Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1.

Rhodopsin is essential for photoreceptor morphogenesis; photoreceptors lacking rhodopsin degenerate in humans, mice, and Drosophila. Here we report that transgenic expression of a dominant-active Drosophila Rho guanosine triphosphatase, Drac1, rescued photoreceptor morphogenesis in rhodopsin-null mutants; expression of dominant-negative Drac1 resulted in a phenotype similar to that seen in rhodopsin-null mutants. Drac1 was localized in a specialization of the photoreceptor cortical actin cytoskeleton, which was lost in rhodopsin-null mutants. Thus, rhodopsin appears to organize the actin cytoskeleton through Drac1, contributing a structural support essential for photoreceptor morphogenesis.

Rhodopsin replacement rescues photoreceptor structure during a critical developmental window.

Rhodopsin is essential for normal photoreceptor development in Drosophila (O'Tousa et al., 1989; Leonard et al., 1992; Kumar and Ready, 1995) and in mice (Humphries et al., 1997). Here we report studies in which a rhodopsin transgene is expressed at restricted stages during the development of Drosophila photoreceptors otherwise lacking rhodopsin. Substantial rescue of normal photoreceptor structure and physiology is effected by rhodopsin expression during the time of the normal onset of rhodopsin synthesis. Expression shortly before or after this critical period does not rescue these deficits. There is a critical developmental period in which rhodopsin plays its key role in photoreceptor morphogenesis.

Glued participates in distinct microtubule-based activities in Drosophila eye development.

A C-terminal truncation of Glued, the Drosophila homolog of the cytoplasmic dynein activating protein, dynactin, results in a severe and complex retinal phenotype, including a roughening of the facet array, malformation of the photosensitive rhabdomeres, and a general deficit and disorder of retinal cells. We have characterized the developmental phenotype in Glued1 and found defects in multiple stages of eye development, including mitosis, nuclear migration, cell fate determination, rhabdomere morphogenesis and cell death. Transgenic flies that express dominant negative Glued under heat-shock control reproduce distinct features of the original Glued1 phenotype depending on the stage of development. The multiple phenotypes effected by truncated Glued point to the multiple roles served by dynactin/dynein during eye development.

Rhodopsin plays an essential structural role in Drosophila photoreceptor development.

Null mutations of the Drosophila Rh1 rhodopsin gene, ninaE, result in developmental defects in the photosensitive membranes, the rhabdomeres, of compound eye photoreceptors R1-R6. In normal flies, Rh1 expression begins at about 78% of pupal life. At approximately 90% of pupal life, a specialized catacomb-like membrane architecture develops at the base of normal rhabdomeres. In ninaE null mutants, these catacombs do not form and developing rhabdomere membrane involutes into the cell as curtains of apposed plasma membrane. A filamentous cytoskeletal complex that includes F-actin and the unconventional myosin, NINAC, decorates the cytoplasmic surface of these curtains.

Integrins and the development of three-dimensional structure in the Drosophila compound eye.

A stereotyped, three-dimensional network of cell-cell contacts mediated by adherens junctions and cell-extracellular matrix contacts mediated by focal adhesions defines the architecture of the Drosophila ommatidium. Developmental reconstruction shows that this network is built in an incremental and generally conservative sequence; contacts established early in eye development typically persist into adulthood. Reconstructions show that photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabdomeres. Rhabdomeres become aligned to the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell-cell and cell-extracellular matrix contacts. Parallel reconstructions of retinal development in integrin mutants show that several eye phenotypes trace their origin to the structural failure of the cone cell plate.

Degeneration of photoreceptors in rhodopsin mutants of Drosophila.

Five different, well-characterized mutants of the R1-6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1-6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1-6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1-6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1-6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1-6 rhodopsin in the ninaE mutants.

Cell death in normal and rough eye mutants of Drosophila.

The regular, reiterated cellular pattern of the Drosophila compound eye makes it a sensitive amplifier of defects in cell death. Quantitative and histological methods reveal a phase of cell death between 35 and 50 h of development which removes between 2 and 3 surplus cells per ommatidium. The timing of this epoch is consistent with cell death as the last fate to be specified in the progressive sequence of cell fates that build the ommatidium. An ultrastructural survey of cell death suggests dying cells in the fly eye have similarities as well as differences with standard descriptions of programmed cell death. A failure of cell death to remove surplus cells disorganizes the retinal lattice. A screen of rough eye mutants identifies two genes, roughest and echinus, required for the normal elimination of cells from the retinal epithelium. The use of an enhancer trap as a cell lineage marker shows that the cone cells, like other retinal cells, are not clonally related to each other or to their neighbors.

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.

The emergence of order in the Drosophila pupal retina.

During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.

Notch is required for successive cell decisions in the developing Drosophila retina.

Mutations in the Notch locus affect a variety of developmental decisions in Drosophila. In this paper, we examine the role of Notch in the developing retina. We reduced Notch activity at successive intervals during development of the retina, and then examined the effect on individual cells. When Notch activity was reduced, cells responded by selecting inappropriate developmental pathways. We found that all cell types appear to require Notch when establishing their fate. To examine further Notch's role in eye development, we examined two alleles of Notch--split and facet-glossy. split flies show defects in the initial clustering of photoreceptors, whereas the defects in facet-glossy flies are due to the misrouting of presumptive primary pigment cells into the secondary pigment cell pathway. Our results suggest that Notch plays a permissive role in the cell-cell interactions used to assemble the eye.

A multifaceted approach to neural development.

The developing compound eye of the fruit fly, Drosophila offers notable advantages for a study of neural differentiation. It is a sensitive amplifier of a simple neural module; each eye is an approximately 700-fold repetition of the unit eye or ommatidium, which is a precise, stereotyped assembly of photoreceptors and accessory cells. The eye develops in a monolayer epithelium, which greatly reduces the complexities of cell-cell interactions often encountered in CNS development, and has permitted a detailed, cell-by-cell description of cell behavior during ommatidial development. Finally, the foundation of fly genetics permits a mutational analysis of eye development and the advanced molecular genetics of Drosophila allows close scrutiny of genes of interest. A recent convergence of cellular, genetic and molecular studies of ommatidial development suggests a model for neural differentiation in the fly eye.

Associative learning modifies two behaviors in the leech, Hirudo medicinalis.

We report that 2 behaviors, stepping and shortening, are modified by associative learning in the leech, Hirudo medicinalis. Experiment 1 explored conditioning of the "stepping" response. Paired presentations of touch to the medial dorsal surface of the leech and shock to the tail of the leech resulted in the development of stepping to the touch. Leeches in control groups experiencing the CS alone, US alone, or explicitly unpaired presentations of the CS and US did not. In experiments 2-4, classical conditioning explored conditioning of the touch-elicited shortening reflex. We found that the reflex was enhanced following paired CS-US presentations but not following CS alone, US alone, or explicitly unpaired presentations of the stimuli. Moreover, the learning was extinguished following 15 unreinforced presentations of the CS but was retained for at least 24 hr without extinction training. Moreover, the associative effect was not evident when the CS and US were presented in a backward relationship. That is, no learning was observed when the US preceded the CS. Lastly, the hand-held stimuli were replaced with implanted electrodes. Using a 3 V pulse that mimicked the touch stimulus (CS), we found that paired CS-US presentations produced a significant enhancement in the shortening reflex. Again, no enhancement was observed following unpaired CS, US presentations.

Cell fate in the Drosophila ommatidium.

Lesions in the Drosophila gene sevenless cause the cell normally destined to differentiate as photoreceptor 7 of the ommatidium to become an accessory lens-secreting cell, the equatorial cone cell. In both P-element- and EMS-induced alleles, the developmental transformation occurs identically. The mutation is cell autonomous, showing that the developmental failure is intrinsic to the transformed cell. Histological and immunological analyses indicate that the cell fails to operate any photoreceptor differentiation machinery prior to adopting the cone cell pathway.

Neuronal differentiation in Drosophila ommatidium.

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.

Ommatidial development in Drosophila eye disc fragments.

We have tested the hypothesis that the leading edge of the growing Drosophila compound eye acts as a template that organizes unpatterned cells of the retinal epithelium into the accurate cellular mosaic of the eye. Unpatterned fragments of the epithelium, not containing the leading edge of the growing field, were transplanted into larval hosts. After hosts pupated, the implants were recovered; most contained ommatidia, demonstrating that the leading edge of the growing eye pattern is not required for its propagation. In a second set of experiments, implants were recovered before hosts pupated and examined for ommatidia using a monoclonal antibody. These implants likewise differentiated ommatidia and the temporal progress of retinal development in the implants mirrored that of normal development. A schedule of ommatidial development thus appears to be mapped onto the retinal epithelium in advance of the leading edge.

Sevenless: a cell-specific homeotic mutation of the Drosophila eye.

Each ommatidium in the compound eye of the Drosophila mutant sevenless lacks photoreceptor number seven (R7) from the normal ommatidial complement of eight photoreceptors. A comparison of mutant and normal development reveals that this deficit is caused by the cell-specific transformation of the cell normally fated to produce R7 into a lens-secreting accessory cell, a cone cell.

Membrane properties and selective connexions of identified leech neurones in culture.

1. Individual, identified neurones, dissected from the central nervous system of the leech and maintained in culture for several weeks, sprouted processes and formed synaptic connexions.2. The action potentials of isolated touch (T), pressure (P), nociceptive (N) cells and Retzius cells resembled those of their counterparts in situ, enabling them to be recognized unambiguously. Their input resistances were approximately 4 times greater than those of corresponding cells within the animal. In T, P and N cells trains of impulses were followed by a pronounced after-hyperpolarization, as in the animal.3. In certain cells, notably the L motoneurones, membrane properties became altered in culture. The current-voltage relation showed novel rectification and action potentials became much larger.4. Numerous neurites often extended for hundreds of micrometres from isolated neurones and ended in typical growth cones. Electron micrographs revealed that many fine axons were braided together to form thicker fascicles. Frequently, the processes were orientated between two neighbouring cells rather than at random. The fine structure of the cytoplasm, nucleus and organelles in cultured cells resembled those of their counterparts in situ. The glial cell that normally surrounds the neurones was, however, absent.5. Pairs of Retzius cells in culture usually became coupled electrically after about 6 days. Similarly L motoneurones became coupled in vitro. These junctions allowed current to pass in both directions and resembled those seen in the animal.6. Selective connexions were made by certain types of cells. Thus, P sensory neurones did not become coupled with Retzius cells but did develop electrical connexions with L motoneurones, as in the animal.7. Novel synaptic interactions not obvious in the animal could appear in culture. Retzius and L cells became electrically coupled and, in some instances where electrical coupling between Retzius cells failed to develop, chemically mediated inhibitory potentials became apparent.8. Isolated, identified leech neurones not only survive but regenerate processes and are capable of forming selective connexions in culture. The ability to define interactions between isolated pairs of cells offers the opportunity to explore in detail problems relating to synapse formation and cell-cell recognition.

Identified neurones isolated from leech CNS make selective connections in culture.

Neurones cultured in vitro offer distinct advantages for studying how processes grow towards their targets and form synaptic connections. In contrast to the complex events occurring during the development of the nervous system, synapse formation in culture can be analysed in a few neurones at a time and under controlled conditions. We have now dissected out and cultured single identified neurones from the central nervous system (CNS) of the adult leech. Various types of sensory cells, motor cells, and interneurones can be identified in leech ganglia--each with a stereotyped set of properties, including: (1) the electrical characteristics of its membrane, (2) the arborisation of its branches and the morphology of its terminals and (3) the pattern of connections it makes with other identified neurones, skin or muscle. Thus, cultured cells can be compared in detail with their counterparts in situ. We have found that isolated cells survive for several weeks, maintain their membrane properties, sprout and form selective connections.