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Background The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. Results We show that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an Otp antibody, a detailed expression analysis during hindgut development was carried out. Otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, we generated the mutant otp allele otp GT by ends-out gene targeting. In addition, we isolated two EMS-induced otp alleles in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. Conclusion Our data suggest that Otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn Otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells.

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84Kmutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84Kmutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in actE84Kmutants. Genetic interactions between actE84Kand neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization. Keywords: Actin; Synapse; Spectrin; NMJ; Cytoskeleton; Neurexin, National Institutes of Health (U.S.) (Grant NS40296)

Brachyury proteins, a conserved subgroup of the T domain transcription factors, specify gut and posterior mesoderm derivatives throughout the animal kingdom. The T domain confers DNA-binding properties to Brachyury proteins, but little is known how these proteins regulate their target genes. We characterized a direct target gene of the Drosophila Brachyury-homolog Brachyenteron. Brachyenteron activates the homeobox gene orthopedia in a dose-dependent manner via multiple binding sites with the consensus (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T. The sites and their A/T-rich flanking regions are conserved between D. melanogaster andDrosophila virilis. Reporter assays and site-directed mutagenesis demonstrate that Brachyenteron binding sites confer in part additive, in part synergistic effects on otp transcription levels. This suggests an interaction of Brachyenteron proteins on the DNA, which we could map to a conserved motif within the T domain. Mouse Brachyury also interacts with Brachyenteron through this motif. We further show that the Xenopus and mouse Brachyury homologs activate orthopedia expression when expressed inDrosophila embryonic cells. We propose that the mechanisms to achieve target gene expression through variable binding sites and through defined protein-protein interactions might be conserved for Brachyury relatives.

Unlike gap genes in the trunk region of Drosophila embryos, gap genes in the head were presumed not to regulate each other's transcription. Here, we show that in tailless (tll) loss-of-function mutants the empty spiracles (ems) expression domain in the head expands, whereas it retracts in tll gain-of-function embryos. We have identified a 304bp element in the ems-enhancer which is sufficient to drive expression in the head and brain and which contains two TLL and two BCD binding sites. Transgenic reporter gene lines containing mutations of the TLL binding sites demonstrate that tll directly inhibits the expression of ems in the early embryonic head and the protocerebral brain anlage. These results are the first demonstration of direct transcriptional regulation between gap genes in the head.

The homeobrain (hbn) gene is a new paired-like homeobox gene which is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells which form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins DRx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and DRx resemble each other. Two other homeobox genes, orthopedia and DRx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, DRx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions.

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent.

Signaling by the secreted hedgehog, decapentaplegic and wingless proteins organizes the pattern of photoreceptor differentiation within the Drosophila eye imaginal disc; hedgehog and decapentaplegic are required for differentiation to initiate at the posterior margin and progress across the disc, while wingless prevents it from initiating at the lateral margins. Our analysis of these interactions has shown that initiation requires both the presence of decapentaplegic and the absence of wingless, which inhibits photoreceptor differentiation downstream of the reception of the decapentaplegic signal. However, wingless is unable to inhibit differentiation driven by activation of the epidermal growth factor receptor pathway. The effect of wingless is subject to regional variations in control, as the anterior margin of the disc is insensitive to wingless inhibition. The eyes absent and eyegone genes encode members of a group of nuclear proteins required to specify the fate of the eye imaginal disc. We show that both eyes absent and eyegone are required for normal activation of decapentaplegic expression at the posterior and lateral margins of the disc, and repression of wingless expression in presumptive retinal tissue. The requirement for eyegone can be alleviated by inhibition of the wingless signaling pathway, suggesting that eyegone promotes eye development primarily by repressing wingless. These results provide a link between the early specification and later differentiation of the eye disc.

The Drosophila homolog of the vertebrate serum response factor (SRF) was isolated by low stringency hybridization. Nucleotide sequence analysis revealed that the Drosophila SRF homolog (DSRF) codes for a protein that displays 93% sequence identity with human SRF in the MADS domain, the region required for DNA binding, dimerization and interaction with accessory factors. The DSRF gene is expressed during several phases of embryonic development. In the egg, both the RNA and the protein are maternal in origin and slowly decrease in amount during gastrulation. After germ band retraction, high levels of zygotic expression are observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. Many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues. The DSRF gene was mapped to position 60C on the second chromosome, and overlapping deficiencies which remove the gene were identified. Analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. Although the initial migration of tracheal cells was not affected in those deficient embryos, many tracheal cells appeared not to maintain their correct position and continued to migrate. Thus, the DSRF gene might play a role in the proper formation and maintenance of the trachea.

The Drosophila Pax-6 homologs eyeless (ey) and twin of eyeless (toy) are expressed in the eyes and in the central nervous system (CNS). In addition to the pivotal functions in eye development, previous studies revealed that ey also plays important roles in axonal development of the mushroom bodies, centers for associative learning and memory. It has been reported that a second intron enhancer that contains several Pax-6 binding sites mainly controls the eye-specific expression, but the DNA sequences that control CNS expression are unknown. In this work, we have dissected transcriptional enhancer elements of the ey gene that are required for the CNS expression in various developmental stages. We first show that CNS expression is independent of the eye-specific enhancer of the second intron. By systematic reporter studies, we have identified several discrete DNA elements in the 5' upstream region and in the second intron that cooperatively interact to generate most of the ey expression pattern in the CNS. DNA sequence comparison between the ey genes of distant Drosophila species has identified conserved modules that might be bound by the upstream regulatory factors of the ey gene in CNS development. Furthermore, by RNA interference and mutant studies, we show that ey expression in the brain is independent of the activity of toy and ey itself whereas in the eye primordia it requires both, supporting the notion that ey and toy are regulated by parallel and independent regulatory cascades in brain development.

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.

The development of the Drosophila compound eye requires the function of a set of evolutionarily conserved genes. Among these, the Drosophila Pax-6 gene eyeless ( ey ) plays a major role. ey has been considered a master control gene of eye development in the animal kingdom because targeted expression of ey and vertebrate as well as invertebrate homologs lead to the formation of ectopic eyes in Drosophila . We demonstrate that an intron of the ey gene contains an enhancer that regulates the eye specific expression of the gene in the eye disc primordia of embryos and in the eye imaginal discs of third instar larvae. Moreover, a 212-bp enhancer element is necessary and sufficient for the enhancer function. It is partially conserved in Drosophila hydei and contains putative Pax-6 Paired domain binding sites. We show that several binding sites are required for the eye specific expression, and, therefore, we propose a Pax-6 -like molecule to be a positive transactivator for the eye specific ey expression. This transactivator recently has been identified as twin of eyeless , the second Pax-6 gene in Drosophila .

The previously detected ebony ( e ) locus ( Caizzi et al., 1987 ) consists of a complex gene structure that is divided into seven exons. An open reading frame encoding the putative Ebony protein of 98.5 kDa exhibits homology to a family of peptide synthetases ( Stachelhaus and Marahiel, 1995 ), in good correlation with the proposed function as β-alanyl-dopamine synthetase. Multiple ebony transcripts are detected throughout development. P-factor mediated transformation of genomic DNA rescues the cuticle, electrophysiological and behavioural phenotypes. Fusion of the ebony reading frame with that of β-galactosidase of E. coli reveals expression in cuticle and nervous system. Strong staining in the first and, to a lesser extent, in the second optic neuropile may reflect the pronounced visual defect observed in ebony mutants. In addition, weak central brain and thoracic ganglion expression is detected in flies. Conservation of a multidomain protein structure known from peptide synthetases should have functional implications on the putative reaction mechanism of peptide bond formation.

Irregular facets ( If ) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel ( Kr ), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. Here, we show that If is a gain-of-function allele of Kr . We then used the If mutation in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr -interacting loci, two were cloned, and we examined whether they also represent components of a natural Kr -dependent developmental pathway of the embryo. We show that the two genes, eyelid ( eld ) and extramacrochaetae ( emc ), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr -expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr -dependent developmental processes.

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.

We have previously isolated a 500 bp-long cDNA clone, NO9-15, which is derived from a nuclear transcript originating from the heat shock locus 2-48B of Drosophila hydei (Peters et al. 1982). Sequence analysis shows that this clone carries 4 complete copies and 1 partial copy of a 115 bp repeat unit. The repeats are closely homologous with a maximal sequence divergence of about 10%. The sequence does not contain an open reading frame. The genomic organization of heat shock locus 2-48B, as probed with the cloned cDNA sequence NO9-15, is highly polymorphic. Four different allelic arrangements have been found in different inbred strains. A number of genomic clones isolated from region 2-48B, both in phage lambda and in cosmid vectors, all differ in length, mainly due to varying numbers of the NO9-15 repeat unit. These differences are found primarily in the proximal region of the locus. The transcribed region of these clones includes the distal sequence flanking the NO9-15 repeat as well as the NO9-15 repeat itself. An oligo A stretch was found between the distal flanking sequence and the NO9-15 repeat region.

The possibility that mechanisms of retinal determination may be similar between vertebrates and Drosophila has been supported by the observations that Pax6/eyeless genes are necessary and sufficient for retinal development. These studies suggest that the function of other gene families, operating during early eye development, might also be conserved. One candidate is the retinal homeobox (Rx) family of transcription factors. Vertebrate Rx is expressed in the prospective eye and forebrain and is required for eye morphogenesis, retinal precursor appearance, and normal forebrain development, indicating that it is an essential regulator of early eye and brain formation. Here, we test the hypothesis that Drosophila Rx (drx) is required for adult and larval eye development. We have isolated a drx null allele and demonstrate that the mutant compound eye and larval visual system is not detectably abnormal. However, we find that drx is required for development of a central brain structure, the ellipsoid body, suggesting that Rx function in the brain may be conserved. Finally, we characterize a novel anterior head phenotype and demonstrate that drx is required for clypeus development. Thus, our data suggest that drx may be required for the regulation of genes involved in brain morphogenesis and clypeus precursor development. We propose that differences in insect and vertebrate eye development may be explained by changes in gene regulation and/or the tissue of origin for eye precursor cells.

Homeodomain interacting protein kinase (Hipk) is a member of a novel family of serine/threonine kinases. Extensive biochemical studies of vertebrate homologs, particularly Hipk2, have identified a growing list of interactors, including proteins involved in transcriptional regulation, chromatin remodeling and essential signaling pathways such as Wnt and TGFβ. To gain insight into the in vivo functions of the single Drosophila Hipk we characterized loss of function alleles, which revealed an essential requirement for hipk. We find that in the developing eye, hipk promotes the Notch pathway. Notch signaling acts at multiple points in eye development to promote growth, proliferation and patterning. Hipk stimulates the early function of Notch in promotion of global growth of the eye disc. It has been shown in the Drosophila eye that Hipk interferes with the repressive activity of the global co-repressor, Groucho (Gro). Here, we propose that Hipk antagonizes Gro to promote the transmission of the Notch signal, indicating that Hipk plays numerous roles in regulating gene expression through interference with the formation of Gro-containing co-repressor complexes.

We report the isolation of seven homeobox-containing genes from the honeybee (Apis mellifera). Sequence analysis of all homeoboxes and some flanking sequences showed that six of seven genes are more than 90% identical to their corresponding Drosophila homologues within the homeobox and, with one exception, also in the flanking sequences. The homologues that were identified include three homeotic selector genes [Sex combs reduced (Scr), Antennapedia (Antp), and abdominal-A (abd-A); the two engrailed (en) genes; and the muscle segment homeobox (msh)]. Surprisingly, no homologue of the segmentation gene fushi tarazu was found in the honeybee. For the remaining bee gene, a Drosophila homologue is not known. This indicates that, with some exceptions, structurally homologous genes are involved in the control of bee and Drosophila development, although Hymenoptera differ significantly in their embryogenesis from Diptera and have evolved separately for some 250 million years.

A comparison of gene structure, sequence, and transcription pattern of heat shock loci 93D of Drosophila melanogaster and 48B of Drosophila hydei has been performed. Both heat shock loci consist of an unique region that is flanked by an internally repetitive element. Different members of these elements are highly conserved, repeat unit length, however, and primary sequence diverged totally. Whereas the overall gene structure in both species is substantially related, sequence conservation is only observed at very few sites in the unique region. These represent primarily sequences that are identified as regulatory elements for faithful transcription and processing. The number and size of transcripts obtained from heat shock locus 48B in third instar larvae closely resembles the pattern of heat shock locus 93D. Thus their quite alike structure and transcription pattern suggest strongly a conserved hitherto unknown function.

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

The Drosophila Pax-6 gene eyeless acts high up in the genetic hierarchy involved in compound eye development and can direct the formation of extra eyes in ectopic locations. Here we identify sine oculis and eyes absent as two mediators of the eye-inducing activity of eyeless. We show that eyeless induces and requires the expression of both genes independently during extra eye development. During normal eye development, eyeless is expressed earlier than and is required for the expression of sine oculis and eyes absent, but not vice versa. Based on the results presented here and those of others, we propose a model in which eyeless induces the initial expression of both sine oculis and eyes absent in the eye disc. sine oculis and eyes absent then appear to participate in a positive feedback loop that regulates the expression of all three genes. In contrast to the regulatory interactions that occur in the developing eye disc, we also show that in the embryonic head, sine oculis acts in parallel to eyeless and twin of eyeless, a second Pax-6 gene from Drosophila. Recent studies in vertebrate systems indicate that the epistatic relationships among the corresponding vertebrate homologs are very similar to those observed in Drosophila.