The embryonic development of the Drosophila visual system

We have used electron-microscopic studies, bromodeoxyuridine (BrdU) incorporation and antibody labeling to characterize the development of the Drosophila larval photoreceptor (or Bolwig's) organ and the optic lobe, and have investigated the role of Notch in the development of both. The optic lobe and Bolwig's organ develop by invagination from the posterior procephalic region. After cells in this region undergo four postblastoderm divisions, a total of approximately 85 cells invaginate. The optic lobe invagination loses contact with the outer surface of the embryo and forms an epithelial vesicle attached to the brain. Bolwig's organ arises from the ventralmost portion of the optic lobe invagination, but does not become incorporated in the optic lobe; instead, its 12 cells remain in the head epidermis until late in embryogenesis when they move in conjunction with head involution to reach their final position alongside the pharynx. Early, before head involution, the cells of Bolwig's organ form a superficial group of 7 cells arranged in a rosette pattern and a deep group of 5 cells. Later, all neurons move out of the surface epithelium. Unlike adult photoreceptors, they do not form rhabdomeres; instead, they produce multiple, branched processes, which presumably carry the photopigment. Notch is essential for two aspects of the early development of the visual system. First, it delimits the number of cells incorporated into Bolwig's organ. Second, it is required for the maintenance of the epithelial character of the optic lobe cells during and after its invagination.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

Sine Oculis Is a Homeobox Gene Required for Drosophila Visual System Development

The so(mda) (sine oculis-medusa) mutant is the result of a P element insertion at position 43C on the second chromosome. so(mda) causes aberrant development of the larval photoreceptor (Bolwig's) organ and the optic lobe primordium in the embryo. Later in development, adult photoreceptors fail to project axons into the optic ganglion. Consequently optic lobe development is aborted and photoreceptor cells show age-dependent retinal degeneration. The so gene was isolated and characterized. The gene encodes a homeodomain protein expressed in the optic lobe primordium and Bolwig's organ of embryos, in the developing adult visual system of larvae, and in photoreceptor cells and optic lobes of adults. In addition, the SO product is found at invagination sites during embryonic development: at the stomadeal invagination, the cephalic furrow, and at segmental boundaries. The mutant so(mda) allele causes severe reduction of SO embryonic expression but maintains adult visual system expression. Ubiquitous expression of the SO gene product in 4-8-hr embryos rescues all so(mda) mutant abnormalities, including the adult phenotypes. Thus, all deficits in adult visual system development and function result from failure to properly express the so gene during embryonic development. This analysis shows that the homeodomain containing SO gene product is involved in the specification of the larval and adult visual system development during embryogenesis.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

Differential regulation of a homologous peptidergic neuron in grasshoppers: evolution of a neural circuit

The vasopressin-like immunoreactive (VPLI) neurons of grasshoppers have paired cell bodies in the suboesophageal ganglion and both anterior and posterior running axons. In non-oedipodine grasshopper species (e.g. Schistocerca gregaria), most of their arborisations are distributed in dorsal and lateral neuropil, while in oedipodine species (e.g. Locusta migratoria), the neurons have additional extensive axonal projections in both the optic lobes and proximal portions of the ganglionic peripheral nerves. This study demonstrates that these morphological differences correlate with their physiology. In L. migratoria, VPLI neuron activity is regulated primarily via a spontaneously active interneuron which descends from the brain. This descending interneuron is inhibited by a light-activated brain extraocular photoreceptor. Regulation of VPLI neuron activity by an extraocular photoreceptor is also seen in the other oedipodine grasshopper investigated. In the four non-oedipodines examined (from two subfamilies), we find no extraocular photoreceptor regulation of VPLI neuron activity. Despite this, VPLI neuron in S.␣gregaria does appear to be driven by a descending interneuron homologous to that in L. migratoria. The descending interneuron in both species receives similar mechanosensory input and excites the VPLI neuron via cholinergic synapses. Histamine injection into the medial protocerebrum of both species causes strong inhibition of the descending interneuron. The evolution of the neural circuitry, by which an extraocular photoreceptor comes to regulate the descending interneuron in oedipodine species, is discussed. Accepted: 6 January 1998     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Transcriptional regulation of atonal required for Drosophila larval eye development by concerted action of eyes absent, sine oculis and hedgehog signaling independent of fused kinase and cubitus interruptus

Bolwig's organ is the larval light-sensing system consisting of 12 photoreceptors and its development requires atonal activity. Here, we showed that Bolwig's organ formation and atonal expression are controlled by the concerted function of hedgehog, eyes absent and sine oculis. Bolwig's organ primordium was first detected as a cluster of about 14 Atonal-positive cells at the posterior edge of the ocular segment in embryos and hence, atonal expression may define the region from which a few Atonal-positive founder cells (future primary photoreceptor cells) are generated by lateral specification. In Bolwig's organ development, neural differentiation precedes photoreceptor specification, since Elav, a neuron-specific antigen, whose expression is under the control of atonal, is expressed in virtually all early-Atonal-positive cells prior to the establishment of founder cells. Neither Atonal expression nor Bolwig's organ formation occurred in the absence of hedgehog, eyes absent or sine oculis activity. Genetic and histochemical analyses indicated that (1) responsible Hedgehog signals derive from the ocular segment, (2) Eyes absent and Sine oculis act downstream of or in parallel with Hedgehog signaling and (3) the Hedgehog signaling pathway required for Bolwig's organ development is a new type and lacks Fused kinase and Cubitus interruptus as downstream components.     

Differential regulation of choline acetyltransferase expression in adult Drosophila melanogaster brain

Choline acetyltransferase (ChAT, E.C.2.3.1.6) catalyzes the synthesis of acetylcholine, and is considered to be a phenotypic marker specific for cholinergic neurons. In situ hybridization using a nonradioactive cRNA probe identified a large number of cell bodies expressing ChAT mRNA in the cortices of wild-type Drosophila melanogaster brain. Strong labeling is remarkable in the cortical regions associated with the lamina and antennal lobe, and also in the median neurosecretory (MNS) cells within pars intercerebralis, suggesting that some of the lamina monopolar neurons, antennal interneurons, and MNS cells are cholinergic. In two temperature-sensitive mutant alleles, Cha^ts1 and Cha^ts2, most hybridization signal disappears after exposure to a restrictive temperature (30°C). Loss of signal is especially evident in the optic lobes. Some centrally located neurons, however, continue to express ChAT mRNA and are thus likely to have expression controlled in a different way than the majority of cholinergic neurons. Immunocytochemistry, using a ChAT specific monoclonal antibody, identified two sets of paired neurons located in the posterior cortex of the brain. These neurons persist in ChAT immunoreactivity even in the Cha^ts mutants exposed to restrictive temperature. ChAT mRNA is also detectable in the corresponding cell bodies when Cha^ts mutants are held at restrictive temperature. Our findings demonstrate some specific cholinergic neurons in Drosophila brain, and indicate that ChAT expression is differentially regulated in particular sets of cholinergic neurons. © 1996 John Wiley & Sons, Inc.     

Fate mapping of Drosophila embryonic mitotic domain 20 reveals that the larval visual system is derived from a subdomain of a few cells.

In an attempt to study the fates of cells in the dorsal head region of Drosophila embryos at gastrulation, we used the photoactivated gene expression system to mark small numbers of cells in selected mitotic domains. We found that mitotic domain 20, which is a cluster of approximately 30 cells on the dorsal posterior surface, gives rise to various ectodermal cell types in the head, including dorsal pouch epithelium, the optic lobe, and head sensory organs, including Bolwig's organ, the larval photoreceptor organ. We found that the optic lobe and larval photoreceptors share the same origin of a few adjacent cells near the center of mitotic domain 20, suggesting that within the mitotic domain, there is a subdomain from which the larval visual system is specified. In addition to the components of the larval visual system, this central region of mitotic domain 20 also generates a part of the eye-antennal disc placode; cells that gives rise to the adult visual system. We also observed that a significant amount of cell death occurred within this domain and used cell ablation experiments to determine the ability of the embryo to compensate for cell loss.     

Disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila

Mutations at the X-linked disconnected locus of D. melanogaster lead to the failure of adult photoreceptor axons to innervate their target cells in the developing optic lobes of the third instar larva, resulting in flies that have rudimentary optic ganglia. The cascade of epigenetic events leading to the adult disconnected phenotype is caused by the misrouting of a larval pioneer nerve, Bolwig's nerve, during embryonic development. In the disconnected mutant this nerve fails to recognize and establish stable connections with its correct synaptic partners. In addition, disconnected affects both the proper aggregation and the movement of the Bolwig neurons to their final location in the embryo. Finally, similar but more subtle defects can be found in a subset of other peripheral neurons in the thoracic and abdominal segments. The different aspects of the phenotype suggest that the disconnected gene plays a role in neuronal cell recognition.     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

Establishment of neuronal connectivity during development of the Drosophila larval visual system

We used confocal microscopy in conjunction with specific antibodies and enhancer trap strains to investigate the development of specific neuronal connections in a simple model system, the larval visual system of Drosophila. We find that the establishment of axonal projections from the larval photoreceptor neurons to their central nervous system targets involves a series of discrete steps. During embryogenesis, the larval optic nerve contacts several different cell types, including optic lobe pioneer (OLP) neurons and a number of glial cells. We demonstrate that OLP neurons are present and project normally in glass (gl) mutant embryos in which the larval optic nerve fails to develop, suggesting that they do not depend on interactions with the larval optic nerve for differentiation and proper axonal projection. The OLPs fail to differentiate properly in disconnected (disco) mutant embryos, where appropriate connections between the larval optic nerve and its targets in the brain are not formed. The disco gene is expressed in the OLPs and may therefore act autonomously to direct the differentiation of these cells. Taken together, our results suggest that the OLPs act as an intermediate target required for the establishment of normal optic nerve projection and connectivity. © 1995 John Wiley & Sons, Inc.     

Novel tissue units of regional differentiation in the gut epithelium of Drosopbila,as revealed by P-element-mediated detection of enhancer

We analysed spatial patterns of expression of a lacZ reporter gene in the gut of Drosophila larvae that had been transformed with a P-element-lacZ vector to identify regional differences in gene expression. lacZ-positive epithelial cells formed distinct domains with discrete transverse and longitudinal boundaries along the gut tube. Boundaries were often found at sites at which morphological boundaries were not obvious. The gut epithelium was subdivided into 36 compartments by the boundaries. We refer to these novel compartments as tissue compartments. The lacZ-positive domain of each strain appeared as a single tissue compartment or as a combination of several tissue compartments. The tissue compartment is considered to be a unit of regional differentiation. The spatial organization of the tissue compartments may represent the floor plan, determined by genes that control the regional differentiation of this nonsegmental organ. Correspondence to: R. Murakami     

果蝇幼虫的视觉系统

视觉对于动物的生存和行为来说是非常重要的。虽然果蝇幼虫的视觉神经系统在组织结构上比成虫简单,但是也具有一定的复杂性和多样性。在最近几年中,随着对果蝇幼虫视觉系统功能的研究取得重要进展,我们对于果蝇幼虫视觉系统的认识更加丰富了。果蝇幼虫视觉系统的结构中,最初级的光感受神经元包括三类,一类是BO/BN(Bolwig's organ/Bolwig's nerve),一类是表达感光分子CRY(cryptochrome)的神经元,另外一类是Ⅳ型DA(classⅣdendriticarborization)神经元;果蝇幼虫视觉系统的次级神经元主要是光节律相关的侧神经元(lateralneurons,LN),它表达Per(period)、Tim(timeless)及Pdf(pigment dispersion factor)等节律相关的蛋白分子;而第三级神经元包括更为下游的、表达果蝇促胸腺激素且直接调控幼虫光偏好的NP394神经元。这三级神经元构成了我们现在所了解的果蝇幼虫视觉神经系统的基本框架。     

Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta)

Using a Biolistic PDS 1000/He system, healthy thalli of Gracilaria changii were bombarded with gold particles coated with plasmid DNA containing the lacZ reporter gene. Transient expression of lacZ was observed in bombarded thalli under the rupture-disc pressures of 4482, 6206, 7584 and 8963 KPa, two days after bombardment. Although G. changii exhibits a slight blue background, positive expression and the background colour can be clearly differentiated. The results indicate that lacZ could be a useful reporter gene and that SV40 promoter could be an effective promoter for Gracilaria transformation.     

Cholinergic neurons in the lamina ganglionaris of the blowfly Calliphora erythrocephala

Summary The distribution of putative cholinergic neurons in the lamina of the blowfly Calliphora erythrocephala was studied by immunocytochemical and histochemical methods. Three different antibodies directed against the AChsynthesizing enzyme, choline acetyltransferase (ChAT), revealed a cholinergic population of fibres running parallel to the laminar cartridges, which have branch-like structures at the distal lamina border. Cell bodies in the chiasma next to the lamina border were also labelled by the anti-ChAT antibodies. Monopolar cell bodies in the nuclear layer were faintly labelled. The distribution of the acetylcholine hydrolyzing enzyme, acetylcholine esterase (AChE), was revealed by histochemical staining and was similar to the ChAT immunocytochemistry. The arrangement of ChAT positive fibres in transverse and longitudinal sections and the distribution of AChE stained fibres indicate that the amacrine cells of the lamina are cholinergic cells.We dedicate this work to Prof. F. Zettler who passed away in fall 1988: K.-H. Datum, I. Rambold     

Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.