibits a complex expression pattern, including a strong stripe dorsal and a weaker stripe ventral to the prospective wing margin. Wing discs of Ser(D) third instar larvae exhibit additional Serrate protein expression in the edge zone of the future wing margin, where it is normally not detectable. In these cells expression of wing margin specific genes, such as cut and wingless, is repressed. By using the yeast Gal4 system to induce locally restricted ectopic expression of Serrate in the edge zone of the prospective wing margin, we can reproduce all aspects of the Ser(D) wing phenotype, that is, repression of wing margin--specific genes, scalloping of the wing margin and enhancement of the Notch haplo-insufficiency wing phenotype. This suggests that expression of the Serrate protein in the cells of the edge zone of the wing margin, where it is normally absent, interferes with the proper development of the margin.