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The number of cells in developing organs must be controlled spatially by extracellular signals. Our results show how cell number can be regulated by cell interactions controlling proliferation and survival in local neighborhoods in the case of the Drosophila compound eye. Intercellular signals act during the second mitotic wave, a cell cycle that generates a pool of uncommitted cells used for most ommatidial fates. We find that G1/S progression to start the cell cycle requires EGF receptor inactivity. EGF receptor activation is then required for progression from G2 to M phase of the same cells, and also prevents apoptosis. EGF receptor activation depends on short-range signals from five-cell preclusters of photoreceptor neurons not participating in the second mitotic wave. Through proliferation and survival control, such signals couple the total number of uncommitted cells being generated to the neural patterning of the retina.

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.

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homologue (DER) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. Their phenotype suggests a possible role for DER in specifying the founder R8 photoreceptor cells for each ommatidium. Here we analyze the basis of Ellipse mutations and use them to probe the role of DER in eye development. We show that Elp mutations result from a single amino acid substitution in the kinase domain which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirmed that the mutation is hypermorphic in vivo, but the DER function was elevated less than by ectopic expression of the ligand spitz. Ectopic spi promoted photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of DER activation were assessed to explore the basis of these distinct outcomes. Elp mutations caused overexpression of the Notch target gene E(spl) mdelta and required function of Notch to suppress ommatidium formation. The Elp phenotype also depended on the secreted protein argos and was reverted in Elp aos double mutants. Complete loss of DER function in clones of null mutant cells led to delay in R8 specification and subsequently to loss of mutant cells. The DER null phenotype was distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands was required for aspects of DER function. In normal development DER protein was expressed in most retinal cells, but at distinct levels. We used an antibody specific for diphospho-ERK as well as expression of the DER target gene argos to assess the pattern of DER activity, finding highest activity in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggested that this activity may not be required for normal ommatidium development. Since we saw distinct phenotypic effects of four different levels of DER activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function, we propose that multiple thresholds separate several aspects of DER function. These include activation of N signaling to repress R8 specification, turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation., (Copyright 1999 Academic Press.)

The scabrous (sca) gene encodes a secreted dimeric glycoprotein with putative coiled-coil domains N-terminally and a C-terminal region related to the blood clot protein fibrinogen. Homozygous sca mutants have extra bristle organs and rough eyes. We describe a GAL4-based expression system for testing rescue of the sca mutant phenotype by altered SCA proteins and for misexpression. We find that deletion of the fibrinogen-related domain (FReD) greatly decreases SCA function, confirming the importance of this conserved region. SCA function could not be restored by FReDs from human fibrinogen chain genes. However, proteins lacking any FReD still showed some function in both rescue and misexpression experiments, suggesting that putative effector-binding regions lie outside this domain. Consistent with this, proteins expressing only the FReD had no rescuing activity but were recessive negative; i.e., they enhanced the phenotype of sca mutations but had no phenotype in the presence of a wild-type sca allele. This suggests that the FReD contributes to SCA function by binding to other components of the bristle determination pathway, increasing the activity of the linked N-terminal region.

The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.

It has been suggested that lateral specification of cell fate by Notch signaling depends on feedback on Notch (N) and Delta (Dl) transcription to establish reciprocal distributions of the receptor and its ligand at the protein level. In Drosophila neurogenesis the predicted reciprocal protein distributions have not been observed. Either this model of lateral specification or the description of N and/or Dl protein distributions must be incomplete. We have reexamined R8 photoreceptor specification in the developing eye to resolve this question for this example of lateral specification. N and Dl protein levels were assessed in the cell as a whole and at the cell surface, where these proteins were mostly found at the intercellular cell junctions. Protein levels did not correspond to Notch signaling in wild type. However, Dl transcription and protein levels did correlate with altered N signaling in mutant genotypes. Our findings suggest the difference relates to the speed of lateral specification in vivo. The time required for N signaling to inhibit ato expression was at most 90 min, but changes in the Dl protein distribution in mutant genotypes arose more slowly. N expression was little regulated by N signaling, but protein encoded by the Nts1 allele was temperature-sensitive for appearance at the cell surface. Some aspects of the pattern of Dl protein appeared to be due to endocytosis. We conclude that feedback of N signaling on Dl transcription does occur but is too slow to account for the pattern of R8 specification. Studies of ommatidia mosaic for a Notch duplication, or for the Nts1 allele at semi-restrictive temperatures, found that cells beginning with less N activity were not necessarily predisposed to be selected for R8 differentiation. Our data argue that other signals may be responsible for the pattern of R8 cell fate allocation by N. Potential relevance to other neurogenic regions is discussed., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved)

Background: . Intercellular signals are major determinants of cell fate during development. Certain signals and receptors are important for many different cell-fate decisions, suggesting that cellular responses to similar signals change during development. Few transitions between such distinct cellular responses have been studied. The Drosophila genes Notch and hedgehog function during intracellular signaling at various stages of development. In the specific case of development of the Drosophila eye, expression of the proneural gene atonal is induced in response to Hedgehog signaling and then becomes subject to autoregulation. The receptor protein Notch has previously been reported to function in the selection of single founder photoreceptor cells (R8 cells) by inhibiting atonal expression. On this basis, complete elimination of Notch gene function would be expected to cause neural hyperplasia in the eye., Results: . Contrary to expectation, we detect a reduction in neural differentiation both in cells expressing a conditional Notch allele and in those lacking expression of either Notch or its ligand Delta. We show here that Notch signaling acts after the initial Hedgehog-driven expression of atonal to enhance proneural competence of the atonal-expressing cells and also to terminate their response to the Hedgehog signals. This occurs before the Notch-induced lateral inhibition of atonal expression within the same cells., Conclusion: . Notch has sequentially opposite effects on the same cells, by first promoting and then inhibiting proneural gene function. This apparently paradoxical sequence of events has two possible consequences. Firstly, coupling of alternative cellular responses to the same receptor may prevent them from occurring simultaneously. Secondly, consecutive regulatory processes become temporally coupled, so that these events follow on from each other, without gaps or overlaps.

R8 photoreceptor cells play a primary role in the differentiation of Drosophila eyes. In scabrous (sca) mutants, the pattern of R8 photoreceptor differentiation is altered. The sca gene is predicted to encode a secreted protein related in part to fibrinogen and tenascins. Using expression in Drosophila Schneider cells, we showed that sca encoded a dimeric glycoprotein which was secreted and found in soluble form in the tissue culture medium. The sca protein contained both N- and O-linked carbohydrates and interacted with heparin. This Schneider cell protein was similar to protein detected in embryos. We showed that sca mutations, along with conditional alleles of Notch (N) and Delta (Dl), each affected the pattern of cells expressing atonal (ato), the proneural gene required for R8 differentiation. In normal development, about 1 cell in 20 differentiates into an R8 cell; in the others, ato is repressed. N and Dl were required to repress ato in the vicinity of R8 cells, whereas sca had effects over several cell diameters. Certain antibodies detected uptake of sca protein several cells away from its source. The overall growth factor-like structure of sca protein, its solubility, and its range of effects in vivo are consistent with a diffusible role that complements mechanisms involving direct cell contact. We propose that as the morphogenic furrow advances, cell secreting sca protein control the pattern of the next ommatidial column.