The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension.

We identified the Drosophila trio gene, which encodes a Dbl family protein carrying two Dbl homology (DH) domains, each of which potentially activates Rho family GTPases. Trio was distributed along axons in the central nervous system (CNS) of embryos and was strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations resulted in the misdirection or stall of axons in embryos and also caused malformation of the MB. The MB phenotypes were attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways. In addition, the localization of Trio in the adult brain suggests its postdevelopmental role in neurite terminals.

The Om (1E) mutation in Drosophila ananassae causes compound eye overgrowth due to tom retrotransposon-driven overexpression of a novel gene.

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, we have carried out molecular analyses. A putative Om(1E) locus cloned by tom tagging and chromosome walking contained two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrated that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affected morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes.

Retrotransposon-induced ectopic expression of cut causes the Om(1A) mutant in Drosophila ananassae.

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The gamma-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.

pox-neuro is required for development of chemosensory bristles in Drosophila.

The gene pox-neuro (poxn), which encodes a possible transcriptional regulator including a paired domain, specifies the differences between monoinnervated and polyinnervated sensory organs in the embryo. A detailed analysis of this gene, and in particular, an analysis of its function in the adult sensory organs, has so far been hampered by the unavailability of loss-of-function mutations. Here, we report the isolation of loss-of-function mutations of poxn and show that the chemosensory bristles are transformed into mechanosensory bristles in mutant flies. The external morphology of putative chemosensory bristles, number of innervating neurons, and cell division pattern are all affected in the mutants, showing that poxn is strictly required for development of the adult chemosensory bristles. In addition, the formation of some precursor cells is suppressed in the mutants, suggesting that poxn is also required for formation of the precursors of chemosensory bristles.

Multiple function of poxn gene in larval PNS development and in adult appendage formation of Drosophila.

The gene pox-neuro (poxn), which encodes a transcriptional regulator including a paired domain, specifies the differences between mono-innervated external sensory (m-es) organs and poly-innervated external sensory (p-es) organs in Drosophila. Here, we analyse the function of poxn in the development of the larval peripheral nervous system (PNS) and in other developmental aspects using a loss-of-function mutant of poxn. We observed that, in addition to the transformation of p-es into m-es organs in the mutant embryo, the external structure of the trichome-like sensilla (hairs) misdifferentiates into that of the campaniform-like sensilla (papillae) in the second and third larval instars. We also observed that POXN is expressed in a cell associated with the external structure of the trichome-like sensilla in the first and second instar larvae. These results imply that poxn is required in two distinct steps in the development of the larval PNS: (1) development of the larval p-es organs during embryogenesis and (2) re-formation of larval sensory hairs after each larval moult. In addition to its expression in the developing PNS, POXN is also expressed in concentric domains of the leg and antennal imaginal discs of early third instar larvae, and in the region of the wing disc that will form the wing hinge. The loss of poxn function results in defects of segmentation of the tarsus and antenna and in a distortion in the wing hinge. These results indicate that the poxn gene plays crucial roles in the morphogenesis of the appendages, in addition to its role in the early specification of neuronal identity.

Genetic analyses of essential genes in cytological region 61D1-2 to 61F1-2 of Drosophila melanogaster.

We performed a systematic mutagenesis screen for lethals in the genomic region 61D1-2 to 61F1-2 on chromosomal arm 3L of Drosophila melanogaster. Our genetic analyses revealed that this region contains eight essential complementation groups including trio, Glut1 and extra macrochaetae (emc). For the trio locus, 22 mutant alleles were identified, and all of the alleles analyzed resulted in defects in the central nervous system of embryos, indicating that trio functions in the control of axon extension or guidance. Western analysis showed that at least three proteins are derived from trio and also suggested that a polypeptide of over 200 kDa plays a crucial role in embryonic or larval development. In addition, a newly identified emc allele was associated with several defects in embryonic morphogenesis, including abnormalities in head involution, gut formation and dorsal closure, thus revealing multiple roles for emc in embryonic development. We also performed preliminary phenotypic analyses on stocks bearing mutations belonging to the other lethal complementation groups. These genes function in essential biological events, but the mutations do not result in gross morphological changes during embryonic stages. The present study extends our knowledge of the Drosophila gene set, by identifying most of the essential genes in the chromosomal region 61D1-2 to 61F1-2.

An eye imaginal disc-specific transcriptional enhancer in the long terminal repeat of the tom retrotransposon is responsible for eye morphology mutations of Drosophila ananassae.

Optic morphology (Om) mutations of Drosophila ananassae are semidominant, neomorphic and nonpleiotropic, map to at least 22 loci scattered throughout the genome, and are associated with the insertion of the tom retrotransposon. Molecular and genetic analyses have revealed that eye morphology defects of Om mutants are caused by the ectopic or excessive expression of Om genes in the eye imaginal discs of third instar larvae. It is therefore assumed that the tom element carries tissue-specific gene regulatory sequences which enhance expression of the Om genes. In the present study, we examined whether or not the long terminal repeats (LTR) of the tom element contain such an eye imaginal disc-specific enhancer, using D. melanogaster transformants containing a lacZ gene ligated to the tom LTR. Analyses of lacZ gene expression in the eye imaginal discs of third instar larvae of 18 independently established transformant lines showed that the tom LTR was capable of enhancing lacZ expression in all the transformant lines, but the degree of enhancement varied between lines. In addition, the effect of the tom LTR lacZ gene evidently changed when the tom LTR construct was relocated to different chromosomal positions. On the basis of these findings, it is hypothesized that ectopic and excessive expression of the Om genes in the eye imaginal discs is induced by an eye imaginal disc-specific enhancer present in the tom LTR, the effect of which may be subject to chromosomal position effects.

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(2D) phenotype in Drosophila ananassae.

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.

An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.