rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila.

The polynucleate myotubes of vertebrates and invertebrates form by fusion of myoblasts. We report the involvement of the Drosophila melanogaster Roughest (Rst) protein as a new membrane-spanning component in this process. Rst is strongly expressed in mesodermal tissues during embryogenesis, but rst null mutants display only subtle embryonic phenotypes. Evidence is presented that this is due to functional redundancy between Rst and its paralogue Kirre. Both are highly related single-pass transmembrane proteins with five extracellular immunoglobulin domains and three conserved motifs in the intracellular domain. The expression patterns of kirre and rst overlap during embryonic development in muscle founder cells. Simultaneous deletion of both genes causes an almost complete failure of fusion between muscle founder cells and fusion-competent myoblasts. This defect can be rescued by one copy of either gene. Moreover, Rst, like Kirre is a myoblast attractant.

Drosophila Pax-6/eyeless is essential for normal adult brain structure and function.

A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed.

Visualization of synaptic markers in the optic neuropils of Drosophila using a new constrained deconvolution method.

The fruitfly Drosophila melanogaster offers compelling genetic advantages for the analysis of its nervous system, but cell size precludes immunocytochemical analysis of wild-type structure and mutant phenotypes beyond the level of neuronal arborizations. For many antibodies, especially when immunoelectron microscopy is not feasible, it would therefore be desirable to extend the resolution limit of confocal microscopy as far as possible. Because high-resolution confocal microscopy suffers from considerable blurring, so-called deconvolution algorithms are needed to remove, at least partially, the blur introduced by the microscope and by the specimen itself. Here, we present the establishment and application of a new deconvolution method to visualize synaptic markers in Drosophila optic neuropils at the resolution limit of light. We ascertained all necessary parameters experimentally and verified them by deconvolving injected fluorescent microspheres in immunostained optic lobe tissue. The resulting deconvolution method was used to analyze colocalization between the synaptic vesicle marker neuronal synaptobrevin and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of light, which could not be distinguished without deconvolution.

Immunostimulation by bacterial components: I. Activation Of macrophages and enhancement of genetic immunization by the lipopeptide P3CSK4.

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also efficient immunoadjuvants in parenteral, oral and nasal immunization either in combination with or after covalent linkage to an antigen. Here we show how alterations in the molecular structure influence their biological properties indicating P3CSK4 as one of the most active members of a lipopentapeptide fatty acid library. This compound resulted in a most pronounced macrophage stimulation as indicated by NO release, activation of NFkappaB translocation, and enhancement of tyrosine protein phosphorylation. Furthermore, P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. Finally we have evidence that P3CSK4 constitutes an effective adjuvant for DNA immunizations, especially increasing weak humoral immune responses. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.

Irregular chiasm-C-roughest, a member of the immunoglobulin superfamily, affects sense organ spacing on the Drosophila antenna by influencing the positioning of founder cells on the disc ectoderm.

We describe a role for Irregular chiasmC-roughest (IrreC-rst), an immunoglobulin (Ig) superfamily member, in patterning sense organs on the Drosophila antenna. IrreC-rst protein is initially expressed homogeneously on apical profiles of ectodermal cells in regions of the antennal disc. During specification of founder cells (FCs), the intracellular protein distribution changes and becomes concentrated in regions where specific intercellular contacts presumably occur. Loss of function mutations as well as misexpression of irreC-rst results in an altered arrangement of FCs within the disc compared to wildtype. Sense organ development occurs normally, although spacing is affected. Unlike its role in interommatidial spacing, irreC-rst does not affect apoptosis during antennal development. We propose that IrreC-rst affects the spatial relationship between sensory and ectodermal cells during FC delamination.

Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on synaptobrevin.

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, we studied the effects of a loss of function of neuronal synaptobrevin, a protein required for synaptic vesicle release. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. We show that targeted toxin expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. Our results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS is discussed.

Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster.

We present the first three-dimensional map of the antennal lobe of Drosophila melanogaster, based on confocal microscopic analysis of glomeruli stained with the neuropil-specific monoclonal antibody nc82. The analysis of confocal stacks allowed us to identify glomeruli according to the criteria shape, size, position, and intensity of antibody labeling. Forty glomeruli were labeled by nc82, eight of which have not been described before. Three glomeruli previously shown exclusively by backfills were not discernible in nc82 stainings. We distinguish three classes of glomeruli: (1) "landmark" glomeruli that are constant in all four criteria mentioned above, (2) less well-demarcated glomeruli that deviate in a single criterion, and (3) poorly defined glomeruli that vary in more than one criterion. All class 2 and 3 glomeruli can be identified by comparison with landmark neighbors. To further aid identification, our model assigns glomeruli to five arrays, each of which is defined by a prominent landmark glomerulus. Six glomeruli consist of distinct, but contiguous structural units, termed "compartments." Glomerular variability observed occasionally between males and females is in the same range as between individuals of the same sex, suggesting the lack of a significant sexual dimorphism in the glomerular pattern. We compare the new model with a previous map and address its potential for mapping activity and expression patterns. An important goal of this work was to create three-dimensional reference models of the antennal lobe, which are accessible on-line.

Glia in the chiasms and medulla of the Drosophila melanogaster optic lobes.

Different classes of glia cells in the optic lobes of Drosophila melanogaster were defined by the enhancer trap technique, using expression of the lacZ reporter gene. At both the outer and inner optic chiasms, there are stacks of glia, arrayed from dorsal to ventral, interpersed between the crossings of axonal fiber bundles. The giant glial cells of both the outer and inner chiasms are similar with respect to their nuclear shapes and positions, indicating similar functions of these cell types. Another class of glia is found in the medulla neuropil. Their cell bodies anchor in the most distal region of the neuropil, and their processes extend into the deeper neuropil layers. Birth dating using BrdU shows that both groups of chiasm glia are born early in larval life; they may participate in the development of the optic lobe. The medulla glia are born later and may be involved primarily in adult functions. In the wild type, and in mutants with structurally altered optic lobes, the numbers of tract-associated glial cells in the outer and inner optic chiasms seem to vary with the number of visual columns, whereas the complement of medulla neuropil glia correlates with the volume of the optic lobe.

Reorganization of membrane contacts prior to apoptosis in the Drosophila retina: the role of the IrreC-rst protein.

The final step of pattern formation in the developing retina of Drosophila is the elimination of excess cells between ommatidia and the differentiation of the remaining cells into secondary and tertiary pigment cells. Temporally and spatially highly regulated expression of the irregular chiasmC-roughest protein, an adhesion molecule of the immunoglobulin superfamily known to be involved in axonal pathfinding, is essential for correct sorting of cell-cell contacts in the pupal retina without which the ensuing wave of apoptosis does not occur. Irregular chiasmC-roughest accumulates strongly at the borders between primary pigment and interommatidial cells. Mutant and misexpression analysis show that this accumulation of the irregular chiasmC-roughest protein is necessary for aligning interommatidial cells in a single row. This reorganisation is a prerequisite for the identification of death candidates. Irregular chiasmC-roughest function in retinal development demonstrates the importance of specific cell contacts for assignment of the apoptotic fate.

Restricted expression of the irreC-rst protein is required for normal axonal projections of columnar visual neurons.

The 104 kDa irreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils, it is present in all columnar domains of specific layers. The number of irreC-rst-positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the irreC-rst protein globally. Epigenesis of the phenotypes can be explained partially on the bases of homophilic irreC-rst interactions.

minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila.

The development of the adult central nervous system of Drosophila requires a precise and reproducible pattern of neuroblast proliferation during postembryonic neurogenesis. We show here that mutations in the minibrain (mnb) gene cause an abnormal spacing of neuroblasts in the outer proliferation center (opc) of larval brain, with the implication that mnb opc neuroblasts produce less neuronal progeny than do wild type. As a consequence, the adult mnb brain exhibits a specific and marked size reduction of the optic lobes and central brain hemispheres. The insufficient number of distinct neurons in mnb brains is correlated with specific abnormalities in visual and olfactory behavior. The mnb gene encodes a novel, cell type-specific serine-threonine protein kinase family that is expressed and required in distinct neuroblast proliferation centers during postembryonic neurogenesis. The mnb kinases share extensive sequence similarities with kinases involved in the regulation of cell division.

The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein.

The axonal projection mutations irregular chiasm C of Drosophila melanogaster comap and genetically interact with the roughest locus, which is required for programmed cell death in the developing retina. We cloned the genomic region in 3C5 by transposon tagging and identified a single transcription unit that produces a major, spatially and temporally regulated mRNA species of approximately 5.0 kb. Postembryonic expression is strong in the developing optic lobe and in the eye imaginal disc. The gene encodes a transmembrane protein of 764 amino acids with five extracellular immunoglobulin-like domains and similarity to the chicken axonal surface glycoprotein DM-GRASP/SC1/BEN. Both known irreC alleles reduce the level of transcription, whereas the roughestCT mutation disrupts the intracellular domain of the protein.

Activity labeling patterns in the medulla of Drosophila melanogaster caused by motion stimuli.

We quantitatively describe 2-deoxyglucose (2-DG) neuronal activity labeling patterns in the first and second visual neuropil regions of the Drosophila brain, the lamina and the medulla. Careful evaluation of activity patterns resulting from large-field motion stimulation shows that the stimulus-specific bands in the medulla correspond well to the layers found in a quantitative analysis of Golgi-impregnated columnar neurons. A systematic analysis of autoradiograms of different intensities reveals a hierarchy of labeling in the medulla. Under certain conditions, only neurons of the lamina are labeled. Their characteristic terminals in the medulla are used to differentiate among the involved lamina monopolar cell types. The 2-DG banding pattern in the medulla marks layers M1 and M5, the input layers of pathway p1 (the L1 pathway). Therefore, activity labeling of L1 by motion stimuli is very likely. More heavily labeled autoradiograms display activated cells also in layers M2, M9, and M10. The circuitry involved in the processing of motion information thus concentrates on pathways p1 and p2. Layers M4 and M6 of the distal medulla hardly display any label under the stimulus conditions used. The functional significance of selective activity in the medulla is discussed.

Additive gene actions on the fiber number in the anterior optic tract of Drosophila melanogaster.

The influence of mutations in seven neurological genes on the number of fibers in the anterior optic tract (AOT) of Drosophila melanogaster has been investigated. It is shown that the number of fibers in the AOT can be drastically reduced in single and especially in multiple mutants. However, no evidence for synergistic interactions between the sample of mutations used in the sine oculis (so), reduced optic lobes (rol), minibrain (mnb), and small optic lobes (sol) genes was obtained at the level of the AOT. The rolKS222 and so mutations eliminate similar fiber sets in the AOT, which are distinctly different from those eliminated by solKS58 and mnb1.

Survival of photoreceptor neurons in the compound eye of Drosophila depends on connections with the optic ganglia.

The importance of retinal innervation for the normal development of the optic ganglia in Drosophila is well documented. However, little is known about retrograde effects of the optic lobe on the adult photoreceptor cells (R-cells). We addressed this question by examining the survival of R-cells in mutant flies where R-cells do not connect to the brain. Although imaginal R-cells develop normally in the absence of connections to the optic lobes, we find that their continued survival requires these connections. Genetic mosaic studies with the disconnected (disco) mutation demonstrate that survival of R-cells does not depend on the genotype of the eye, but is correlated with the presence of connections to the optic ganglia. These results suggest the existence of retrograde interactions in the Drosophila visual system reminiscent of trophic interactions found in vertebrates.

The optic lobe of Drosophila melanogaster. II. Sorting of retinotopic pathways in the medulla.

We present a quantitative evaluation of Golgi-impregnated columnar neurons in the optic lobe of wild-type Drosophila melanogaster. This analysis reveals the overall connectivity pattern between the 10 neuropil layers of the medulla and demonstrates the existence of at least three major visual pathways. Pathway 1 connects medulla layer M10 to the lobula plate. Input layers of this pathway are M1 and M5. Pathway 2 connects M9 to shallow layers of the lobula, which in turn are tightly linked to the lobula plate. This pathway gets major input via M2. Pathways 1 and 2 receive input from retinula cells R1-6, either via the lamina monopolar cell L1 (terminating in M1 and M5) or via L2 and T1 (terminating in M2). Neurons of these pathways typically have small dendritic fields. We discuss evidence that pathways 1 and 2 may play a major role in motion detection. Pathway 3 connects M8 to deep layers of the lobula. In M8 information converges that is derived either from M3 (pathway 3a) or from M4 and M6 (pathway 3b), layers that get their major input from L3 and R8 or L4 and R7, respectively. Some neurons of pathway 3 have large dendritic fields. We suggest that they may be involved in the computation of form and colour. Possible analogies to the organization of pathways in the visual system of vertebrates are discussed.

Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.

Neuronal architecture of the antennal lobe in Drosophila melanogaster.

Computer reconstruction of the antennal lobe of Drosophila melanogaster has revealed a total of 35 glomeruli, of which 30 are located in the periphery of the lobe and 5 in its center. Several prominent glomeruli are recognizable by their location, size, and shape; others are identifiable only by their positions relative to prominent glomeruli. No obvious sexual dimorphism of the glomerular architecture was observed. Golgi impregnations revealed: (1) Five of the glomeruli are exclusive targets for ipsilateral antennal input, whereas all others receive afferents from both antennae. Unilateral amputation of the third antennal segment led to a loss of about 1000 fibers in the antennal commissure. Hence, about 5/6 of the approximately 1200 antennal afferents per side have a process that extends into the contralateral lobe. (2) Afferents from maxillary palps (most likely from basiconic sensilla) project into both ipsi- and contralateral antennal lobes, yet their target glomeruli are apparently not the same as those of antennal basiconic sensilla. (3) Afferents in the antennal lobe may also stem from pharyngeal sensilla. (4) The most prominent types of interneurons with arborizations in the antennal lobe are: (i) local interneurons ramifying in the entire lobe, (ii) unilateral relay interneurons that extend from single glomeruli into the calyx and the lateral protocerebrum (LPR), (iii) unilateral interneurons that connect several glomeruli with the LPR only, (iv) bilateral interneurons that link a small number of glomeruli in both antennal lobes with the calyx and LPR, (v) giant bilateral interneurons characterized by extensive ramifications in both antennal lobes and the posterior brain and a cell body situated in the midline of the suboesophageal ganglion, and (vi) a unilateral interneuron with extensive arborization in one antennal lobe and the posterior brain and a process that extends into the thorax. These structural results are discussed in the context of the available functional and behavioral data.

Genetic and developmental analysis of irreC, a genetic function required for optic chiasm formation in Drosophila.

Irregular chiasm C (irreC) is an X-linked genetic function necessary for the correct projection of visual fibers in the optic chiasms of Drosophila optic ganglia. In addition to a severe disorganization of the inner optic chiasm irreC mutants display a subtle phenotype in the outer optic chiasm, in which some bundles of axons that leave the posterior equatorial part of the lamina on their way to the anterior medulla take a long detour before eventually finding their specific targets in the medulla neuropile. Deletion and recombination mapping of two irreC alleles (one P-element induced, the other associated with an inversion) have yielded a precise cytogenetic location in 3C4-5. A complex complementation pattern between roughest (rst) and irreC alleles indicates that both genetic functions are structurally and/or functionally closely interrelated. Flies in which the irreC locus is completely deleted by overlapping deficiencies are viable and their defects in the optic chiasms are similar to those seen in the two alleles. The defects in the outer and inner optic chiasms are not epigenetically connected and mosaic analyses have shown them to be independent from the genotype of the compound eye. Although the larval visual nerve looks normal, we have found that in the optic lobes of irreC mutants a group of early differentiating larval neurons is misplaced, suggesting a pioneering function of these cells during organization of the outer optic chiasms.