Cell-fate determination in the developing Drosophila eye: role of the rough gene.

The homeobox-gene rough is required in photoreceptor cells R2 and R5 for normal ommatidial assembly in the developing Drosophila eye. We have used several cell-type-specific markers and double mutant combinations to analyze cell-fate determination in rough. We show that the cells that would normally become R2 and/or R5 express a marker, a lacZ insertion in the seven-up (svp) gene, which is indicative of the R1/3/4/6 cell fate. In addition, the analysis of mitotically induced svp,ro double mutant clones in the eye indicates that in rough all outer photoreceptors are under the genetic control of the svp gene. These results show that, in the absence of rough function, R2 and R5 fail to be correctly determined and appear to be transformed into cells of the R3/4/1/6 subtype. This transformation and the subsequent developmental defects do not preclude the recruitment of R7 cells. However, the presence of ommatidia containing more than one R7 and/or R8 cell in rough implies a complex network of cellular interactions underlying cell-fate determination in the Drosophila retina.     

The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes.

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.     

Drosophila atonal controls photoreceptor R8-specific properties and modulates both receptor tyrosine kinase and Hedgehog signalling

During Drosophila eye development, the proneural gene atonal specifies founding R8 photoreceptors of individual ommatidia, evenly spaced relative to one another in a pattern that prefigures ommatidial organisation in the mature compound eye. Beyond providing neural competence, however, it has remained unclear to what extent atonal controls specific R8 properties. We show here that reduced Atonal function gives rise to R8 photoreceptors that are functionally compromised: both recruitment and axon pathfinding defects are evident. Conversely, prolonged Atonal expression in R8 photoreceptors induces defects in inductive recruitment as a consequence of hyperactive EGFR signalling. Surprisingly, such prolonged expression also results in R8 pattern formation defects in a process associated with both Hedgehog and Receptor Tyrosine Kinase signalling. Our results strongly suggest that Atonal regulates signalling and other properties of R8 precursors.     

EGF signaling and ommatidial rotation in the Drosophila eye

The ommatidia of the Drosophila eye initiate development by stepwise recruitment of photoreceptors into symmetric ommatidial clusters. As they mature, the clusters become asymmetric, adopting opposite chirality on either side of the dorsoventral midline and rotating exactly 90 degrees (Figures 1A and 1B, ). The choice of chirality is governed by higher activity of the frizzled (fz) gene in one cell of the R3/R4 photoreceptor pair and by Notch-Delta (N-Dl) signaling. The 90 degrees rotation also requires activity of planar polarity genes such as fz as well as the roulette (rlt) locus. We now show that two regulators of EGF signaling, argos and sprouty (sty), and a gain-of-function Ras85D allele, interact genetically with fz in ommatidial polarity. Furthermore, we find that argos is required for ommatidial rotation, but not chirality, and that rlt is a novel allele of argos. We present evidence that there are two pathways by which EGF signaling affects ommatidial rotation. In the first, typified by the rlt phenotype, there is partial transformation of the "mystery cells" toward a neuronal fate. Although most of these mystery cells subsequently fail to develop as neurons, their partial transformation results in inappropriate subcellular localization of the Fz receptor, a likely cue for regulating ommatidial rotation. Secondly, reducing EGF signaling can specifically affect ommatidial rotation without showing transformation of the mystery cells or defects in polarity protein localization.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Mutation of the Apc1 homologue shattered disrupts normal eye development by disrupting G1 cell cycle arrest and progression through mitosis

The shattered1 (shtd1) mutation disrupts Drosophila compound eye structure. In this report, we show that the shtd1 eye defects are due to a failure to establish and maintain G1 arrest in the morphogenetic furrow (MF) and a defect in progression through mitosis. The observed cell cycle defects were correlated with an accumulation of cyclin A (CycA) and String (Stg) proteins near the MF. Interestingly, the failure to maintain G1 arrest in the MF led to the specification of R8 photoreceptor cells that undergo mitosis, generating R8 doublets in shtd1 mutant eye discs. We demonstrate that shtd encodes Apc1, the largest subunit of the anaphase-promoting complex/cyclosome (APC/C). Furthermore, we show that reducing the dosage of either CycA or stg suppressed the shtd1 phenotype. While reducing the dosage of CycA is more effective in suppressing the premature S phase entry in the MF, reducing the dosage of stg is more effective in suppressing the progression through mitosis defect. These results indicate the importance of not only G1 arrest in the MF but also appropriate progression through mitosis for normal eye development during photoreceptor differentiation.     

Specification of cell fate in the developing eye of Drosophila.

Determination of cell fate in the developing eye of Drosophila depends on a precise sequence of cellular interactions which generate the stereotypic array of ommatidia. In the eye imaginal disc, an initially unpatterned epithelial sheath of cells, the first step in this process may be the specification of R8 photoreceptor cells at regular intervals. Genes such as Notch and scabrous, known to be involved in bristle development, also participate in this process, suggesting that the specification of ommatidial founder cells and the formation of sensory organs in the adult epidermis may involve a similar mechanism, that of lateral inhibition. The subsequent steps of ommatidial assembly, following R8 assignment, involve a different mechanism: Undetermined cells read their position based on the contacts they make with neighbors that have already begun to differentiate. The development of the R7 photoreceptor cell, one of the eight photoreceptor cells in the ommatidium, is best understood. An important role seems to be played by sevenless, a receptor tyrosine kinase on the surface of the R7 precursor. It transmits the positional information--most likely encoded by the boss protein on the neighboring R8 cell membrane--into the cell via its tyrosine kinase, which activates a signal transduction cascade. Constitutive activation of the sevenless kinase by overexpression of an N-terminally truncated form results in the diversion of other ommatidial cells into the R7 pathway suggesting that activation of the sevenless signalling pathway is sufficient to specify R7 development. Genetic dissection of this pathway should therefore identify components of a signalling cascade activated by a tyrosine kinase.     

Genetic mosaic analysis of the equatorial-less mutation in Drosophila mezunogaster

eql (equatorial-less) is a recessive lethal mutation on the second chromosome of Drosophila melanogasfer. J. Campos-Ortega found that eql clones in somatic mosaic flies have reduced numbers of photoreceptor cells, and he suggested that only the R1, R6, and R7 photoreceptor cells were missing in this mutant. These photoreceptor cells help to define the inverted orientation of ommatidial facets along the equatorial midline of the fly eye, hence the mutation was named “equatorial-less”. We have conducted a detailed analysis of the eql mutation, by serial section reconstruction of eql clones marked with bw or w^? in somatic mosaic flies. We found that all photoreceptor cell types (Rl–R8) could be deleted by the eql mutation, and in rare cases the number of photoreceptor cells was increased. The apparent lack of photoreceptor cell type specificity was confirmed by our analysis of genetically mosaic facets, which indicated that no single photoreceptor cell, or subset of photoreceptor cells, was uniquely required to express eql Rather, eql appears to function in all photoreceptor cells, and possibly in all eye precursor cells. The distribution of photoreceptor cell numbers in w eql facets was consistent with the hypothesis that each photoreceptor cell was deleted independently of the others. The eql gene is located on the right arm of chromosome 2 at map location 2 ? 104.5 ± 0.7 and lies between the polytene chromosome bands 59D8 and 60A7. © 1995 Wiley-Liss, Inc.     

ten-a overexpression causes abnormal pattern in the Drosophila compound eye

Ten-a is one of the two Drosophila proteins that belong to the Ten M protein family. This protein is a type Ⅱ transmembrane protein and is expressed mainly in the embryonic CNS, in the larval eye imaginal disc and in the compound eye of the pupa. Here, we investigate the role of ten-α during development of the compound eye by using the Gal4/ UAS system to induce ten-α overexpression in the developing eye. We found that overexpression of ten-α can perturb eye development during all stages examined. In an early stage, overexpression of ten-α in eye primordial cells caused small and rough eyes and interfered with photoreceptor cell recruitment, resulting in some ommatidia having fewer or extra photoreceptor cells. Conversely, ten-α overexpression daring ommatidial formation caused severe eye defects due to absence of many cellular components. Interestingly, overexpression of ten-α in the late stage developing ommatidial cluster affected the number of pigment cells, caused cone cells proliferation in many ommatidia, and caused some photoreceptor cell defects. These results suggest that ten-α may be a novel gene required for normal eye morphogenesis.     

The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.

Events in the morphogenetic furrow set the stage for all subsequent compound eye development in Drosophila. The periodic pattern of the adult eye begins in the furrow with the spaced initiation of ommatidial rudiments, the preclusters. A wave of mitosis closely follows the furrow. A cell-by-cell analysis reveals details of these events. Early stages of ommatidial assembly can be resolved using a lead sulfide stain. Overt ommatidial organization begins in the morphogenetic furrow as cells gather into periodically spaced concentric aggregates. A stereotyped sequence of cell rearrangements converts these aggregates into preclusters. In the furrow, new rows of ommatidia are initiated at the equator and grow as new clusters are added to the peripheral ends. Mitotic labeling using BrdU feeds shows that all cells not incorporated into a precluster divide. BrdU injections show that cells divide roughly simultaneously between two adjacent rows of ommatidia.     

Diversification of cell types in the Drosophila eye by differential expression of prepattern genes

According to Freeman (Development, 124 (1997) 261), reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Here we show evidence that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up- or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes was regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression.     

semang affects the development of a subset of cells in the Drosophila compound eye

semang (sag), a mutation isolated as a suppressor of Drosophila Src42A, has previously been shown to affect some receptor tyrosine kinase mediated embryonic processes. Here we show that sag specifically affects the development of R1, R6 and R7 photoreceptor cells in a cell-autonomous manner. These cells are absent in the mutant at the time when they normally appear in the ommatidial pre-clusters. Genetic analyses suggest that sag functions downstream of, or parallel to, Mapk and Yan in the photoreceptor differentiation pathway. The autonomous requirement of sag for R1/R6/R7 development could be explained by a selective impairment of the late, but not early, rounds of Egfr-induced precursor cell assembly by the sag mutations. Egfr signaling is highly regulated by autocrine or paracrine mechanisms in different cells. Knowing that the photoreceptor cluster formation is a complex process involving dynamic changes in cell-cell contact, our hypothesis is that the sag alleles affected certain special aspects of Egfr-signaling that are unique for the recruitment of R1/R6/R7 cells.     

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal.

The peripheral nervous system (PNS) of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal (ato) or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Here, we show that dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation.