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, cells secreting sca protein control the pattern of the next ommatidial column.Cell-cell signals mediated both by diffusible factors and by direct cell contact are thought to be important during development. Studies of the Drosophila eye have contributed examples of interactions between neighboring cells (49). Definitive proofs of diffusible signaling have been harder to obtain. Here we have characterized the scabrous protein, a candidate secreted signal acting in eye development.The Drosophila retina is made up of hundreds of 20-cell ommatidia, precisely arranged in a hexagonal array. The origin of the array during development is not fully understood. As the wave of differentiation crosses the eye imaginal disc from posterior to anterior, the ommatidia in each new column form out of phase with those in the previous column. Within each ommatidium, R8 photoreceptor cells are the first cell type to differentiate, and each R8 cell is required for differentiation of the other ommatidial cells. R8 cell determination is thus important for the entire retinal pattern (24,33,43).Several lines of evidence suggest that determination of the R8 cells shares features with determination of neural cells from undifferentiated ectoderm in other parts of the nervous system. R8 differentiation requires the basic helix-loop-helix protein atonal (ato), similar to the proneural genes required for other neural cells (24). Loss of Notch (N) or Delta (Dl) gene function results in supernumerary R8 cells, similar to the neural hypertrophy that these neurogenic mutations cause elsewhere (3, 5, 32). Retinal differentiation is delayed by the helixloop-helix genes hairy and extramacrochaetae, a...

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.

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.

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