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Background Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. Results In this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions. Conclusion This analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future. Supplementary Information The online version contains supplementary material available at 10.1186/s41065-021-00209-6.

Homeodomain-interacting protein kinase (Hipk), the Drosophila homologue of mammalian HIPK2, plays several important roles in regulating differentiation, proliferation, apoptosis, and stress responses and acts as a mediator for signals of diverse pathways, such as Notch or Wingless signalling. The Paired box protein 6 (Pax6) has two Drosophila homologues, Twin of eyeless (Toy) and Eyeless (Ey). Both stand atop the retinal determination gene network (RDGN), which is essential for proper eye development in Drosophila. Here, we set Hipk and the master regulators Toy and Ey in an enzyme-substrate relationship. Furthermore, we prove a physical interaction between Toy and Hipk in vivo using bimolecular fluorescence complementation. Using in vitro kinase assays with different truncated Toy constructs and mutational analyses, we mapped four Hipk phosphorylation sites of Toy, one in the paired domain (Ser121 ) and three in the C-terminal transactivation domain of Toy (Thr395 , Ser410 and Thr452 ). The interaction and phosphorylation of the master regulator Toy by Hipk may be important for precise tuning of signalling within the RDGN and therefore for Drosophila eye development.

The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

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.

Hox genes are known to control the identity of serially repeated structures in arthropods and vertebrates. We analyzed the expression pattern of the Hox genes Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), and Ultrabithorax/abdominal-A (Ubx/abd-A) from the honey bee Apis mellifera. We also cloned a cDNA with the complete coding region of the Antennapedia gene from Apis. Comparison with Antp proteins from other insect species revealed several regions of homology. The expression patterns of the isolated Hox genes from Apis showed that the original expression patterns of Dfd, Scr, and Antp appear between late blastoderm and early germ band stage in a temporal and spatial sequence. Each of them shows up as a belt, spanning approximately two segment anlagen, Dfd in the anterior gnathal region, Scr in the posterior gnathal and anterior thoracic region, and Antp in the thoracic region. Following expansion of the Antp domain in the abdomen as a gradient towards the posterior, Ubx/abd-A expression appears laterally in the abdomen. During gastrulation and in the germ band stage the domains of strong expression do not overlap any more, but touch each other. After gastrulation the borders of the expression domains partly correlate with parasegment and partly with segment boundaries. Laterally, gaps between the domain of each gene may show no expression of any of the genes examined.

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.

A Drosophila gene that contains both a paired box and a homeobox and has extensive sequence homology to the mouse Pax-6 (Small eye) gene was isolated and mapped to chromosome IV in a region close to the eyeless locus. Two spontaneous mutations, ey2 and eyR, contain transposable element insertions into the cloned gene and affect gene expression, particularly in the eye primordia. This indicates that the cloned gene encodes ey. The finding that ey of Drosophila, Small eye of the mouse, and human Aniridia are encoded by homologous genes suggests that eye morphogenesis is under similar genetic control in both vertebrates and insects, in spite of the large differences in eye morphology and mode of development.

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.

Using low stringency hybridisation with a Drosophila melanogaster EF-1α gene fragment we have isolated a genomic DNA clone encoding elongation factor 1α (EF-1α) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1α sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1α protein family.

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.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. By high-resolution neuroanatomy, we show that eyeless ( ey ), twin of eyeless , and dachshund (dac ), which are implicated in eye development, also are expressed in the developing MBs. Mutations of ey completely disrupted the MB neuropils, and a null mutation of dac resulted in marked disruption and aberrant axonal projections. Genetic analyses demonstrated that, whereas ey and dac synergistically control the structural development of the MBs, the two genes are regulated independently in the course of MB development. These data argue for a distinct combinatorial code of regulatory genes for MBs as compared with eye development and suggest conserved roles of Pax6 homologs in the genetic programs of the olfactory learning centers of complex brains.

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.

We have isolated recombinant DNA clones coding for female specific proteins from Drosophila melanogaster. By screening with 32P-(A)+RNA from male and female flies, respectively, we were able to isolate a set of 100 cDNA clones which showed a positive hybridization signal for RNA from female flies. These clones have been rescreened with RNA isolated from fat body of two day old male and female flies. We obtained four positive cDNA clones. Isolation of the corresponding genomic sequences, construction of the physical map and comparing it with the restriction maps published by Barnett et al. (1) led us to conclude that we had isolated the genes coding for two of the three known yolk protein precursors (vitellogenins), YP I and YP II. The sequence of the YP I gene was determined. It gives rise to a protein of 48 700 dalton MW which might be cleaved to a MW of 46 700 during transport. The coding sequence is interrupted by a single intron of 75 bases in length. The proposed leader sequence starts at a region homologous to six heat shock gene sequences at the site of initiation of transcription, suggesting the existence of an 11 bp cap specific consensus sequence for Drosophila melanogaster.

The heat-shock locus 93D from Drosophila melanogaster has recently become available for a molecular analysis. We established a restriction site map of a recombinant DNA clone covering the major part of heat-shock locus 93D. This clone includes part of a repetitive Taq I region and neighbouring unique sequences. The portion of the Taq I repeat analysed consists of tandemly arranged sequence blocks of about 280 base pairs (bp) in length. Using genomic and cDNA as hybridization probes we examined the transcription of 93D in 2- to 4-day-old flies. We identified two major RNA classes enhanced after heat shock, namely nonpolyadenylated transcripts of heterogeneous length derived from the repetitive region and one discrete polyadenylated transcript in spliced and unspliced form from the neighbouring unique region. The occurrence of a highly heterogeneous poly(A)- transcript and high levels of an unspliced discrete poly(A)+ species suggests unusual mechanisms of transcription regulation in the 93D region.

We have characterized two previously cloned genes, F1 and F2 (1) that code for elongation factor EF - 1 alpha of Drosophila melanogaster. Genomic Southern blot hybridization revealed that they are the only gene copies present. We isolated cDNA clones of both transcripts from embryonal and pupal stage of development that cover the entire transcription unit. The 5' ends of both genes have been determined by primer extension and for F1 also by RNA sequencing. These start sites have been shown to be used consistently during development. Comparison of cDNA and genomic sequences revealed that EF - 1 alpha,F1 consists of two and EF - 1 alpha,F2 of five exons. The two described elongation factor genes exhibit several regions of strong sequence conservation when compared to five recently cloned eucaryotic elongation factors.

Screening a cDNA library of 2- to 3-day-old flies with poly(A) + RNA from male and female flies, we were able to isolate a small number of clones hybridizing preferentially with RNA from female flies. Four cDNA clones, derived from a single mRNA species, proved to be highly abundant in female flies. When we screened a genomic library with the longest cDNA, we obtained two genomic clones, F1 and F2 ; F1 was a direct copy of the cDNA and F2 was obtained by cross-hybridization. A detailed analysis of these genomic clones revealed two independent genes coding for proteins of 50 kDa that are >90% homologous. RNA analysis with gene-specific probes from the 3′ untranslated region showed an expression of F1 in all stages of development with a 5- to 10-fold overexpression of this RNA in female flies compared with males. In contrast to F1, F2 is mainly expressed in late pupae and is expressed only at low levels in adult flies.

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.

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.

We have isolated and characterized a homeoboxcontaining gene from the honeybee Apis mellifera. Its homeobox region shows a high degree of sequence similarity to the homeobox of the Drosophila gene Deformed (Dfd). At the DNA level 82% of the basepairs are the same, whereas the putative amino acid sequences are identical between the bee and the fruitfly genes. Similarity is also present 5′ and 3′ to the homeobox. Using this isolate as a probe we have performed in situ hybridization on sections from blastoderm-stage embryos of the honeybee Apis mellifera. In early blastoderm stages we found a rather irregular pattern of labelled nuclei. In middle stages we found silver grains over each nucleus and also over the cytoplasm in a belt of blastoderm cells in the prospective gnathal region. These results indicate that the Deformed genes from honeybee and fruitfly are homologous both with respect to their DNA sequence and their spatial and temporal pattern of expression during embryogenesis.

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.