Identifying targets of the rough homeobox gene of Drosophila: evidence that rhomboid functions in eye development.

In order to identify potential target genes of the rough homeodomain protein, which is known to specify some aspects of the R2/R5 photoreceptor subtype in the Drosophila eye, we have carried out a search for enhancer trap lines whose expression is rough-dependent. We crossed 101 enhancer traps that are expressed in the developing eye into a rough mutant background, and have identified seven lines that have altered expression patterns. One of these putative rough target genes is rhomboid, a gene known to be required for dorsoventral patterning and development of some of the nervous system in the embryo. We have examined the role of rhomboid in eye development and find that, while mutant clones have only a subtle phenotype, ectopic expression of the gene causes the non-neuronal mystery cells to be transformed into photoreceptors. We propose that rhomboid is a part of a partially redundant network of genes that specify photoreceptor cell fate.     

Central brain postembryonic development in Drosophila: implication of genes expressed at the interhemispheric junction

Postembryonic brain development of Drosophila has become recently a subject of intense investigations. In particular, the linotte (lio) mutants display strong structural defects in the mushroom bodies and the central complex. The Lio kinase is expressed in a glial structure at the interhemispheric junction of late larval and young pupal brain. With the aim of identifying new genes involved in the formation of adult central brain structures, 821 enhancer-trap Gal4 lines were generated and screened for late larval expression. We identified 167 lines showing expression at or near the interhemispheric junction of third-instar larval brain, an area from which the central complex differentiates. Adult brains from 104 of these 167 lines were analyzed through paraffin sections. This secondary screen allowed the recovery of five central brain mutants. Of 89 control lines showing various patterns of expression excluding the interhemispheric junction, only one anatomical mutant was isolated. These six mutations, which have been thoroughly characterized, affect the midline area of the adult brain with phenotypes of split central complex structures and/or fused mushroom body lobes. This work opens the way for further analysis of the molecular and cellular events involved in central brain reorganization during metamorphosis.     

Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila.

Drosophila melanogaster has been a premier genetic model system for nearly 100 years, yet lacks a simple method to disrupt gene expression. Here, we show genomic cDNA fusions predicted to form double-stranded RNA (dsRNA) following splicing, effectively silencing expression of target genes in adult transgenic animals. We targeted three Drosophila genes: lush, white, and dGq(alpha). In each case, target gene expression is dramatically reduced, and the white RNAi phenotype is indistinguishable from a deletion mutant. This technique efficiently targets genes expressed in neurons, a tissue refractory to RNAi in C. elegans. These results demonstrate a simple strategy to knock out gene function in specific cells in living adult Drosophila that can be applied to define the biological function of hundreds of orphan genes and open reading frames.     

Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos

We describe a new strategy for producing tissue-specific cDNA libraries and subsequently identifying tissue-specific clones. This method was used to screen for cDNA clones corresponding to RNAs expressed in the Drosophila head that cannot be detected in the early embryo. RNA blots were used to assess the spatial and temporal patterns of expression of these RNAs. The ensemble of 436 head-not-embryo clones identified roughly 700 distinct RNAs that are differentially expressed in the Drosophila head. The RNA expression patterns can be classified into five major categories. it is argued that this ensemble of clones represents a large fraction of all genes differentially expressed in the adult head, but not detected in the early embryo. Many of these genes are likely to encode eye- and nervous system-specific products.     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Expression of evolutionarily conserved eye specification genes during Drosophila embryogenesis

Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. We examined the expression patterns of four eye specification genes, eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac) throughout all time points of embryogenesis and show that only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. We also compare the expression of Ey with that of a putative antennal specifying gene Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field.     

A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ

The Drosophila adult external sensory organ, comprising a neuron and its support cells, is derived from a single precursor cell via several asymmetric cell divisions. To identify molecules involved in sensory organ development, we conducted a tissue-specific gain-of-function screen. We screened 2293 independent P-element lines established by P. Rorth and identified 105 lines, carrying insertions at 78 distinct loci, that produced misexpression phenotypes with changes in number, fate, or morphology of cells of the adult external sensory organ. On the basis of the gain-of-function phenotypes of both internal and external support cells, we subdivided the candidate lines into three classes. The first class (52 lines, 40 loci) exhibits partial or complete loss of adult external sensory organs. The second class (38 lines, 28 loci) is associated with increased numbers of entire adult external sensory organs or subsets of sensory organ cells. The third class (15 lines, 10 loci) results in potential cell fate transformations. Genetic and molecular characterization of these candidate lines reveals that some loci identified in this screen correspond to genes known to function in the formation of the peripheral nervous system, such as big brain, extra macrochaetae, and numb. Also emerging from the screen are a large group of previously uncharacterized genes and several known genes that have not yet been implicated in the development of the peripheral nervous system.     

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.     

Development and organization of the Drosophila olfactory system: an analysis using enhancer traps.

Drosophila uses different olfactory organs at different developmental stages. The larval and adult olfactory organs are morphologically dissimilar and have different developmental origins: the antenno-maxillary complex (AMC), which houses the larval olfactory organ, is histolyzed during metamorphosis; the third antennal segment--the principal adult olfactory organ--derives from an imaginal disc. A screen for genes expressed in both larval and adult olfactory organs, but in relatively few other tissues, has been carried out. Seven enhancer trap lines showing reporter gene expression in both the larval AMC and in certain subsets of the adult antenna are described. The antennal staining pattern of one line shows a striking change over the first few days of adult life, with a time course comparable to that of the development of sexual maturity. A pronounced sexual dimorphism in antennal staining pattern is seen in another line. Some staining patterns resemble the patterns of certain classes of antennal sensilla; others show expression restricted to only a small number of cells. Some lines also show expression associated with other chemosensory organs at either the larval or adult stage, including the maxillary palps, labellum, and anterior wing margin. One line, which also shows staining in the male reproductive tract, is male sterile. The significance of these results is considered in terms of (1) the molecular organization of the olfactory system; (2) the recruitment of olfactory genes for use in two developmental contexts; (3) the sharing of genes among different sensory modalities; (4) the role of olfaction in sexual behavior; and (5) posteclosional changes in the olfactory system.     

Changes in mortality patterns and temperature dependence of lifespan in Drosophila melanogaster caused by inbreeding

After an inbreeding event, lifespan can be curtailed through the expression of deleterious alleles. This will impact on both mortality patterns and interactions with the environment as visualised in reaction norms. We have established the effects of inbreeding on the temperature dependence of lifespan and on mortality patterns in Drosophila melanogaster. Four inbred lines displaying severely decreased lifespan and five outbred controls were assessed for male adult survival at three temperatures. As expected, all inbred lines showed a shorter lifespan than noninbred lines. The mechanisms behind this, however, appeared to be very diverse. Two inbred lines showed a significantly decreased temperature dependence of lifespan compared to the control lines. Analysis of variance on the mortality parameters over all lines showed that inbreeding changes the age-independent mortality but not the age-dependent mortality, whereas temperature does the opposite. This suggests that gene-by-environment interaction caused by inbreeding is the result of changes in the processes of lifespan determination. Importantly, for the two other inbred lines, a particular temperature regime triggered the expression of conditional lethal alleles. Mortality was concentrated in short lethal phases early in adult life. These conditionally expressed lethal alleles affecting lifespan demonstrate line specificity for inbreeding depression and will help ageing studies as such alleles may serve as candidate genes for ageing processes and age-related pathologies in humans.     

Heat shock gene expression and function during zebrafish embryogenesis

Recent work in the zebrafish, Danio rerio, indicates that heat shock genes are expressed in unique spatial patterns under non-stress conditions. In particular, hsp90alpha is expressed during the normal differentiation of striated muscle fibres, and hsp70-4 is expressed during normal lens development in the eye. Furthermore, disruption of the activity of either of these genes or their protein products gives rise to unique embryonic phenotypes that result from failures in proper somitic muscle development and lens development, respectively. Embryonic hsp70-4 expression is also activated in a cell-specific manner following heavy metal exposure. This has allowed for the development of a hsp70-4/eGFP reporter gene system in stable transgenic zebrafish that serves as a reliable yet extremely quick indicator of cell-specific toxicity in the context of the multicellular, living embryo.     

Drosophila single-minded gene and the molecular genetics of CNS midline development.

Our goal is to understand the molecular mechanisms that govern the formation of the central nervous system. In particular, we have focused on the development of a small group of neurons and glia that lie along the midline of the Drosophila CNS. These midline cells possess a number of unique attributes which make them particularly amenable to molecular, cellular, and genetic examinations of nervous system formation and function. In addition, the midline cells exhibit distinctive ontogeny, morphology, anatomical position, and patterns of gene expression which suggest that they may provide unique functions to the developing CNS. The single-minded gene encodes a nuclear protein which is specifically expressed in the midline cells and has been shown to play a crucial role in midline cell development and CNS formation. Genetic experiments reveal that sim is required for the expression of many CNS midline genes which are thought to be involved in the proper differentiation of these cells. In order to identify additional genes which are expressed in some or all of the midline cells at different developmental stages, a technique known as enhancer trap screening was employed. This screen led to the identification of a large number of potential genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. Further molecular, genetic, and biochemical analyses of sim and several of the enhancer trap lines are being pursued. This should permit elucidation of the genetic hierarchy which acts in the specification, differentiation, and function of these CNS midline cells.     

Expression of two novel mouse Iroquois homeobox genes during neurogenesis

Members of the Drosophila Iroquois homeobox gene family are implicated in the development of peripheral nervous system and the regionalization of wing and eye imaginal discs. Recent studies suggest that Xenopus Iroquois homeobox (Irx) genes are also involved in neurogenesis. Three mouse Irx genes, Irx1, Irx2 and Irx3, have been previously identified and are expressed with distinct spatio-temporal patterns during neurogenesis. We report here the cloning and expression analysis of two novel mouse Irx genes, Irx5 and Irx6. Although Irx5 and Irx6 proteins are structurally more related to one another, we find that Irx5 displays a developmental expression pattern strikingly similar to that of Irx3, whereas Irx6 expression resembles that of Irx1. Consistent with the notion that Mash1 is a putative target gene of the Irx proteins, all four Irx genes display an overlapping expression pattern with Mash1 in the developing CNS. In contrast, the Irx genes and Mash1 are expressed in complementary domains in the developing eye and olfactory epithelium.     

斑马鱼肌肉高表达基因近端非编码元件分析及功能检测

为鉴定鱼类肌肉组织特异性顺式调控元件,通过分析斑马鱼多个组织的转录组数据,筛选出肌肉高表达基因及低表达基因.通过MEME对肌肉高表达基因和低表达基因非编码区序列特征进行分析,在5个肌肉高表达基因的转录起始位点上游发现了序列保守的DNA区域,包含6个排列顺序一致的DNA基序.将其中一段目标片段插入具有Tol2转座子元件的...